DNA Wrapping and the Nature of Interaction Between E. Coli ...

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DNA Wrapping and the Nature of InteractionBetween E. Coli RNA Polymerase and PromoterDNA.Parisa JazbiLouisiana State University and Agricultural & Mechanical College

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DNA WRAPPING AND THE NATURE OF INTERACTION BETWEEN K coli RNA POLYMERASE AND PROMOTOR DNA

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and

Agricultural and Mechanical College in partial fulfilment of the

requirements for the degree of Doctor of Philosophy

in

The Department of Microbiology

byParisa Jazbi

B.S., Shiraz University, 1991 December 1997


UMI Number: 9820725

UMI Microform 9820725 Copyright 1998, by UMI Company. All rights reserved.

This microform edition is protected against unauthorized copying under Title 17, United States Code.

UMI300 North Zeeb Road Ann Arbor, MI 48103


In the name of God most merciful most gracious

DEDICATION

To my parents for their love and faith in me

ii


ACKNOWLEDGMENTS

I am greatly indebted to my major adviser who has been a significant mentor. His

constant support, guidance, and encouregment made this work possible.

I would also like to thank the members of my committee, Dr. Randall Gayda, Dr.

Ronald Siebeling, Dr. Gregory Jarosik, Dr. Gregg Pettis, Dr. Ding Shih, and Dr. David

Senior for their invaluable and sensible advice.

My thanks also to my fellow graduate students in the microbiology department for

their friendship and encouragement.

My brother, Ali deserves a special thank you for being my inspiration and incentive

to finish this work.

Most of all, I am grateful to my husband, without whose love and belief in me I

never could have completed this degree. He believed in me when I had a hard time believing

in myself.

iii


TABLE OF CONTENTS

DEDICATION................................................................................................................ ii

ACKNOWLEDGMENTS ............................................................................................iii

LIST OF TABLE............................................................................................................vi

LIST OF FIGURES......................................................................................................vii

ABSTRACT.................................................................................................................... ix

INTRODUCTION.......................................................................................................... 1DNA-dependent RNA polymerase....................................................................... 1Promoters............................................................................................................. 5Transcription initiation kinetics............................................................................ 8Regulators of transcription................................................................................. 10DNA curvature................................................................................................... 13DNA curvature in prokaryote promoters............................................................15

MATERIALS AND M ETHODS................................................................................ 18Materials..............................................................................................................18Plasmid transformation and isolation.................................................................. 18Construction of Alul56 derivative promoters.....................................................18Primer labeling .............................................................................................21PCR amplification...............................................................................................21RNA polymerase isolation..................................................................................22DNase I footprinting analysis............................................................................. 22Gel retardation analysis...................................................................................... 23KMn04 footprinting...........................................................................................24Single-round run-off transcription assay............................................................ 24

RESULTS..................................................................................................................... 26DNase I footprint analysis of the interaction between E. coliRNA polymerase and Alul56 and Ball29 and their derivatives..........................26Nature of the interaction between RNA polymerase and DNA upstreamof the -35 region................................................................................................. 41The relationship between open promoter complex formation andDNA wrapping................................................................................................... 54Test for DNA wrapping at different stages of transcription initiation................ 54Effect of AT-rich regions in DNA wrapping and transcription efficiency.......... 63

iv


Effect of a mutation in the C-terminal domain of a subunit on the interaction between RNA polymerase and DNA upstream of the -35 region.......................74

DISCUSSION................................................................................................................ 79Effect of DNA curvature immediately upstream of the -35 region on DNAwrapping by the RNA polymerase...................................................................... 79Model for the role of DNA curvature in RNA polymerase binding andtranscription initiation........................................................................................80The nature of the interaction between E. coli RNA polymerase and DNAupstream of the -35 region.................................................................................. 82Existence of unwrapped open promoter complexes............................................83DNA wrapping in the absence of open promoter complex formation.................84Comparison of the effect of AT-rich regions and curved DNA on wrappingand transcription initiation...................................................................................85Contribution of the a subunit of RNA polymerase on wrapping........................ 86Summary of the model........................................................................................ 86

REFERENCES .............................................................................................................87

VITA............................................................................................................................. 98

v


LIST OF TABLES

1. Promoters................................................................................................................. 21

vi


LIST OF FIGURES

1. Nucleotide sequence of the Alul56 and Ball 29 promoters..................................... 27

2. Nucleotide sequence of the Alul56, Ball29, and their derivatives..........................28

3. DNase I footprint analysis of the Alul56 promoter..................................................30

4. DNase I footprinting of the AluExt promoter bound by RNA polymerase..............33

5. The summary of DNase I footprint analysis on both strands o f theAlul56 (A) and the AluExt (B) promoters...............................................................

6. DNase I footprint analysis of Ball29 promoter........................................................ 37

7. DNase I footprint analysis of BalExt promoter........................................................ 39

8. The summary of DNase I footprinting results on both strands ofBall29 (A) and BalExt (B )........................................................................................42

9. DNase I footprint analysis of the Alul56 promoter bound to RNA polymeraseas a function of NaCl concentration........................................................................... 45

10. Effect of NaCl concentration on DNase I footprint pattern of AluExt.....................48

11. DNase I footprint analysis of the Ball29 promoter as a function ofNaCl concentration....................................................................................................50

12. Effect of NaCl concentration on the DNase I footprint analysis of theBalExt promoter...................................................................................................... 52

13. Open promoter complex formation at different salt concentrationsfor Ball29 (A) and Alul56 (B )................................................................................. 55

14. Effect of temperature on open complex formation of Alul56 promoter.................. 57

15. DNase I footprint analysis of Alul56 as a function o f temperature..........................58

16. Effect of heparin on the footprint analysis of the Alul56 promoter boundto RNA polymerase at 37°C and 0 °C .................................................................... 61

17. Nucleotide sequence of the Alul56, AluExt, AluDel, and AT-rich derivatives 64

vii


18. DNase I footprint analysis of the Alul56, AluUnc, AluUp, andAlu27AT promoters................................................................................................. 65

19. DNase I footprint analysis of the Alul56, AluExt, Alu8AT, andATDel promoters..................................................................................................... 67

20. Gel retardation analysis of Alul56, AluUnc, AluUp, and Alu27AT.........................70

21. Gel retardation analysis of Alul56, AluExt, Alu8AT, and AluDel...........................71

22. Transcribable promoter complexes formed as a function of temperature................. .72

23. DNase I footprint analysis of the AIuPr, BalP^ and APR promoters as afunction of wild-type (lane 2) and mutant (lane 3) RNA polymerase.......................76


ABSTRACT

Regulation of transcription most often occurs at the stage of initiation. RNA

polymerase binding to the transcription start site, the promoter, is influenced by many

nucleotide sequence elements. The predominant recognition sequences are those bound by

the a subunit of RNA polymerase located at -10 and -35 relative to initiation site of most

promoters. Another element involved in this regulation is intrinsic DNA curvature. This

study examined the contribution of intrinsically curved DNA immediately upstream of the

promoter to the interaction between Escherichia coli RNA polymerase and this DNA

DNase I footprinting analysis confirmed that RNA polymerase wraps DNA upstream of the

promoter around the enzyme. The nature of interaction between DNA upstream of

promoter and RNA polymerase was explored using addition of NaCl. The wrapped

complex was not observed at NaCl concentration above 150 mM suggesting the

electrostatic, sequence-independent nature of the interaction. Study of the effect of

temperature on DNA wrapping and open promoter complex formation demonstrated the

existence of closed, wrapped complexes. No wrapped complexes survived a 30 second

heparin challenge indicating the absence of wrapped open complexes. The above data

provided evidence that DNA wrapping occurs prior to open complex formation. Promoters

containing an AT-rich region or the UP element of ribosomal RNA promoter rm BPl were

constructed. Using a gel retardation assay, the relative affinity of RNA polymerase for these

promoters was compared to that observed for curved DNA-containing promoter. The

promoter containing curved DNA displayed the highest binding to RNA polymerase. The

ix


presence of curved DNA favored the formation of the wrapped complex. A run-off

transcription assay limited to a single round of initiation examined the efficiency of

transcription for these promoters as a function of temperature. Relative to promoters

lacking curved DNA, the promoter with curved DNA formed significantly more heparin-

resistant, closed complexes at low temperature. These complexes could quickly isomerize

to open complex at 37°C. We propose that curved DNA facilitates wrapping of DNA

around RNA polymerase and enhances the transition from a heparin sensitive closed

complex to a heparin resistance closed promoter complex.

x


INTRODUCTION

The regulation of gene expression is essential to the efficiency and economy of

bacterial growth. Control of the activity of bacterial genes usually starts at the stage of

transcription. The controlled step in this process is commonly the initiation of transcription.

The least amount of energy and other resources are used by controlling the very first step

of transcription.

Several elements are involved in the multiple step process o f initiation of

transcription. These components are common among several species of bacteria, including

Escherichia coli and Bacillus subtilis. DNA dependent RNA polymerase binds to the

promoter, a specific sequence of DNA, with or without the regulatory proteins that repress

or activate transcription. Most of the interactions between the RNA polymerase and the

promoter DNA are mediated through consensus DNA sequence elements, such as those

commonly found at -10 and -35 relative to the site of transcription initiation. These

interactions are involved in the separation of the DNA strands. Other DNA elements, such

as intrinsically curved DNA, have been shown to affect the initiation of transcription.

Involvement of these elements in regulation of transcription initiation can be further studied

using in vitro systems.

DNA-dependent RNA polymerase. RNA polymerase is the cell's central processing unit.

This enzyme was discovered in 1959 by Weiss and Gladstone. RNA polymerase copies the

information from a DNA template to RNA molecule specifically. It synthesizes an RNA

molecule that is complementary to the DNA template. The RNA polymerase of E. coli is

composed of a core enzyme with the subunit structure of (Chamerlin, 1982; McClure,

1


2

198S) and one of the several species of a subunit which are involved in the specific

promoter recognition (Helmann and Chamberlin, 1988). The catalytic site of RNA

polymerase is located on the 3 (150,619 Daltons) subunit (Glass et aL, 1982), while RNA

polymerase binds to the DNA nonspecifically via the 3' (155,162 Daltons) subunit (Fukuda

and Ishihama, 1979).

Alpha (a) subunits are involved in assembly of the RNA polymerase (Ishihama,

1981), and also in protein protein interactions with positive regulators (Ishihama, 1992).

E. coli RNA polymerase with deletion in the C-terminal one third of the a subunit is

enzymatically active, however, some activator dependent promoters cannot be transcribed

by this mutant RNA polymerase. This suggests that the C-terminal region of a interacts

with some transcription factors (Igarashi and Ishihama, 1991). The cyclic AMP receptor

protein (CRP) contacts the a subunit of RNA polymerase when activating transcription at

“class I promoters” in which the CRP binding site is located upstream of the -35 region.

The targets for CRP interaction also reside in the C-terminal domain (CTD) of the a subunit

(Chen et aL 1994; Zhou, et aL, 1994; Zou et aL, 1992). In addition, for a number of other

bacterial activators, such as AraC, OxyR, PhoB, FNR, and integration host factor (IHF), it

has been shown that they mediate their effect via the a CTD (Ishihama, 1992). The function

of the C-terminal domain of the a subunit of E. coli RNA polymerase in basal expression

and integration host factor-mediated activation of the early promoter of bacteriophage MU

has been recently studied by Vanulsen et aL(1997). The results of this study indicates that

interaction of the a CTD with DNA is involved not only in the IHF mediated activation of


3

early promoter but also in maintaining the basal level of transcription from this promoter.

It was shown in rm B P 1 promoter that sequences between -40 and -60, the so called “UP

element”, increases transcription by interacting with the a subunit of RNA polymerase (Ross

et aL, 1993; Gaal et aL, 1996). RNA polymerase lacking the C-terminal domain of the a

subunit was unable to contact the UP element, and therefore, transcription from rm B PI

promoter was less efficiently initiated (Ross et aL, 1993). In the same study, it was shown

that purified a binds specifically to the UP element. The binding was diminished when

mutated a was used. Therefore, it can be concluded that the UP element represents a third

promoter recognition region and that a acts directly in promoter binding.

