Scotty Merrell Department of Microbiology and Immunology B4140 dmerrell@usuhs.mil

Scotty MerrellDepartment of Microbiology and Immunology

B4140dmerrell@usuhs.mil

Regulation of Gene Expression I

1. Why does the expression of genes need to be regulated?

QUESTIONS

3. How is the expression of genes regulated?

2. Why is it important to study gene regulation?

4. How do we study gene regulation?

Pathogenic bacteria:External reservoir Host

Infection site #1 Infection site #2

Bacteria experience different conditions depending on environment

1. Why does the expression of genes need to be regulated?

QUESTIONS

3. How is the expression of genes regulated?

2. Why is it important to study gene regulation?

4. How do we study gene regulation?

Pathogenic bacteria produce virulence factors whenthey sense they are inside of a host

Vibrio cholerae, the cause of cholera, produces toxin insideof the host. Understanding regulation of expression of this toxin

is a means of understanding ways to prevent its production.

ICDDR,B

1. Why does the expression of genes need to be regulated?

QUESTIONS

3. How is the expression of genes regulated?

2. Why is it important to study gene regulation?

4. How do we study gene regulation?

DNAPromoteroperator

Attenuator Stop signal

RNA polymeraseRegulatory proteins aa-tRNAs RNA polymerase

Transcription

Transcriptional control(a) Transcription initiation: positive/negative(b) Transcription termination: attenuation/anti-termination

Regulation of gene expression

mRNA

Degradation

Translational controlTranslation initiation: positive/negative

Ribosomebindingsite

Stop signal

Regulatory proteinsAntisense RNAs

Translation

Protein

Post-translational control(e.g., proteolysis)

Regulation of Gene Expression

DNAPromoteroperator

Attenuator Stop signal

RNA polymeraseRegulatory proteins aa-tRNAs RNA polymerase

Transcription

Transcriptional control(a) Transcription initiation: positive/negative(b) Transcription termination: attenuation/anti-termination

Regulation of gene expression

mRNA

Degradation

Translational controlTranslation initiation: positive/negative

Ribosomebindingsite

Stop signal

Regulatory proteinsAntisense RNAs

Translation

Protein

Post-translational control(e.g., proteolysis)

Regulation of Gene Expression

’

5’ppp

Promoter

Polymerase binds topromoter region, forminga closed complex

Polymerase unwindsDNA, forming anopen complex

Transcription begins

Core enzyme

Holoenzyme

TTGACAAACTGT

TATAATATATTA

-6-13-30-37 +1-10 region-35 region

mRNA

’

’

Transcription initiation

RNA polymerase-promoter interactions

Some promoters contain UP elements that stimulate transcriptionthrough direct interaction with the C-terminal domains of the

subunits of the RNA polymerase

Promoter with a full UP element containing two consensus subsites.

Promoter with an UP element containing only a consensus proximal subsite.

Promoter with an UP element containing only a consensus distal subsite.

Arrangement of   subunits on UP elements

Genes come in two main flavors:

1. Constitutively expressed (transcription initiationis not regulated by accessory proteins)

2. Regulated (transcription initiationis regulated by accessory proteins)

a. Negatively Regulated--Repressor Proteinb. Positively Regulated--Activator Protein

Mechanisms of Regulation of Transcription Initiation:Negative Regulation

RNA Polymerase

Mechanisms of Regulation of Transcription Initiation:Negative Regulation

Repressor

Repressor

Co-repressor

Repressor

Inactivator

The lac operona model for negative regulation

A bacterium's prime source of food is glucose, since it does not have to be modified to enter the respiratory pathway. So

if both glucose and lactose are around, the bacterium wants to turn off lactose metabolism in favor of glucose metabolism.

There are sites upstream of the lac genes that respond to glucose concentration.

This assortment of genes and their regulatory regions is called the lac operon.

H O

L a c t o s e

O

H O C H 2O

O H

O H

H O C H 2O

O H

O H

O H

H O C H 2O

O H

O H

O H

G a l a c t o s e

H O C H 2O

H OO H

O H

O H

G l u c o s e

+

- G a l a c t o s i d a s e

- G a l a c t o s i d a s e

OH OH O C H 2

O

O H

O H

C H 2O

O H

O H

O H

H O

A l l o l a c t o s e

Structural genes:lacZ encodes -galactosidase lacY encodes -galactoside permeaselacA encodes -galactoside transacetylase

Regulatory gene and elements:lacI --- encodes repressor proteinlacO --- operatorlacP --- promoter

lacIPi P O lacYlacZ lacA

The lac operon

The lac promoter and operator regions

Lac Repressor(monomer) (tetramer)

The Lac Repressor is constitutively expressed

Repressor binding prevents transcription

When lactose is present, it acts as an inducer of the operon. It enters the cell and binds to the Lac repressor, inducing a conformational

change that allows the repressor to fall off the DNA. Now the RNA polymerase is free to move along the DNA and RNA can be made from

the three genes. Lactose can now be metabolized.