Six different a subunits have been found in association with E. coli polymerase. All

these sigma factors play an important role in the specificity of the transcription initiation.

Alternative a subunits control the transcription of coordinately regulated sets of genes

distinct from those recognized by the primary o factor (Doi and Wang, 1986; Losick et aL

1986). In exponentially growing cells, most genes are transcribed by a 70 (70,263 Daltons).

Genes for heat shock proteins are transcribed by a32 (Grossman et aL, 1984; Cowing et aL,

1985; Fujita et aL, 1987). A second heat shock sigma factor, a24, has been identified as a

requirement for survival of high temperatures. The a54 is required for transcription of the

genes which are controlled by the availability of nitrogen source (Garcia et aL, 1977). The

promoters of the genes that are expressed only in stationary growth phase can be recognized

by a 3* (Tanaka et aL, 1993; Heggearonis, 1993). Genes for flagellar synthesis and

chemotaxis are controlled by a28 (Amosti et aL, 1989). Recently, evidence for contact


between CRP and a™ subunit of E coli RNA polymerase has been published for class II

promoters (Ruzhonsl et aL, 1995). The data indicates the role of the a 70 subunit in

transcription activation.

Unlike E. coli, B. subtilis undergoes a unique developmental process, called

sporulation. This process is directed by a cascade of sigmas which lead to the temporal

activation of different sets of genes during sporulation (Losick and Pero, 1981; Doi, 1982).

B. subtilis RNA polymerase is very similar in core subunit composition to that of-El coli.

However, B. subtilis also contain an additional polypeptide, the 8 subunit (20,400 Dalton)

(Doi, 1982; Lampe et aL, 1988) and two © subunits. The 8 subunit is responsible in

promoter discrimination. It appears that the 5 subunit allows RNA polymerase to

differentiate between strong and weak promoters (Achberger and Whiteley, 1982; Whiteley

et aL, 1982). The a subunit may be required for rapid recycling of the core RNA polymerase

after termination (Juang and Helmann, 1994). The majority of cellular transcription is

controlled by a*, the major vegetative sigma of B. subtilis (Losick et aL, 1986). The o° is

involved in flagellar synthesis, chemotaxis, and nutrient stress responses (Helmann and

Chamberlin, 1987; Helmann et aL, 1988). In addition to a * , or B, a c(Wiggs et aL, 1981)

and oD (Haldemwang and Losick, 1980) are associated with vegetatively growing cells.

Recently, three minor sigmas, a* , a Y, and o w, involved in the control of gene expression

in response to environmental stresses were discovered (Huang et al., 1997). There are other

sigmas associated with sporulating B. subtilis, including a", a F, o E, a °, and a K factors

(Losick and Stragier, 1992). During sporulation the cell undergoes an asymmetric septation


5

that gives rise to a small cell destined to become the endospore and a larger, terminally

differentiated mother cell. The a ” is a minor sigma factor involved in the transcription of

both vegetative and sporulating genes. In this process, the inactive form of c f is transcribed

by o” in the mother cell before the asymmetric cell division. Activation of o* after septation

directs the expression of oG. The (9 is produced in forespore and involved in

transcription of forespore specific genes. The active sigma in mother cell, a 2, is required

for prespore formation. Endospore coat proteins are the product of cot genes. The 0 s is

necessary for expression of these genes in the mother cell.

Alternative sigma factors also have been found in phage SP82 or SP01 infected

B. subtilis cells. During the infection process, phage gene expression is controlled by a

cascade of sigmas. The host a* RNA polymerase is required for expression of early phage

genes including gp28, which encodes a®28. The a®2* displaces a* on the RNA polymerase

shutting down host and early phage gene expression. The middle phage gene promoters are

transcribed by o®28 RNA polymerase. Among the middle genes are the gp33 and gp34, the

gene products of which form a sigma, o®3334, these new sigmas displace <3®28 from the RNA

polymerase shutting down middle phage gene expression and permitting late phage gene

expression. Thus, the temporal gene expression during phage development is regulated by

alternate o factors (Duffy et aL, 1975; Talkington and Pero, 1978; Lee and Pero, 1981;

Geidushik and Ito, 1982).

Promoters. Promoters are specific sequences of DNA located upstream of transcription

start sites. RNA polymerase recognizes and binds to the promoter and initiates


6

transcription. The level of expression o f a gene is greatly affected by the sequence of the

promoter (Galas et aL 1985). The sequence comparison of many promoters has generated

consensus sequence for particular RNA polymerase. The consensus sequence for K coli

ct70 holoenzyme is TATAAT (the -10 region) located about 10 base pairs upstream of the

transcription start she and TTGACA (the -35 region) located 17 base pairs upstream of the

-10 region (Rosenberg and Court, 1979; Siebenlist et aL 1980; Hawly and McClure, 1983;

Harley and Reynolds, 1987). The role of -10 and -35 regions in the initiation of

transcription has been studied. The -10 region is involved in DNA melting, while the -35

region plays a role in initial binding of RNA polymerase. In addition to binding affinity, the

rate of open promoter complex formation is also affected by base substitution in -35 region

(Hawley and McClure, 1982; Koboyashi et aL, 1990). Genetic studies have shown that two

regions of most a factors are involved in interaction with the -10 and -35 regions of

promoters (Waldburger et aL, 1990; Siegle, et aL, 1989; Kenney and Moran, 1991). In feet,

amino acid substitution in the conserved sequences of a which interact with the -35 region

can alter promoter specificity. Thus, holoenzyme containing such a mutant a recognizes

promoters which normally are not transcribed by wild-type holoenzyme (Schmidt et al.,

1990). It has been also demonstrated that holoenzyme containing alternate o subunits

recognizes unique promoters with different consensus sequences (Doi and Wang, 1986).

Several elements are responsible for promoter strength or the relative

transcriptional effectiveness. Promoter strength is profoundly affected by mutation in the

-10 and -35 region. Mutations causing divergence from the consensus sequence reduce the

promoter strength, while mutations increasing the level of homology exhibit enhanced


7

promoter activity (McClure, 1985; Hawley and McClure, 1983; Harley and Reynold, 1987).

The actual sequence of spacer DNA does not play a major role in promoter strength,

however the distance between two consensus regions is critical for productive interaction

between RNA polymerase and promoter.

Investigators have identified an additional conserved promoter sequence immediately

upstream of the -10 region (Moran et aL, 1982). A sequence around the +1 promoter

region has been also identified to affect transcription in B. subtilis but not in E. coli (Henkin

and Sonenshein, 1987).

Recently it has been established that there is a third important sequence element, in

addition to the -35 and -10 elements, at some E. coli promoters (Ross et aL, 1993). The

exceptional strength of these promoters (ribosomal RNA genes) is due to the UP element,

an AT-rich sequence of 20 base pairs located immediately upstream of the -35 region or the

UP element. Fredrick et al. (1995) also demonstrated that transcription from the B. subtilis

felagellin promoter is stimulated by an UP element both in vivo and in vitro. It is likely that

promoter strength is a function of all promoter elements, with very strong promoters having

near consensus elements while weaker promoters deviate significantly from the consensus.

Although the major forms of the RNA polymerase from E. coli and B. subtilis

recognize the same consensus sequences (Moran et aL,1982; Galas et aL,1985; Graves and

Rabinowitz, 1986), some differences have been reported for B. subtilis RNA polymerase.

B. subtilis RNA polymerase generally requires DNA sequences that are more similar to the

consensus sequence. In addition, inefficient utilization of E. coli promoters has been

observed when they are introduced to B. subtilis (Wigg etal.,\919\ Lee et al., 1980; Henkin


8

and Sonenshein,1987; Whipple and Sonenshein,1992). In contrast, B. subtilis promoter can

be utilized efficiently in £ coli. Investigators have identified an AT-rich sequence upstream

of the -35 region in many B. subtilis promoters, including promoters for early gene

expression in phage SPO1, which may be responsible for functional differences between K

coli and B. subtilis (Moran et aL,1982). To be fully functional, many promoters need

additional sequences where regulatory proteins bind. These proteins can act as repressors

or activators and regulate promoter function (Pabo and Sauer, 1984).

Transcription initiation kinetics. E. coli is the traditional system for the study of

transcription initiation kinetics. Transcription initiation by RNA polymerase is a

complicated process comprised of a series of defined biochemical intermediates

(Chamberlin, 1974; VonHipple et aL,1984; McClure, 1985). Two intermediates were

involved in the original model. In this model, after binding of RNA polymerase (R) to the

promoter (P), first a closed promoter complex is formed (RPc). The close complex then is

isomerized to open promoter complex (RP0) in which the DNA strands of the promoter

region are melted (Sienbenlist, 1979; Kirkegaad et aL, 1983), exposing the template strand

to RNA polymerase for RNA synthesis.

R + P '*— *“ RPC T— *“ RP0 ^ R N A synthesis

The existence of at least two intermediate complexes before open promoter complex

was documented (Rosenberg et aL,1982; Roe et aL,1984; Roe, 1985; Buc and McClure,

1985; Duval-valentin and Ehrlich, 1987). In most recent kinetic scheme RNA polymerase

first binds to promoter and forms a closed complex. A conformational change, possibly


9

leading to partial unwinding of the helix, forms a second closed complex, PR^ . Although

closed promoter complexes are normally sensitive to heparin, this complex is heparin

resistant. Heparin is a polyanion with high affinity for RNA polymerase (Walter et

aL,1967). Isomerization later on leads to open promoter complex formation in a

temperature-dependent process. Increasing the temperature activates the isomerization and

drives the open promoter complex formation. In & coli abortive RNA synthesis has been

identified as an intermediate step between open promoter complex formation and productive

RNA synthesis (Carpouis and Gralla, 1980). RNA polymerase of£. coli can go through

multiple cycles of abortive initiation. After each cycle, a short RNA oligomer is released

from the transcription complex. Usually after synthesis o f 9 to 11 bases of RNA, promoter

clearance occurs and the RNA polymerase complex enters the productive state (Grachev

and Zaychikov, 1980; Streney and Crother, 1985; Garpouisis and Gralla, 1985). After the

RNA polymerase clears the promoter, the o subunit is released, and the elongation phase

begins. The process ends with RNA chain termination.

There are many useful techniques to study thermodynamics and kinetics of specific

RNA polymerase-promoter interactions. Binding assays, run-off transcription (Strauss et

aL,1980; Rose et aL, 1984) and abortive initiation (Hawley and McClure, 1982) are among

the widely used techniques in this field. Chemical probes have been very useful to

investigate RNA polymerase-DNA complexes. DNA melting has been studied using

potassium permanganate which is known to react prefentially with pyrimidines in single

stranded DNA. This chemical probe can be used to detect DNA melting and open promoter

complex formation both in vitro and in vivo (Sasse-Dwight and Gralla, 1989; Kainz and


10

Roberts, 1992). Information about DNA conformation and accessibility to solvent and the

presence of single-stranded DNA in open promoter complex has been accumulated using

1,10-phenanthroline-copper. There are some other chemical probes, such as hydroxy radical

which has been used in the investigation. DNase I footprinting analysis has helped to

demonstrate the interaction between RNA polymerase and promoter region and to define

the kinetic intermediates of transcription initiation. DNase I cleavage patterns are different

for open and closed complexes. Gel retardation analysis (Crothers, 1987; Gamer and

Revzin, 1986) also known as gel shift assay can be used to determine the binding affinity of

RNA polymerase for a promoter.

Regulators of transcription. Regulation of transcription initiation can be influenced by

components in addition to the -10 and -35 regions. These components include proteins or

DNA structural factors which either increase or decrease promoter activity and the

initiation of transcription. & coli CRP is a structurally characterized transcription activator

protein (Kolb et aL, 1993). In the presence of the allosteric effector cyclic AMP, CRP binds

to specific DNA sites located near or in CRP-dependent promoters. Indeed, the binding of

RNA polymerase to the promoter is enhanced by contact with bound CRP. CRP also

stimulates transcription by bending the DNA. The bend induced by CRP, is estimated to

be about 100° to 130° (Tompson and Landy,1988; Zinkle and Crothers, 1990). The

binding of CRP and of RNA polymerase are cooperative because they bend DNA in the

same direction. There are two classes of CRP-dependent promoters. In class I , the DNA

site for CRP is located upstream of the DNA site for RNA polymerase. The best

characterized class I promoter is the ZoePl promoter. In class II, however, the binding site


11

for CRP overlaps the DNA site for RNA polymerase. The gal? I promoter is an example

of class n. Amino acids 156-164 of CRP constitute an activator region essential for

transcription activation at both class I and class II CRP-dependent promoters, but it is not

essential for DNA binding and DNA bending (Bell et aL,1990; Eschenlauer and

Reznikoff 1991; Zhou et aL, 1993; Niu et al,1994; Niu et aL, 1996). For both class I and

class II promoters, the activating region functions through protein-protein interaction with

RNA polymerase. It is now believed that CRP interacts with the RNA polymerase a subunit

C-terminal domain (aCTD) and facilitates the binding o f aCTD to DNA adjacent to CRP

(Igarashi and Ishihama, 1991; Kolb et aL,1993; Chen et aL, 1994; Belyaeva et aL,1996).

For class I promoters, the interaction between the activating region and aCTD appears to

be the entire basis of transcription activation. The CRP-induced bend in class I tends to

become localized at an apical loop of supercoiled D N A , thus helping the DNA to wrap

around the promoter- bound RNA polymerase. In class n , the transcription activation

requires not only the interaction between the activating region and aCTD, but also an

interaction between a second site in the activating region of CRP and the RNA polymerase

a subunit N-terminal domain (Niu et aL, 1996). This new finding establishes that an

activator can interact with multiple targets within the transcription machinery and thereby

affect multiple steps of transcription initiation.

Another example of a regulatory protein which can bend the DNA is the P4 protein

of B. subtilis phage ((>29 . This protein is responsible for the switch from early to late

transcription in the bacteriophage development. Protein P4 is produced at an early stage

o f infection and activates the transcription from the promoter for late genes called PA3.


12

This promoter is inactivated in the absence of protein P4. Activation of transcription in the

presence of P4 is via stabilizing the binding of B. subtilis RNA polymerase to the late

promoter as a closed complex (Nuez et aL, 1992). There is also evidence that P4 protein

interacts directly with RNA polymerase at the PA3 promoter (Nuez et aL, 1991; 1992;

Serrano et al,1991).

It has been shown recently that N4ssB, a single stranded DNA binding protein

encoded by bacteriophage N4, interacts with the carboxyl terminus of the RNA polymerase

P' subunit(Miller et aL, 1997). N4ssB activates transcription by the K coli a70 RNA

polymerase at the N4 late promoter.

The level of transcription activation is also influenced by DNA supercoiling. It has

been observed that supercoiling may either increase or decrease transcription activation in

some genes. Nevertheless, many other genes are not influenced by these phenomena (Pruss

and Drelica, 1989). DNA supercoiling may facilitate binding of RNA polymerase to the

promoter and activator protein to the DNA Transcription initiation at the lacPl promoter

of E. coli is assisted by DNA supercoiling in a CRP-dependent fashion (Meikleijohn and

Gralla, 1989).

Negative regulation of transcription can be also mediated through regulatory

proteins. In 1961 Jacob and Monad proposed that a regulator gene produces a repressor

that can interact with a DNA segment called the operator. Upon this interaction, initiation

of transcription is prevented. This type of negative control has been observed for the lac

operon. The product of the lac I gene is an allosteric repressor protein with two binding

sites. One binding site is for the operator region and the other is for the inducer molecule


13

(allolactose). Upon binding of the lac repressor to the operator region, RNA polymerase

is prevented from initiating transcription. However, in the presence of an inducer molecule,

this molecule binds to the repressor and alters the structure of the repressor so it no longer

binds to the operator. Thus the operator is unoccupied and the RNA polymerase can bind

the promoter and start transcription.

There are other factors which influence how proteins interact with the DNA during

transcription. DNA looping plays an important role in transcriptional control. This

phenomena is mediated by binding of regulatory proteins to two distinct sites. The gal

operon is one of the best characterized operons involving DNA looping. There are two

operator sequences in gal operon, Oe and O j. 0 E is found upstream of two overlapping

promoters and Ot is located downstream of the promoters (Irani et aL,1983; Adhya,1987;

Mandal et aL,1990). The loop structure is formed when a single repressor complex binds

both operators and consequently RNA polymerase is constrained from binding to the

structured promoters. An analogous mechanism has been observed in lac operon

regulation. However, in the lac operon the operators are located downstream of the

promoter (Mossing and Record, 1986; Kramer et aL, 1987).

Finally, it has been noted that sequence-dependent DNA curvature afreets many

processes in which the DNA is structured, such as DNA bending, wrapping and looping.

DNA curvature has been associated with many promoters.

DNA curvature. There are many unusual forms of DNA Among those, intrinsic DNA

curvature has been most studied the most and its biological significant has been shown.

Intrinsic DNA curvature is a phenomena that plays an important role in a variety of DNA


14

transactions. While there are various methods for detecting altered DNA structure, the most

sensitive and convenient method is polyacrylamide gel electrophoresis (Diekmann, 1987).

Curved DNA tends to migrate more slowly in an acrylamide gel than non-curved DNA of

equal length. Aberrant mobility is influenced by acrylamide concentration, temperature, and

salt concentration in the gel (Diekmann and Wang, 1985; Diekmann, 1987).

Curved DNA was first identified in electrophoretic studies of Idnetoplast minicircle

DNA from Leishmtmia tarantolate (Marini et aL, 1982). The first clue that kinetoplast DNA

might be bent came from the observation that a restriction fragment from a Ltaratolate

minicircle migrated anomalously slowly during electrophoresis on a polyacrylamide gel.

It was shown that runs of adenine, or an adenine tract (A-tract), would produce a small

bend in DNA helix. Intrinsically curved DNA is commonly characterized by runs of 4-6

adenine residues in phase with periodicity of B-form DNA This periodicity places the A

tracts on the same side of the helix and allows the angle of deflection from the helical axis

resulting from each A tract to be additive, thus leading to a large overall bend in the DNA

(Hagerman,1985; Diekmann, 1986; Koo et aL,1986).

Several theoretical models have been proposed to explain intrinsic DNA bending on

the molecular level. Among those, the first proposed model still is widely accepted. This

model, called the “wedge model” (Trifonove and Sussman, 1980), assumes smooth global

bending as a result of small additive wedges. The combination of tilt and roll cause

formation of a wedge or angle between adjacent AT base pairs in the DNA helix (Trifonov

and Sussman, 1980; Ulanovsky, 1987; Bolshoy et al., 1991). Such deformation in-phase

with the helical repeat cause a long-range curvature. It has been recently proposed that the


15

DNA helix in an A-tract is characterized by base inclination in form of a negative roll

(Haran et aL, 1994). Progressive narrowing in the minor groove of the helix is the result

o f this negative roll.

In addition to A-tracts, there are other sequences which cause bending of DNA.

Sequences with specific periodic dinucleotide, such as AG, CG, GA, or GC, have been

shown to contribute in bending of DNA (Bolshoy et aL, 1991). Compared to A-tracts, they

bend the DNA to smaller degree (Fujimura,1988; Milton et aL,1990; Bolshoy et aL,1991).

Strong gel-mobility anomaly has been noticed for GGGCCC-containing DNA in the

presence of divalent ions (Brukner et aL, 1994). Perhaps the sequence dependent dynamic

feature of DNA is influenced by metal ions. Another intrinsically curved sequence appears

to be the GGCC element. This element is bent toward the major groove (Goodsell et al.,

1993).

DNA curvature in prokaryote promoters. DNA curvature generally is generated by an

A-tract sequence located upstream of the -35 region (Tanaka et aL,1991). Promoters

containing this upstream sequence are a well documented phenomena in prokaryotes. It has

been suggested that there is a relationship between intrinsic curved DNA and transcriptional

activity in certain E. coli ribosomal and tRNA promoters (Nishi and Itoh, 1986; Bauer et

aL, 1988). Reduced activity has been observed when the curved DNA is deleted in several

other promoters such as the ompF (Verda et aL, 1981), the bla promoter from pUC19

(Ohyama et aL,1992), the his and 1PP promoters (Verda et aL, 1981), and the Alul56

promoter from 5. subtilis phage SP82 (McAllister and Achberger, 1988).


16

The effect o f CRP in the activation of gal promoter in E. coli has been well

documented. It has been shown that if the CRP binding site is replaced with synthetic or

natural curved DNA, the transcriptional activity can be restored (Bracco et aL,1989).

Similar results were reported using synthetic curved DNA in the lac promoter (Gartenberg

and Crothers, 1991). The addition of distamycin, a drug which is able to relax the DNA,

to gal p i promoter, which has a curved sequence upstream of promoter, caused a

significant reduction in transcriptional activity in the absence of the cAMP-CAP complex

(Lavigne et aL,1992). These results suggest that similar to protein induced bending,

sequence-specific DNA curvature enhances gene expression.

In studies from our laboratory, it was shown that sequence dependent DNA

curvature immediately upstream of the -35 region can enhance RNA polymerase binding to

promoters. Furthermore, it has been reported that deletion of curved DNA dramatically

decreased promoter utilization by the RNA polymerase from B. subtilis in vivo and in vitro.

It was also documented that one region of curved DNA will substitute for another when

properly aligned, and the rotational orientation (on the helix) of the curve relative to the

promoter was more important to function than the distance between the curved DNA and

the promoter (McAllister and Achberger, 1988, 1989). Hybrid promoters were created by

placing curved DNA from B. subtilis bacteriophage promoters on E. coli phage APL and

PR promoters. The addition of curved DNA influenced the binding of the RNA polymerase

from either B. subtilis or E. coli (McAllister, 1988). Wrapping of promoter DNA around

the E coli RNA polymerase was documented for one series of these promoters (Nickerson

and Achberger, 1995). B. subtilis RNA polymerase also wrapped the DNA upstream of the


17

promoter around itself (Cheng, 1996). A model was proposed for all these finding. It was

proposed that curved DNA enhances promoter function by facilitating the wrapping of the

DNA around the RNA polymerase. This structured DNA-RNA polymerase complex allows

the DNA helix to be untwisted and the two strands separated for transcription initiation.

The focus of this research was to test for the interaction of E. coli RNA polymerase

with DNA upstream of the promoter consistent with DNA wrapping and to investigate the

nature of this phenomenon. In this study, the Alul56 and Ball29 promoters from the B.

subtilis bacteriophage SP82 were chosen for analysis. For each promoter, the nucleotide

sequence upstream from -35 region contains intrinsic DNA curvature. A DNase I

footprinting assay was used to study the interaction between promoter DNA and RNA

polymerase. In addition, the effect of temperature, salt concentration, and heparin on RNA

polymerase complex formation and wrapping was investigated. This study shows that E.

coli RNA polymerase wraps the DNA upstream of the promoter around itself. A model

for the nature of this interaction and its relation to the initiation of transcription is proposed.


MATERIALS AND METHODS

Materials. PCR reagents were supplied by Perkin Elmer. Restriction enzymes were

purchased from either Bethesda Research Laboratories or New England Biolabs.

DNase I was purchased from Boehringer Manheim GmbH. Permanganate was purchased

from Sigma Chemical Company. The [y32P] ATP and [c^P] ATP were purchased from

New England Nuclear, Dupont. All other materials used were of the highest quality

available.

Plasmid transformation and isolation. Escherichia coli strain DH5a McR was grown in

LB medium (Miller, 1972) at 37°C. E coli cells were made competent using CaCl2 washes

(Lederberg and Cohen, 1974). Competent cells were mixed with 20-50 ng plasmid in the

test tube, placed on ice for 20 minutes and heat shocked at 42 °C for 2 minutes. The cells

were cooled briefly on ice and diluted with L-broth followed by incubation at 37°C with

shaking for 90 minutes. Between 0.2-1 ml of cells were concentrated and spread onto the

agar plates containing 50 mg/ml ampicillin to select for plasmid containing cells. Selected

colonies were isolated and cultured for plasmid isolation. Alkaline-lysis method of

Bimboim and Doly(1979) was used in order to isolate plasmid DNA To isolate highly

purified plasmid DNA large scale isolation was carried out followed by cesium chloride

density gradient separation in the presence of ethidium bromide. The purified DNA was

quantified by spectrophotometry.

Construction of Alul56 derivative promoters. An Alul56 promoter derivative which

contain uncurved DNA upstream of -35 region was constructed. In order to disrupt the

I S


19

intrinsic DNA curvature, Thymines were inserted into the middle of each A tract using site

directed mutagenesis (Chen and Przybyla, 1994). In this method, two rounds of PCR were

performed. In the first round, the AluUnc primer (5'-GCTAATATTCCTGAATA

ATATTGCAATAAGTTGTTGAC-3') and the M13/pUC reverse sequencing primer (-48)

24-mer (New England Biolabs) were used to incorporate the mutations into the promoter.

The template used in this round was plasmid pUC8 containing the original Alul56

promoter. The first PCR amplified fragment was gel purified and used directly as a primer

together with the M13/pUC sequencing primer

(-47) 24-mer (New England Biolabs) to direct a second round of DNA amplification using

the Alu 156 DNA template. Both rounds of PCR were performed in a Perkin Elmer Model

480 thermal cycle for 25 cycle at 94°C for 1 min, 60°C for 2 min, and 72°C for 3 min. The

products from the second round of amplification were digested with EcoRl and HindUI.

The restriction fragments were then gel purified and ligated to pUC8 digested with the same

enzymes. The promoter was named AluUnc.

To study the role of AT rich regions upstream of the promoter, two promoters in

which AT rich DNA was substituted for the curved DNA were constructed using PCR For

the first promoter the AT-rich region immediately upstream of the E. coli rmBP promoter,

called the UP-element (Ross et al., 1993), was inserted upstream of -35 region. To

construct this promoter, the M13/pUC sequencing primer (-47) 24-mer (New England

Biolabs) and AluUp primer (5-GCGAATTCAGAAAATTATTTTAAATTG

TTGTTGACTTTCTCTACGAGGTGTG-3') were used for DNA amplification with the

plasmid pUC8 containing the wild-type Alul56 promoter as template. The amplified


20

products were digested with EcoBl and H indSl and gel purified. Purified fragments were

ligated to pUC8 digested with the same enzymes. The promoter was named AluUp.

The second promoter contained 27 nucleotide long AT rich region upstream of the

promoter. The same procedures were used except that the 27AT primer (51-

GCGAATTC AT AATT AAT AATT AATTCGTTGACTTT-3') was used instead o f AluUp

primer. This promoter was named Alu27AT. In order to replace the DNA upstream of

the curved DNA in Alu27AT and AluUP with heterologous DNA in other promoters used

in this study, EcoRI DNA fragment from AluExt promoter DNA was inserted at the EcoRI

site of these promoter. AIu27At, AluUp, and AluUnc primers were synthesized in the Gene

Lab, Louisiana State University.

To study the effect of AT-rich region, two other promoters were constructed one

with a short stretch of AT and the other without any AT-rich region upstream of -3 5 region.

For construction of the first promoter, AluExt which does not possess any curved DNA

upstream of promoter was cut with EcoRI. The S' extension was made flush using Klenow

fragment and the gap was ligated. This promoter was named AT.

The last promoter lacked any AT-rich region upstream of promoter. In order to

construct this promoter, after cutting the AluExt with EcoRI, the S' over hang was digested

with S1 nuclease to remove the single stranded DNA and then ligated. This promoter was

named AluDel.

The promoter constructs were sequenced using the Circumvent Thermal Cycle

Sequencing kit (New England Biolabs) to insure that there were no changes in the promoter


21

sequence other than the intended mutations. Other promoters used in this study are listed

in Table 1. All of these promoters are from our laboratory collection.

Table 1. Promoters

Name Description Source

Alu 156Early gene promoter from phage SP82 containing curved DNA, Alul56 is 82% homologous to E. coli promoter consenses sequence

Laboratorycollection

AluExt Alul56 derivatives in which the curved DNA was replaced by a fragment of pBR322, base pairs 376-467


Ball29B. subtilis bacteriophage SP82 promoter containing curved DNA, Ball29 is 90% homologous to E. coli promoter consenses sequence


BalExt Ball29 derivatives in which the curved DNA was replaced by a fragment of pBR322, base pairs 376-467


^Pr Bacteriophage X promoter Laboratorycollection

AlupR XpR derivatives in which curved DNA from Alul56 was inserted upstream of phage promoter


BalpR XpR derivatives in which curved DNA from Ball29 was inserted upstream of phage promoter


Primer labeling. Each primer was 5'end labeled using T4 polynucleotide kinase (New

England Biolabs) and 30 mCi [g-32? ATP] (Dupont, New England Nuclear) at 37°C for 30

min. In each 25 ml reaction 20 pmol of primer and 10 units of kinase were used. The

reaction was then denatured at 95 °C for 5 min and stored at - 20°C.

PCR amplification. Two primers, M13/pUC reverse sequencing primer (-48) 24-mer and

the M13/pUC sequencing primer (-47) 24-mer (New England Biolabs) used for

amplification. Only one of the two primers was labeled with [g-32P] ATP as it was.


22

Drivitives of pUC8 containing the various promoter constructs were used as templates in

amplification reactions. Each reaction contained, 2.5 ml of supplied 10X PCR buffer, 2ml

of a mixture containing 2.5 mM of each deoxynucleotide substrate, 2 ml 25 mM MgClj, 0.1

ml AmpliTaq DNA polymerase, 4ml 5'end-labeled primer (0.8 mM) and 1ml second primer

(20 mM), 2 ml (0.2 ng/ml) DNA template and 11.5 ml water. PCR was performed in Perkin

Elmer DNA Thermal Cycler Model 480 for 25 cycles of the following, 94 °C for 1 min,

60°C for 2 min, and 72°C for 3 min. Labeled products were purified through a Sephadex

G-50 (Pharmacea Biotech) spin column (Neal and Florini, 1973).

RNA polymerase isolation. RNA polymerase was isolated from E. coli MRE600 as

described by Spiegelman et al. (1978) with minor modifications (Achberger and Whitely,

1980; McAllister and Achberger, 1988). Sonication was used to lyse the cells, and RNA

polymerase was purified by the sequential steps of polyethylene glycol-dextran phase

partitioning, ammonium sulfate precipitation, gel filtration chromatography, and DNA

cellulose chromatography. RNA polymerase activity was examined using an in vitro

transcription assay (Spiegelman et al., 1978). SDS-PAGE electrophoresis was used to

analyze subunit composition and purity of RNA polymerase (Lammli, 1970). The Bio-Rad

protein assay was used to calculate RNA polymerase concentration. RNA polymerase was

stored at -20°C in 35% glycerol.

DNase I footprinting analysis. To examine promoter-RNA polymerase interactions, a 190

pi of reaction mixture containing 40 mM Tris-HCl(pH 8.0), 10 mM MgCl^ 50 mM NaCl,

and 100,000cpm end-labeled DNA was incubated at 37°C with or without 2 mg RNA

polymerase. After 5 min incubation, 0.04 pg of DNase I(Boehringer Manheim Corp.) was


23

added to mixture. Following a 30 sec digestion with DNase I, 20 pi of salt solution

containing 3 M sodium acetate and 0.05 pg/pl yeast tRNA was added and two volumes of

phenol/chloroform mixture were added to stop the reaction. After mixing with a vortex

mixer, the phases were separated by centrifugation. The aqueous layer containing the

DNA was ethanol precipitated, rinsed with 80% ethanol, and dried under reduced pressure.

The DNA pellet was resuspended in 5 pi of loading buffer made with 10 ml deionized

formamide, 10 mg xylene cyanol, 10 mg bromophenol blue, and 0.2 ml 0.5 M EDTA, pH

7.0. Samples were heated to 95° C for 5 min and immediately transferred to ice. DNA

banding patterns was visualized by autoradiography after electrophoresing the samples on

6% polyacrylamide (acrylamide to bisacrylamide, 30:1.5) gel containing 7M urea in TBE

buffer at 1700 volts. Following electrophoresis the dried gels were analyzed by

autoradiography.

Gel retardation analysis. To analyze the relative afl&nity of E. coli RNA polymerase for

different promoters, gel retardation analysis was performed as described by Ausubel et al.,

1989 with minor modifications. End-labeled DNA fragments were incubated with or

without RNA polymerase in buffer containing 40 mM Tris-HCl (pH 7.8), 10 mM MgCl^

50 mMNaCl and 1 pg of non-specific competitor DNA at 37°C for 10 min. Following the

addition of 4 pi of loading dye (26% Ficoll, 0.1% Bromophenol blue), DNA-RNA

polymerase complexes were resolved in 4% polyacrylamide gel (acrylamide to

bisacrylamide, 30:2 ) with high ionic strength buffer (50 mM Tris, 400 mM Glycine, and 2

mM EDTA pH 8.5). The the gel was electrophoresed at 150 volts at room temperature for


24

two hours. Then gel was transferred to Whatman 3 MM paper , dried and

autoradiographed. Both free and RNA polymerase bound DNA were quantified by

densitometry of autoradiograms.

K M n04 footprinting. To detect open promoter complex formation, the basic reaction

conditions were the same used for DNase I footprinting. After 5 min incubation at 37°C

with or without RNA polymerase, 5 pi of 80 mM KMn04 was added to the mixture. The

reactions were stopped by adding 10 pi 2-mercaptoethanol and 15 pi of 3.0 M sodium

acetate after 5 min. The samples were extracted with two volume of a phenol/chloroform

mature and precipitated with ethanol. DNA pellets were washed with 80% ethanol, dried,

and resuspended in 5 pi formamide loading buffer as described before. After heating the

samples to 95 °C for 5 min, they were analyzed by polyacrylamide gel electrophoresis as

described for DNase I footprinting. Following electrophoresis the dried gels were analyzed

by autoradiography.

Single-round run-off transcription assay. All DNA templates used for the experiment

were synthesized by PCR amplification. For each reaction, an equal amount of DNA

template was used as determined by densitometry of ethidium bromide stained

polyacrylamide gels. In addition to template DNA, each reaction contained 0.5 pi of K coli

RNA polymerase (1.5 mg/ml), 2 pi of 10X transcription buffer (400 mM Tris-HCl, 100 mM

MgCl, 500 mMNaCl2), and water for the total reaction volume of 17.5 pi. The reactions

were mixed on ice and then transferred in duplicate to desired temperature for 5 min. Then

2 pi of nucleotides (20 mM GTP, 20 mM CTP, 20 mM UTP, 4 mM ATP, and 0.5 mCi/ml,

3000 Ci/mmol [a32?] ATP) and 1 pi of heparin (1 mg/ml) was added to each reaction at the


25

same time. Half of the reactions were shifted to 37°C after 30 second and the rest remained

at incubated temperature. After 5 min., all of the reactions were stopped by adding urea to

a final concentration of 5M.

Transcription products were analyzed by electrophoresing the samples on 6%

polyacrylamide (acrylamide to bisacrylamide, 30:1.5) gel containing 7M urea in TBE buffer

at 500 volts. After electrophoresis, the gels were dried and exposed to X-Ray film (Kodak,

X-OMAT). Resulting bands were quantified by densitometry.


RESULTS

The present work focuses on the role of intrinsic DNA curvature upstream of -35

promoter region in DNA wrapping and investigates the nature of interaction between £ coli

RNA polymerase and curved DNA The Alul56 and Ball29 promoters from the B. subtilis

bacteriophage SP82 are 83% and 90% homologous to the consensus sequence ofE. coli

promoters, respectively. These two promoters are recognized by £ coli RNA polymerase

and both have intrinsically curved DNA sequences upstream of -35 region (Figure 1).

Previous studies have demonstrated that upstream DNA curvature is required for efficient

utilization o f these promoters by B. subtilis RNA polymerase (McAllister and Achberger,

1988:1989). Curved DNA also appears to play a role in DNA structuring by £ coli RNA

polymerase. When curved DNA was spliced onto the phage XPR promoter, £ coli RNA

polymerase wrapped the curved DNA around the enzyme (Nickerson and Achberger,

1995). To study the phenomena of DNA wrapping, theAlul56andBall29 promoters

and their derivatives have been used in this study.

DNase I footprint analysis of the interaction between £ coli RNA polymerase and

Alul56 and Ball29 and their derivatives. It was of interest to determine if DNA

wrapping could occur in promoters other than those used in a previous study in our

laboratory. To investigate this problem, Akil56 and Ball29 and their derivatives was used.

The AluExt and BalExt are derivatives of Alul56 and Ball29, respectively, in which the

curved DNA upstream of promoter was replaced by DNA with no curvature (Figure 2).

The DNase I footprint analysis on both strands of all these promoters was performed. This

26


Reproduced

with perm

ission of the

copyright ow

ner. Further

reproduction prohibited

without

permission.

-60 -50 -40 -30 -20 -10 +1A 1u 156 I I I I I I ICTGCTAAAATTCCTGAAAAATTTTGCAAAAAGTTGTTGACTTTCTCTACGAGGTGTGGCATAATAATCTTA

-60 -50 -40 -30 -20 -10 +1Ball29 I I I I I I IAAGAAAAAATATCTACAGAAAATATGAAAAAGTTGTTGACATTTCTTCCCATCCATGCTATAATAAAGTCA

Figure 1: Nucleotide sequence of the Alu 156 and Bal 129 promoters. Bases representing the +1 transcription start site,the -35 and the -10 regions are in bold type. The runs of adenine in the DNA upstream of the -35 region are underlined.

to-o

Reproduced

with perm

ission of the

copyright ow

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without

permission.

-35 -10 +1Alu156 | | |CTGCTAAAATTCCTGAAAAATTTTGCAAAAAGTTGTTGACTTTCTCTACGAGGTGTGGCATAATAATCTTA

AluExtTGGGGAAGATCCCCGGGAATTCCCCCAGTGAATTGTTGACTTTCTCTACGAGGTGTGGCATAATAATCTTA

-35 -10 +1Ball 29 | | |AAGAAAAAATATCTACAGAAAATATGAAAAAGTTGTTGACATTTCTTCCCATCCATGCTATAATAAAGTCA

BalExtCGATGGGGAAGATCCCCGGGAATTCCCCCAGTGAATTGACATTTCTTCCCATCCATGCTATAATAAAGTCA

Figure 2: Nucleotide sequence of the Alul56, Ball 29, and their derivatives. Bases representing the +1 transcription start,the -35 and -10 regions are in bold type. The runs of adenine in the DNA upstream of the -35 region are underlined. Nucleotide sequence downstream of the -35 region is the same for each wild-type promoters and its derivative.

29

assay can locate the protein binding site. Bound protein will protect the DNA from cleavage

with DNase I. DNase I cutting is also sensitive to protein-induced changes in the DNA

conformation. DNA conformation changes are often observed as DNase I hypersensitive

sites. DNase I digestion pattern indicative of wrapping is identified as a series of enhanced

cleavages (dark bands) followed by protection (light bands) with a 10 base pair periodicity.

Figure 3 corresponds to an autoradiogram after DNase I footprint analysis of both strands

of Alul56. Lanes marked by a minus sign are control reactions where the DNA fragment

containing the promoter was digested with DNase I in the absence of RNA polymerase.

When RNA polymerase was bound to the Alul56 promoter (lanes marked by plus sign), a

large protection area typical of results with prokaryote RNA polymerases was observed

from +24 to -55 in lower strand of DNA (Figure 3 A). The nucleotides are numbered

relative to the transcription start site at position +1. Hypersensitive sites, represented by

dark bands relative to control lane, were observed at -58, -59, -68, -69, -71, -92, -97, -101,

-102, and from -108 to -110. The curved DNA region from -40 to -65 is weakly cleaved

by DNase I because of the altered DNA conformation in this region. Small regions of

protection from DNase I were observed following these hypersensitive areas. For the upper

strand o f this promoter, the pronounced protected region was observed from +21 to -56

(Figure 3B). This region was interrupted with hypersensitive sites at -24, -44, and -47.

DNase I hypersensitive area upstream of promoter were around -57, -58, -59, -60, -67, -68,

-78, -79, -80, -81, -86, -87, -98, -99, -112 and -113 followed by protected regions. This

pattern of alternating protection and enhanced cleavages with 10 base pair periodicity in

both strands of Alul56 was indicative of wrapping.


30

Figure 3: DNase I footprint analysis of the Alul56 promoter. Panel A represents thefootprinting of the lower (template) strand and panel B correspond the upper strand DNA footprint. DNase I digestion patterns in the absence (lanes with minus sign) and presence (lanes with plus sign) of RNA polymerase are shown. The bands are numbered relative to the transcription start site at position+1.


31

A. B.saW

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- 6 0 * -

- 5 0 * -

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32

The DNase I pattern for AluExt, which lacks the curved DNA, was similar to that

of Alul56 promoter in the protected region from DNase I cleavage from +24 to -46 in

lower strand (Figure 4A) and +20 to -56 in upper strand (Figure 4B). Enhanced cleavages

were observed around the -60 region of both strands. Evidence of protein-DNA interaction

consistent with DNA wrapping was less obvious for this promoter. Figure 5 represents the

summary of the DNase I footprinting results on both strands o f Alul56 and AluExt

promoters compiled from three separate experiments.

Analysis of E. coli RNA polymerase binding to the Ball29 promoter has also

provided evidence for DNA wrapping. The footprint for lower strand of Ball29 extended

from +17 to -47, which was typical for other E. coli promoters (Figure 6A). Enhanced

cleavages upstream of promoter were observed at -49, -48, -58, -59, -60, -69, -70, -71,

-73 and -93, and -94. For upper strand of Ball29 (Figure 6B) the footprint region was from

+20 to -43 with enhanced cleavages at -23 and a gap at -21, and -22 . Hypersensitive areas

to DNase I were observed at -44 to -46, and -53. Protected regions were observed around

-50, -60, and -70. For technical reasons, footprint data upstream of -72 was unavailable for

the upper strand. This pattern of DNase I cleavage for both strands of Ball29 was in

agreement with presence of DNA wrapping.

The general footprint for BalExt was the same as Ball29 promoter downstream of

-40 except enhancement cleavages at -32, and -37 to -39 in upper strand and -37 in lower

strand (Figure 7). Almost no upstream interaction was detected for this promoter.

Evidence of DNA wrapping for BalExt was not obvious. This suggests that the curved


33

Figure 4: DNase I footprinting of the AluExt promoter bound by RNA polymerase.Footprint of the lower (template) strand of DNA is shown in panel A and panel B represents the footprinting of upper strand of the promoter. Presence and absence of RNA polymerase are indicated by plus and minus signs, respectively. The DNA bands are numbered relative to the transcription start site at position +1.


34

A. B.

- +

_100i.90* .80* _70»-| .60*

_50H

_40*

.30*

_20»-|

.10*

+ 1 *

+ 10*

+20.

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-30>

-40*-

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-80*

-90*-

. 100*

. 110*


35

Figure 5: The summary of DNase I footprint analysis on both strands of the Alul56(A) and the AluExt (B) promoters. The -10 and -35 regions are in bold type. The line over the upper strands and below the lower strands represent the area protected from DNase I cleavage. Hypersensitive regions are marked by arrows. The results were compiled from three separate experiments.


Reproduced

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- 1 2 0 - 1 1 0 - 1 0 0 - 9 0 - 8 0 - 7 01

GAAAGATATCCTAACAGCACAAGAGCGGAAACACGfl’TfGTTCTACATcjtGAACAACCTCTicT CTTTCTATAGGATTGTCGTGTTCTCGCCTTTGTGCAAAACAAGATGTAGGTCTTGTTGGAGACGA AAA AA A A------------- A A A

- 6 0 - 5 0 - 4 0 - 3 0 - 2 0 - 1 0 +1_ l w _ _ _ _ _ _ _ I_ _ _ f f T T - - - J_ _ _ _ _ _ _ _ _ _ _ _ I_ _ _ _ _ _ T _ _ _ _ I_ _ _ _ _ _ _ _ _ _ _ _ I_ _ _ _ _ _ _ _ _ _ _ _ It t c c t g a a a a a t t t t g c a a Aa a g ttg ttg a c tttc tc ta c g a g g t g tg g c a ta a ta a tc ttaAAAA'

t t t t a aggact t t t t a a a a c g t t t t t c a a ca actg aaa g a g a tg c tc c a c a c c g ta tta tta g a a t

+10 + 20 +301__________ Lt t t t t t t t tIttt

a c a a c a g c a g g a c g c t a g g a c g g a t c c g g g g a a t t cTGTTGTCGTCCTGCGATCCTGCCTAGGCCCCTTAAG ----------------A A

B - 6 0 - 5 0 - 4 0 - 3 0 - 2 0 - 1 0 +1

TCACCGATGgGGAAGATCCCCGGGAiTTCGTTGACTTTCTCTACGAGGtfGtfGGCATAMAATCTTAAGT^CTACCCCTTCTAGGGGCCCTTAAGCAACTGAAAGAGATGCTCCACACCGTATTATTAGAAT

+10 +20 +30________ I______ ^ I t t t t t t t t tItttACAACAGCAGGACGCTAGGACGGATCCGGGGAATTC TGTTGTCGTCCTGCGATCCTGCCTAGGCCCCTTAAG ------------------------------AA

u>On

37

Figure 6: DNase I footprint analysis o f Ball29 promoter. Lower (template) strand(panel A) and upper strand (panel B) of the promoter are footprinted in the absence (lanes with a minus sign) and presence (lanes with a plus sign) of RNA polymerase. The DNA bands are numbered relative to transcription start she a t+1.



39

Figure 7: DNase I footprint analysis of BalExt promoter. Lower (template) strand(panel A) and upper strand (panel B) of the promoter are footprinted in the absence (lanes with a minus sign) and presence (lanes with a plus sign) of RNA polymerase. The DNA bands are numbered relative to transcription start site a t+1.


40

A. B.

1 0 0 ^

m

_ 10j

+1»

+.10*Ks

, 2 0w «

r l

-90>

> 110*


41

DNA contributes to the interaction of RNA polymerase with DNA upstream of the -35

region. Figure 8 represents the summary of footprint analysis for both strands of BaI129

and BalExt.

There is an excellent agreement between the results for both promoters. DNase I

digestion pattern indicative of DNA wrapping was more obvious for both wild-type

promoters relative to the altered promoter lacking curved DNA There was a periodicity

of approximately 10 base pairs in the pattern of enhanced cleavages and protection in both

wild-type promoters. This pattern provided evidence that the upstream curvature of Alul5 6

and Ball29 was bent when wrapped around the RNA polymerase. It should be noted that

the possibility of second RNA polymerase binding to upstream region was ruled out by

footprinting the promoters at different RNA polymerase concentrations. At the lowest

concentration of RNA polymerase where binding to the promoter was observed, wrapping

was present. The DNase I pattern indicative of wrapping disappeared at the same point as

the main footprint (+20 to -50 region). This indicates that wrapping was not the result of

binding of another RNA polymerase to upstream sequence.

Nature of the interaction between RNA polymerase and DNA up stream of the -35

region. Electrostatic interactions between RNA polymerase and the backbone of DNA are

essential for sequence-independent DNA binding. Salts such as NaCl affect sequence-

independent binding between RNA polymerase and promoter DNA by competing with RNA

polymerase for charged phosphate residues on the DNA backbone (Roe and Record, 1985;

Suh et al., 1992). To explore the nature of interaction between RNA polymerase and DNA

upstream of the curved DNA, NaCl was used to disrupt electrostatic interactions. DNase


42

Figure 8: The summary ofDNase I footprinting results on both strands of Ball29 (A)and BalExt (B). The -10 and -35 regions are in bold type. The lines over the upper strand and below the lower strand represent the area protected from DNase I cleavage. Hypersensitive regions are marked by arrows. The results were compiled from at least two experiments for each strand.


Reproduced

with perm

ission of the

copyright ow

ner. Further


without

permission.

- 9 0 - 8 0 - 7 0

GAATTCCCCGGATCCGTCACCCCTAAGAA CTTAAGGGGCCTAGGCAGTGGGGATTCTT M - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A A A - - - - -

- 6 0 - 5 0 - 4 0 - 3 0 - 2 0 - 1 0 +1

d^TACAGAAlATATGLuuJ^GTTGTTGACATTTCTTCCCATCCATGCTA' 'AATAAACTclAAAATATCTACAGITTTTA^AGATGTCTTTTATACTTTTTCAACAACTGTAAAGAAGGGTAGGTACGATATTATTTGAGT

+10 +20 +30

ta g a g a a c a a c a c ta tc aI atg a a^ J a^ I^ a t tATCTCTTGTTGTGATAGTTTACTTACCTCTCTAA - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A A A - - - - - - A A A A A A A

B- 6 0 - 5 0 - 4 0 - 3 0 - 2 0 - 1 0 +1

GGGAAGATCCCCGGGAATTCCCCCAGTGAATTGACATTTCTTCCCATCCATGCTATAATAAACTCACCCTTCTAGGGGCCCTTAAGGGGGTCACTTAACTGTAAAGAAGGGTAGGTACGATATTATTTGAGT

+10 +20 +30

t A c J A g a a c I a c a c t a t c a I a t g a a t g g a g a g a t t a t c t c t t g t t g t g a t a g t t t a c t t a c c t c t c t a a

- - - - - - - - - - - - - - - - - - - A A A A A A A A A A

44

I footprinting analysis was performed for both wild-type promoters and their derivatives

using binding buffer containing various concentrations of NaCl. Since the results of DNase

I footprinting analysis were in agreement for both strands of each promoter, only the

interactions with lower strands were analyzed in this study.

Figure 9 demonstrates the effect of different salt concentration on DNA wrapping

for the lower strand of Alul56. Lane 1 and 7 represent control reactions which DNase I

footprinting was performed in the absence o f RNA polymerase. In lanes 2 to 6, salt

concentrations were incrementally increased from 50 mM to 200 mM, respectively. The

major footprint area (i.e., +20 to -56) consistent with the RNA polymerase bound to the

promoter, was observed at every salt concentration tested. Some differences were detected

among various salt concentrations. For example, bases -38 and -58 were not protected from

DNase I at salt concentrations greater than 100 mM. This indicates that RNA polymerase-

DNA complexes formed at lower salt concentrations were different from those at higher salt

concentrations. Evidence of DNA wrapping progressively faded with increasing NaCl

concentration. Visible changes were observed at different regions upstream of promoter.

For example, enhanced cleavages at -61, and -62 disappeared by increasing the salt

concentration to 100 mM. Obvious differences were displayed at the regions around -70,

-80, -90, -100, -110, and -120. Even bases very far upstream exhibit evident differences

among various salt concentration. Basically, enhanced cleavages and protection areas

indicative of DNA wrapping were lost gradually by increasing the salt concentration in

binding buffer.


45

Figure 9: DNase I footprint analysis of the Alul56 promoter bound to RNApolymerase as a function o f NaCl concentration. Lanes 1 and 7 represent the DNase I digestion pattern in the absence o f RNA polymerase at 50 and 200 mM NaCl concentration. Lanes 2 to 6 correspond to the footprint of the promoter bound by RNA polymerase at 50, 75, 100, 150, and 200 mM NaCl respectively.


46

i

Reproduced with permission o f the copyright owner. Further reproduction prohibited without permission.

47

The AluExt promoter was also footprinted as a function of salt concentration

(Figure 10). Footprint area from +20 to -53 was observed in all lanes in Figure 10 except

two control lanes. Minor protection around -50 and enhanced cleavages around -60

disappeared as the salt concentration increased. Even in the absence of detectable DNA

wrapping, the RNA polymerase-promoter complex that predominate at 50 mM NaCl

differed from those at 150 to 250 mM.

Effect of salt concentration on Ball29 promoter was also examined (Figure 11). As

observed for last two promoters, the large footprint area from +19 to -50 remained the same

at all salt concentrations except for changes at -40 and -48 regions consistent with the

presence of different complexes at various salt concentrations. Upstream interactions

indicative of wrapping were diminished by increasing the salt concentration. Enhanced

cleavages around -60, -71, and -91 disappeared at lane 5, which represents 150mMNaCl

concentration. Protected areas around -50, -60, -80, and -100 were diminished at the same

salt concentration. At 150 mM NaCl or greater, no changes in the DNase I pattern were

observed upstream of -59.

In the case of the BalExt promoter, as observed for AluExt, the limits of the

footprint at the promoter region were similar at all salt concentrations (Figure 12). Minor

changes observed upstream of promoter around -60 were diminished when salt

concentration was increased. For each promoter tested, protection at +16 to +20 increased

with increase of NaCl concentration.


48

Figure 10: Effect of NaCl concentration on the DNase I footprint pattern o f AluExt.DNase I digestion patterns in the absence of RNA polymerase at SO and 250 mM NaCl concentration are shown in lanes 1 and 8. Lanes 2 to 7 correspond to the footprint of the promoter at SO, 75, 100, ISO, 200, and 250 mMNaCl, respectively.



50

Figure 11: DNase I footprint analysis of the Ball29 promoter as a function of NaClconcentration. DNase I digestion patterns in the absence of RNA polymerase at 50 and 250 mMNaCl concentration are shown in lanes 1 and 8. Lanes 2 to 7 correspond to the footprint of the promoter at 50,75,100, 150, 200, and 250 mMNaCl, respectively.


51

a 4 5 6 7 _8_

120 ►

t i l S_110 —

M M_ 9 0 > - iia_60*"

1

Ifi l lm m

Reproduced with permission o f the copyright owner. Further reproduction prohibited without permission.

52

Figure 12: Effect ofNaCl concentration on the DNase I footprint analysis of the BalExtpromoter. DNase I digestion pattern in the absence o f RNA polymerase at 50 and 250 mM NaCl concentration are shown in lanes 1 and 8. Lanes 2 to 7 correspond to the footprint o f the promoter at 50, 75, 100,150,200, and 250 mMNaCl.



54

In general, as the salt concentration increased, wrapping decreased. DNase I

digestion patterns consistent with the RNA polymerase bound to the promoter were

observed at every NaCl concentration tested. Based on our results it can be concluded that

wrapping is more favorable at low salt concentration suggesting the electrostatic nature of

the phenomenon. This would be expected if these DNA-RNA polymerase interactions were

sequence independent.

The relationship between open promoter complex formation and DNA wrapping.

Potassium permanganate was used to probe the RNA polymerase-promoter complexes

formed at various NaCl concentration for both Alul56 and Ball29 (Figure 13). Potassium

permanganate is an oxidizing agent, which preferentially nicks at T and C residues in single

stranded DNA (Sasse-Dwight and Gralla, 1988). It was noted that while open promoter

complexes decreased at high salt concentration (i.e., 200 mM), they were still observed.

DNA wrapping as visualized by DNase I footprinting was lost at high NaCl concentrations.

This indicates that there are open complexes in which the DNA is no longer wrapped.

Test for DNA wrapping at different stages of transcription initiation. It is known that

at low temperature, the conformational changes leading to the open promoter complex do

not occur (Cowing et al., 1989; Mecsas et al., 1991). When Alul56 was tested for open

promoter complex formation at different temperatures, it was observed that at 0°C almost

no open complexes were formed (Figure 14). Upon raising the temperature, significant

increase in the amount of open promoter complex formation was observed. RNA

polymerase-promoter complexes at 0°, 5°, 15°, 22°, and 37°C were examined by DNase

I footprinting (Figure 15). The results were consistent with the presence of DNA wrapping


55

Figure 13: Open promoter complex formation at different salt concentrations forBall29 (A) and Alul56 (B). Potassium permanganate probe was used to detect open promoter complexes formed in the absence (lane with minus sign) and presence of RNA polymerase. The salt concentration used in each reaction is marked above the lines. The sequence of the open promoter complex regions are marked.


56

A .

N a C l , o i n o in o o( m M ) I « ® w w g

' T

I*G

B .

i 8K s- r

f t f I fT AT T A T T

\ G


57

Figure 14: Effect of temperature on open promoter complex formation for the Alu 156promoter. Absence and presence of RNA polymerase in each reaction are shown by minus and plus sign respectively. The temperature at which each reaction was performed is shown. The sequence of open complex region is marked.


58

Figure 15: DNase I footprint analysis of Alul56 as a function of temperature. Presence or absence of RNA polymerase is represented by plus and minus sign. The temperature of each reaction is shown.


59


60

of all temperatures. Basically, the protected region indicative of sequence-specific RNA

polymerase-promoter interaction was detected at every temperature. Wrapping occurred

at low temperature in the absence of open promoter complexes. RNA polymerase footprint

was shorter at 0°C. Protection from the +1 region to +20 is significantly reduced at 0°C.

This is a signature footprint for closed promoter complexes.

To confirm the presence of wrapped closed complexes, heparin was used in DNase

I footprinting analysis. Heparin is strong DNA competitor that rapidly binds free RNA

polymerase but not enzyme stably bound to the DNA or enzyme engaged in RNA synthesis

(Walter et al., 1967). Closed complexes with short half lives are sensitive to effect of

heparin (i.e., they irreversibly dissociate in the presence of heparin). The Alul56 promoter

was footprinted at 37°C and 0°C (Figure 16) with and without a heparin challenge. RNA

polymerase was allowed to bind the AlulS6 promoter at the indicated temperature. To one

sample at each temperature, heparin was added for 30 second prior to DNase I treatment.

At 37° C, addition of heparin virtually eliminated the DNase I pattern consistent with DNA

wrapping. Since heparin eliminates sensitive closed promoter complexes, this indicated that

unwrapped heparin resistant open complexes were formed at 37°C. In addition, wrapped

complexes appeared to be closed complexes. It was previously demonstrated that at 0°C

almost no open promoter complexes were formed. DNase I digestion pattern consistent

with wrapping was observed in the absence of heparin at 0°C. Upon addition of heparin,

in addition to loss o f wrapping, the footprint was diminished too. This suggests that all

wrapped complexes were closed complexes.


61

Figure 16: Effect of heparin on the footprint analysis of the Alul56 promoter bound toRNA polymerase at 37°C and 0°C. The minus sign represents the DNase I digestion pattern in the absence of RNA polymerase. Lane marked with the plus sign (+) represent the binding of RNA polymerase without the heparin challenge. Lanes in which the reactions were challenged with heparin are designated by “+H”.


62

3 7 ° c 0 ° c

- + + H

100**_90»-

_80*-

_60>-

%T' ss*<- 3

- + + H

#


63

Effect of AT-rich regions in DNA wrapping and transcription efficiency. The a

subunit of RNA polymerase is known to bind an AT-rich sequence upstream of the -35

region (Ross et al., 1991). Since curved DNA is AT rich, the contribution o f the AT-rich

sequence was examined. To compare the role of AT-rich region upstream of promoter and

curved DNA in DNA wrapping and transcription efficiency, five different promoter-

containing fragments were constructed. In one promoter, AluUp, the DNA upstream of -35

region in AluExt promoter was replaced with the a subunit binding site, the so called “UP

element”, of.E. coli ribosomal RNA promoter rmB PI. In second promoter, AluUnc, a

non-curved AT-rich region was constructed by insertion of T’s in the middle of each A-tract

upstream of Alul56 promoter. These mutations eliminate curvature while preserving the

AT-rich nature of this promoter. Both Alu8AT and Alu27AT promoters contained AT-rich

regions upstream of the -35 region of AluExt promoter. However, the length of this region

in Alu8AT was shorter than that of Alu27AT. The AluDel promoter was constructed as a

control with no AT-rich region upstream of AluExt promoter. The relevant nucleotide

sequences of these promoters are listed in Figure 17 in comparison with the sequence of the

Alul56 and AluExt promoters.

To examine the specific interactions between E. coli RNA polymerase and the AT-

rich promoters, DNase I footprinting analysis was performed. This approach was used to

determine the role of AT-rich region in DNA wrapping. Each promoter sequence was

digested with DNase I in the absence and presence of RNA polymerase. Figures 18 and 19

illustrate the DNase I digestion patterns for the lower strands of each promoter. The DNase

I digestion pattern indicative of wrapping that was observed for Alul56 was reduced in the


Reproduced

with perm

ission of the

copyright ow

ner. Further


without

permission.

-35 -10 +1AluI56 | | |TAAAATTCCTGAAAAATTTTGCAAAAAGTTGTTGACTTTCTCTACGAGGTGTGGCATAATAATCTTA

AluUncTAATATTCCTGAATAATATTGCAATAAGTTGTTGACTTTCTCTACGAGGTGTGGCATAATAATCTTA

* * * *AluExtGAAGATCCCCGGGAATTCCCCCAGTGAATTGTTGACTTTCTCTACGAGGTGTGGCATAATAATCTTA

AluUpCGAATTCAGAAAATTATTATTTTAAATTGTTGTTGACTTTCTCTACGAGGTGTGGCATAATAATCTTA

AluSATCACAGCTATGACATGATTACGAATTAATTCGTTGACTTTCTCTACGAGGTGTGGCATAATAATCTTA

Alu27ATTATCGCGAATTCATAATTAATAATTAATTCGTTGACTTTCTCTACGAGGTGTGGCATAATAATCTTA

AluDelCAACAGCAGGACAGCTATGACCATGATTACGTTGACTTTCTCTACGAGGTGTGGCATAATAATCTTA

Figure 17: Nucleotide sequence of the Alu 156, AluExt, AluDel, and AT-rich drivatives. AT-rich regions areunderlined. The -10, -35, and +1 regions are in bold type. An asterisk is placed under the bases in AluUnc that differ from Alu 156.

65

Figure 18: DNase I footprint analysis of the Alu 156, AluUnc, AluUp, and Alu27ATpromoters. Lanes depicting DNase I digestion in the presence and absence of RNA polymerase are indicated by plus and minus sign. The DNA bands are numbered relative to the transcription start site at position +1.


the copyright owner. Further

R e p ro d u c e d Witt' p e rm iss io n

reproduction prohibited without permission.

67

Figure 19: DNase I footprint analysis of the Alul56, AluExt, Alu8AT, and ATDelpromoters. DNase I patterns generated in the presence and absence of RNA polymerase are indicated by plus and minus sign. The DNA bands are numbered relative to the transcription start site at position +1.



69

AluUnc promoter (Figure 18). For the rest of promoters, evidence of protein-DNA

interactions consistent with DNA wrapping were not obvious. The DNase I footprinting

pattern for all promoters was similar to that of the original Alul56 in the region protected

from DNase I cleavage from +20 to -57. The AluUp promoter displayed a strongly

protected region between -37 and -58 corresponding to the UP element. The AluExt and

AluDel, which lacked AT-rich regions and curved DNA, displayed no significant DNA

wrapping. Likewise, DNA wrapping was not observed for Alu8AT promoter (Figure 19).

Gel retardation analysis was performed to determine the relative binding affinity of

the RNA polymerase for the original Alul56 and each of the promoters listed in Figure 17.

The binding reactions were carried out in binding buffer with equivalent amount of 32P end-

labeled promoter. For each concentration of RNA polymerase used, the DNA fragment

containing the Alu 156 promoter was efficiently bound by E. coli RNA polymerase (Figure

20 and 21). This suggest that RNA polymerase binding is enhanced by DNA curvature

upstream of promoter. An unexpected result was observed for AIu27AT promoter. RNA

polymerase displayed a dramatically low affinity for this promoter.

In order to compare the promoter strength in vitro, the single-round run-off

transcription assay was performed. This assay measures the number of the transcribable

complexes at the time of assay. Equal amount of DNA for each promoter was incubated

in the presence of RNA polymerase at each temperature tested. Heparin and mixture of all

four nucleotides triphosphates including [a 32P] ATP were added to each reaction. After

10 minutes, the reactions were stopped by adding urea and heating to 95°C for 5 minutes,

the sample were analyzed by polyacrylamide gel electrophoresis. The relative amount of


70

Alu156 UncRNA

BO UN

27 AT

FREE

m

-r.—X-tV?}. ... / ^ «. £*^.

Figure 20: Gel retardation analysis of Alul56, AluUnc, AluUp, and Alu27AT.Equivalent amounts of32? end-labeled DNA fragments were incubated with various amounts of RNA polymerase at 37°C for 10 minutes, followed by electrophoresis. Lanes marked with a minus sign represent the absence of RNA polymerase. The RNA polymerase concentration was increased as it is marked by the symbol. The RNA polymerase amounts were 0.005, 0.016, and 0.05 pg. The DNA bands representing the RNA polymerase bound complexes and free DNA are marked.


71

A l u 1 5 6 A l u E x t 8 A T A T D e l

RNAn

BOUND-

« • • - | * * * * # *4 * - | *

FREE —

Figure 21: Gel retardation analysis of Alu 156, AluExt, Alu8AT, and AluDel.Equivalent amounts of32? end-labeled DNA fragments were incubated with various amounts of RNA polymerase at 37°C for 10 minutes, followed by electrophoresis. Lanes marked with a minus sign represent the absence of RNA polymerase. The RNA polymerase concentration was increased as it is marked by the symbol. The RNA polymerase amounts were 0.02,0.07, and 0.2 pg. The DNA bands representing the RNA polymerase bound complexes and free DNA are marked.


72

Figure 22: Transcribable promoter complexes formed as a function of temperature.Relative number of transcripts for Alu 156(9), AluExt(■), AluUnc(A), AluUp(V), and Alu27AT(^) without (A) and with (B) temperature shift were determined by densitometry from an autoradiograph of a polyacrylamide gel containing 32P labeled transcripts. Both panels contain representative data for single-round, run-off transcription in the presence of heparin.


73

B:

COQ-O03CCO

<D.OE3z<D.>

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10 15 20 25 30Temperature (°C)

15 20 25Temperature ( C)


74

transcripts formed for each promoter was determined by densitometry. The results are

shown in Figure 22 A. As expected, very little transcription was observed at 0°C, with

increased temperature, transcription increased for all promoters. At low temperature, the

Alu 156 displayed slightly better transcription relative to the other promoters. The Alu27AT

displayed very low transcription at all temperatures. For second set of reactions, 30 seconds

after addition of heparin and nucleotides, they were transferred to 37°C for 10 minutes prior

to stopping the reactions. This temperature shift allows the heparin resistance complexes

to form open complexes and start transcription. The relative number of transcripts as a

function of temperature are shown in Figure 22B. The Alu27AT was the weakest promoter

in all temperatures. This result confirms the result from gel retardation assay where RNA

polymerase exhibited a very low affinity for this promoter. In the temperature shift

reactions, significant transcription at low temperature was observed for the original Alu 156

promoter in contrast to the results observed for panel A, where small number of transcripts

where formed even at 10°C for all promoters tested. At temperatures above 30°C no

significant difference was observed among promoters (data not shown). This indicates that

the step in transcription initiation aided by curved DNA is normally inefficient at low

temperatures. The presence of curved DNA stimulates the formation of a heparin resistant,

closed complex. In the single round transcription assay, open complex formation (i.e.,

strand separation) appears to be the overall rate limiting step for Alul56 and its derivatives.

Effect of a mutation in the C-terminal domain of a subunit on the interaction between

RNA polymerase and DNA upstream of the -35 region. To study the role of a subunit

in the DNA wrapping, RNA polymerase with a deletion starting at position 235 of C-


75

terminal domain (CTD) of a subunit was used. RNA polymerase with and without the CTD

mutation were a gracious gift from Dr. Richard Gourse at University of Wisconsin-Madison.

It has been shown that protection of UP element DNA by the CTD mutant RNA polymerase

is severely reduced in footprinting experiment (Ross et al., 1993). Three hybrid promoters

A1uPr , BalPR, and AP R (McAllister, 1988) were used in footprinting study with mutant

RNA polymerase. The phenomenon of DNA wrapping has been documented for both

A1uPr and BalPR (Nickerson and Achberger, 1995). These two promoters contain the

curved region of Alul56 and BaI129 upstream of APR promoter. APR lacks the curved

DNA immediately upstream of the promoter and no obvious evidence of wrapping was

observed for this promoter.

The results of footprinting with the mutant RNA polymerase are illustrated in Figure

23. For each promoter, the DNase I digestion pattern in the absence of RNA polymerase

is shown in lane 1. Lane 2 represents the footprint of each promoter using wild-type RNA

polymerase and lane 3 shows the footprinting analysis using the CTD mutant RNA

polymerase. Evidence of DNA wrapping was observed for AluPR and BalPR when wild-

type RNA polymerase was used. For APR no obvious wrapping was observed. This result

was in agreement with published data. Addition of mutant RNA polymerase to APr did not

change the pattern except that an enhanced region was observed around -50 region, which

is part o f a binding site. Since mutation in a prevents binding of the subunit to this region,

mutant RNA polymerase can not protect this region from DNase I cutting. This “a

signature” was observed for other two promoters as well. DNA wrapping was diminished


76

Figure 23: DNase I footprint analysis of the AluP*, BalPR, and promoters as afunction of wild-type (lane 2) and mutant (lane 3) RNA polymerase. The nucleotide are numbered approximately relative to transcriptiion start sit at position+1.


BalPB Xi77

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78

for both AIuPr and BaIPR when mutant RNA polymerase was used. This indicates that the

curved DNA is an a binding site and binding of a subunit to its binding site aids DNA

wrapping in the promoters containing curved DNA


DISCUSSION

Effect of DNA curvature immediately upstream of the -35 region on DNA wrapping

by the RNA polymerase. In a previous study using hybrid promoters containing curved

DNA, R coli RNA polymerase was shown to wrap the curved DNA around the enzyme

(Nickerson and Achberger, 1995). The hybrid promoters were made by splicing the curved

DNA found upstream of the Alu 156 and Ball 29 promoters of I?, subtilis phage SP82 onto

X PR promoter (McAllister, 1988). hi this limited study, the phenomenon of wrapping was

examined on one strand of those promoters. One of the first questions asked in the present

study was whether DNA wrapping could be observed for other promoters containing

curved DNA. To explore this question, the Alul56 and Ball29 promoters were used.

Both of these promoters were isolated from B. subtilis bacteriophage SP82 and contain

curved DNA immediately upstream of -35 region. DNase I footprinting was performed to

investigate the interaction between the R coli RNA polymerase and these promoters. Large

region of protection demonstrating the tight binding of RNA polymerase to -10 and -35

regions was observed for both promoters. The DNase I footprint patterns for these two

promoters exhibited enhanced cleavages and sites protected from DNase I repeated almost

every 10 base pair in the DNA upstream of promoters. This pattern was observed for both

strands of both promoters. This feature is indicative of DNA wrapping around RNA

polymerase. A similar pattern was observed when DNA is wrapped around proteins such

as nucleosomes (Shaw et al., 1976; Prunell et al., 1984). The DNase I patterns of both

strands combine to describe interactions characterized by region of DNA wrapped around

79


so

the enzyme and larger region of protection from -70 to -90 consistent with the DNA bound

within a fold or cleft of the RNA polymerase. For derivatives of Alu 156 and Ball29 which

lack curved DNA, the footprint of the promoter region was the same as wild-type

promoters. However, there was no obvious evidence of the DNase I cleavage pattern

consistent with DNA wrapping upstream o f these promoters. This indicates that curved

DNA facilitated the wrapping of DNA upstream of the promoter around the RNA

polymerase. Similar results were observed using the RNA polymerase from B. subtilis

(Cheng, 1996).

Model for the role of DNA curvature in RNA polymerase binding and transcription

initiation. Sequence-directed and protein-induced DNA curvature has been found in

promoters of both prokaryotes and eukaryotes. It has been documented that intrinsically

bent DNA plays a role in modulation of transcription initiation (Lamond and Travers, 1983;

Bossi and Smith, 1984; Gourse et al., 1986; McAllister and Achberger, 1988). Based on

previous studies in our laboratory, a model was proposed for the role of curved DNA in

transcription initiation. It was proposed that curved DNA enhances promoter function by

facilitating the wrapping of the DNA around the RNA polymerase. The structured DNA-

RNA polymerase complex allows the DNA helix to be untwisted and the two strands

separated for transcription initiation.

The model addressed the fact that there are multiple RNA polymerase promoter

complexes identified with conformational changes in the DNA and enzyme. Among these

complexes are the initial closed RNA polymerase-promoter complexes (RPCi)> a second

closed complex in which the strands of the DNA have been partially untwisted but remain


81

base paired (RPq), and the open promoter complex in which DNA strand separation has

occurred between the -10 region and +1 start site (RP0 ). Previous studies with B. subtilis

RNA polymerase have demonstrated that curved DNA associated with promoters aids

binding and the formation of open promoter complex (McAllister and Achberger, 1988;

Stemke, 1993). More recently DNase I footprinting studies with B. subtilis RNA-

polymerase demonstrated the presence of a closed complex in which the upstream DNA is

wrapped around the RNA polymerase (Cheng, 1996). This suggested that curved DNA

stimulates a step prior to RP0. Prior to the present study, little evidence, other than the

existence of DNA wrapping for hybrid promoters, was available to address this model to the

E. coli RNA polymerase.

The model proposed in our laboratory is similar to a model proposed for the role of

E. coli CRP, a DNA bending regulatory protein, in transcription activation. It has been

demonstrated that curved DNA can replace the CRP binding she for the lacP l promoter and

gcdpl promoters (Barco et al., 1989; Gartenberg and Crothers, 1991). Based on the model

proposed by Gartenberg and Crothers (1991), curved DNA localizes at the end of a

superhelical domain and facilitates the wrapping of DNA around RNA polymerase bound

to the promoter. This structure favors the transcription initiation.

Based on present the study we were able to relate K coli RNA polymerase to the

existing model and advance it. The presence of an additional closed promoter complex

between the initial closed complex and the open promoter complex has been identified in

many studies (Kadesch et al. 1982; Buc and McClure, 1985; Straney and Crothers, 1985).

The RPq is stable at low temperature (i.e., the predominant complex formed below 20°C)


82

and resistant to RNA polymerase competitors such as heparin and poly d(AT) (Buc and

McClure, 1985; Spascky et al. 1985; Schickor et al., 1990). The transition step from RPq

to RP0 became the rate limiting step below 20°C (Buc and McClure, 1985; Spassky et al.

1985). We now have evidence that the presence of curved DNA affects the formation of

RPq. A single-round transcription assay was used in a temperature shift format. RNA

polymerase was allowed to form complexes with various promoters at 0°, 10°, 20°, and

30 °C prior to the addition of a mixture of the four nucleotide triphosphates and heparin.

The nucleotides allow RNA syntheses from RPq, and the heparin binds free RNA

polymerase. RPC1 is said to be heparin sensitive since it dissociates rapidly and free RNA

polymerase is bound by heparin. If the reactions were shifted to 37°C after addition of

heparin and nucleotides, transcription from the low temperature complexes increased

dramatically for Alul56 relative to promoters without curved DNA. Curved DNA on

Alu 156 allowed formation of RPq at low temperatures (0° and 10°C) which quickly

isomerized to RP0 at 37°C. Taking this result in consideration, our updated model

proposes that by facilitating the wrapping of DNA around RNA polymerase (i.e., assisting

the RNA polymerase to structure the DNA), curved DNA enhances the transition from

heparin sensitive closed complex (RPC]) to a heparin-resistant, closed complex (RI^ ),

which is easily isomerized to form the open promoter complex. This allows more efficient

transcription initiation when this step is rate limiting. Since the DNA in RPa is believed

to be partially untwisted, curved DNA appears to aid DNA untwisting by RNA polymerase.

The nature of the interaction between K coli RNA polymerase and DNA upstream of

the -35 region. The interaction between RNA polymerase and DNA upstream of the


83

curved DNA would be sequence independent if salt can be used to disrupt these

interactions. In other words, addition of salts such as NaCl interfere with the electrostatic

interactions between RNA polymerase and the phosphate charges on the DNA As the salt

concentration increased to ISO or 200 mM, wrapping diminished for both Alu 156 and

Ball29 promoters. The DNase I pattern consistent with RNA polymerase bound to the

promoter was observed at every salt concentration. This indicates that the population of

DNA-RNA polymerase complexes formed at low salt concentration were different to those

formed at high salt concentration. The wrapped DNA complex was formed at low salt

concentration suggesting that electrostatic interactions predominant.

Existence of unwrapped open promoter complexes. Since presence of different DNA-

RNA polymerase complexes were shown in various salt concentrations, it was important to

show if any of these complexes represent open promoter complexes. Potassium

permanganate was used to test for open promoter complex formation as a function of salt

concentration. While open promoter complexes were formed at all salt concentrations

tested, open promoter complex formation decreased with increasing the NaCl concentration.

Considering that no wrapped DNA complexes were observed above ISO mMNaCl there

were open promoter complexes in which the DNA is no longer wrapped. In order for RNA

polymerase to engage in active transcription wrapping must be released at some point.

Presence of unwrapped open promoter complexes are in agreement with this proposal.

Consistent with the proposed model, this indicates that wrapping must occur in steps prior

to open promoter complex formation.


84

DNA wrapping in the absence of open promoter complex formation. According to our

model, for DNA wrapping to aid strand separation, wrapping must exist prior to formation

of the open promoter complex in which the strands are separated. The step of strand

separation is rate limiting at low temperatures. Thus the effect of temperature on the

structured complex of Alul56 was studied. At 0°C, as expected, almost no open promoter

complexes were found. Open promoter complexes were observed at S°C, and the

formation of these complexes increased when temperature was raised. Although minor

differences were observed in the DNase I digestion patterns upstream of the curved DNA

at each temperature, DNA wrapping was obvious at all temperatures. The minor changes

in the footprint indicates that there were different complexes formed at various

temperatures. The evidence supports the presence of DNA wrapping in the absence of open

promoter complex formation. This suggests that wrapping can occur in complexes

preceding the open promoter complex.

In our study, we went one step further and confirmed the presence of closed,

wrapped promoter complexes. This would be expected if indeed wrapping occurs prior to

strand separation. Many closed complexes are sensitive to effect of heparin. Heparin

challenge was used for complexes formed at 37°C and 0°C. Results indicate that at 37°C

all wrapped complexes were eliminated upon addition of heparin. Heparin challenge at 0°C

confirmed the idea that wrapped complexes were heparin sensitive, closed complexes. Most

o f the complexes formed at 0°C dissociated in the presence of heparin including all the

wrapped complexes. Since few open complexes were observed at 0°C, all wrapped


85

complexes were closed, and heparin sensitive. This agrees well with the model that the

wrapped complex is the RPC1 complex.

Comparison of the effect of AT-rich regions and curved DNA on wrapping and

transcription initiation. It has been established that the a subunit of & coli RNA

polymerase binds a specific AT-rich region upstream of the -35 region. It has been

suggested that A-tract curvature simply functions as an a binding site (Ross et al., 1993).

A collection of promoters containing AT-rich region upstream of promoter were studied and

compared to Alu 156 promoter. Based on the results of DNase I footprints from these

promoters, wrapping was more evident for wild-type Alu 156. However, small degree of

wrapping was observed for AluUnc and AluUp promoters. This supports the idea that the

curved DNA may stabilize a transient complex, formed by RNA polymerase with all

promoters, long enough to be detected by DNase I footprinting. The results from gel

retardation assay suggested that binding of E. coli RNA polymerase was strongest with the

promoter with curved DNA This suggests that there are more tight binding complexes in

the population of RNA polymerase-promoter complexes formed in the presence of curved

DNA. Some researchers have suggested that A-tract curved DNA is simply an AT-rich

binding site for a subunit of RNA polymerase. Although we agree with part of that

suggestion that curved DNA could act as an a binding site, our study showed that none

of the AT-rich DNA containing promoters, even the AluUp promoter, which contain the

so called UP element, were able to perform as well as Alul56. In addition, we showed that

not all AT-rich regions are beneficial. In our Alu27AT promoter, the long stretch of AT


86

seemed to be detrimental to promoter function. Perhaps for an AT-rich region to be good

a binding site, proper alignment of the a binding site relative to the promoter is necessary.

Contribution of a subunit of RNA polymerase on wrapping. The £ coli RNA

polymerase containing C-terminal truncation of 94 amino acids is stable in vivo and

assembles into holoenzyme. In other studies, this enzyme failed to protect the UP element

DNA of ribosomal RNA promoter rm BPl. hi other words, mutant a subunit could not bind

to its AT-rich binding site (Ross et al., 1993). We used this enzyme to explore the effect

of a subunit on DNA wrapping. DNase I footprints of this enzyme to the promoter region

was the same as that of wild-type enzyme. However, DNA wrapping for promoters

containing curved DNA was diminished when the mutant enzyme was used. This indicates

that A tracts involved in DNA curvature may function as a subunit binding site and

contribute to DNA wrapping.

Summary of the model. Based on the present study, we concluded that the C-terminal

domain of a contacts the A-tract curvature and promoters DNA structuring by the RNA

polymerase. Wrapping appears to be limited to RPC1, a heparin sensitive, closed complex.

The curved DNA promotes the transition from RPC1 to RPo, a heparin resistant, closed

complex. The RPq can quickly isomerize to RPq, an open promoter complex lacking DNA

wrapping. The effect of DNA curvature on overall transcription will only be observed under

conditions in which the RPC1 to RP^ transition is the rate-limiting reaction.


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VITA

Parisa Jazbi received her bachelor of science degree in Zoology from Shiraz

University in Shiraz, Iran. In August 1993, she joined the graduate program in the

Department of Microbiology at Louisiana State University. She has worked as a teaching

assistant for past four years. Her research focuses on investigating the role o f DNA

wrapping in initiation of transcription using K coli RNA polymerase. Parisa is currently

attending Louisiana State University, where she is a candidate for the doctor of philosophy

degree in Microbiology with a minor in Biochemistry.

98


DOCTORAL EXAMINATION AND DISSERTATION REPORT

Candidate: Parisa Jazbi

Major Field: Microbiology

Title of Dissertation: DNA Wrapping and the Nature of Interaction

between E.coli RNA Polymerase and Promoter DNA

Approved:IM

Major Professor and Chairman

EXAMINING COMMITTEE:

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Date of Examination:

October 17, 1997


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