Remember, the repressor actsas a tetramer

When the inducer (lactose) is removed, the repressor returns to its original conformation and binds to the DNA, so that RNA polymerase

can no longer get past the promoter to begin transcription. No RNA and no protein are made.

Remember, the repressor actsas a tetramer

1. Mutation in the regulatory circuit may either abolish expression of the operon or cause it to occur without responding to regulation.

2. Two classes of mutants: A. Uninducible mutants: mutants cannot be expressed at all. B. Constitutive mutants: mutants continuously express genes that do not respond to regulation.

3. Operator (lacO): cis-acting element Repressor (lacI): trans-acting element

How to identify the regulatory elements?

cis-configuration: description of two sites on the same DNA molecule (chromosome) or adjacent sites.

cis dominance: the ability of a gene to affect genes next to it on the same DNA molecule (chromosome), regardless of the natureof the trans copy. Such mutations exert their effect, not because of altered products they encode, but because of a physical blockage or inhibition of RNA transcription.

trans-configuration:description of two sites on different DNA molecules (chromosomes) or non-contiguous sites.

Definitions:

Constitutive mutants: do not respond to regulation.

l a c I -P i P O l a c Yla c Z l a c A

N o n b in d i n gr e p r e s s o r

m R N A m R N AX

M u t a t i o n s t h a t i n a c t i v a t e t h e l a c I g e n e ( l a c I - ) c a u s e t h e o p e r o n t o b e c o n s t i t u t i v e l y e x p r e s s e d , b e c a u s e t h e m u t a n t r e p r e s s o rp r o t e i n c a n n o t b i n d t o t h e o p e r a t o r .

Would this be a cis-dominant or recessive mutation?

lacI+Pi

mRNA

lacI-Pi P O lacYlacZ lacA

mRNA

Constitutive mutants in the lacIgene are recessiveConstitutive mutants can be recessive

lacI+

mRNA

lacI-Pi P O lacYlacZ lacA

mRNA mRNAX

Constitutive mutants can also be dominant if the mutant allele produces a “bad” subunit, which is not only itself unable to bind to

operator DNA, but is also able to act as part of a tetramer to prevent any “good” (wild type LacI) subunits from binding.

et al.

Think about how you could determinewhether a mutation was dominant or

recessive.

Questions about negativeRegulation of lac ?

Mechanisms of Regulation of Transcription Initiation:Positive Regulation

RNA Polymerase

Mechanisms of Regulation of Transcription Initiation:Positive Regulation

RNA Polymerase

Activator

The lac operona model for positive regulation

When levels of glucose (a catabolite) in the cell are high, a molecule called cyclic AMP is inhibited from forming. So

when glucose levels drop, more cAMP forms. cAMP binds to a protein called CAP (catabolite activator protein), which is

then activated to bind to the CAP binding site. This activates transcription, perhaps by increasing the affinity of the site for

RNA polymerase. This phenomenon is called catabolite repression, a misnomer since it involves activation, but

understandable since when it was named, it seemed that the presence of glucose repressed all the other sugar

metabolism operons.

CAP --- a positive regulator

1. Catabolite repression: the decreased expression of many bacterial operons that results from addition of glucose. Also known as “glucose effect” or “glucose repression”.

2. E. coli catabolite gene activator protein (CAP; also known as CRP, the cAMP receptor protein).

3. CAP-cAMP activates more than 100 different promoters, including promoters required for utilization of alternative carbohydrate carbon sources such as lactose, galactose, arabinose, and maltose.

Inactive CAP

CAP --- a positive regulator

A. under catabolite-respressing conditions cAMP level is very low

crp Target operon

cAMP

B. Under non-catabolite-respressing conditions cAMP level is very high

InactiveCAP

CAP-cAMPActive CAP

crp Target operon

RNAP RNAP

cAMPAut

oreg

ulat

ion

Act

ivat

ion

-O-P~O-P~O-P-O-CH2

=-

O

O

=-

O

O

=-

O

OO

C

--

H C

--

OHC

--

OH

C

--

HH H

Adenine

ATP

O-CH2 OC

--

H C

--

OC

--

OH

C

--

HH H

O=PO-

Adenine

cAMP

Adenylate cyclase

PTSGlucose Glucose-6-P

IIAGlc-P

IIAGlc

OUT IN

How does glucose reduce cAMP level?

1. IIAGlc-P activates adenylate cyclase.2. Glucose decreases IIAGlc-P level, thus reducing cAMP production.3. Glucose also reduces CAP level: crp gene is auto-regulated by CAP-cAMP.

PTS - phosphoenolpyruvate-dependent carbohydrate phosphotransferase systemIIAGlc - glucose-specific IIA protein, one of the enzymes involved in glucose transport.

Activation of expression of the lac operon

E. coli CAP (CRP) --- 209 amino acids

NH2- -COOH

140-209

DNA-bindingHelix-turn-helix

AR1156-164

His19His21

Glu96Lys101

AR2

Dimerization and cAMP-binding

1-139

Transcription activation by CAP at class I CAP-dependent promoters

(-62)

Transcription activation:1. Interaction between the AR1 of the downstream CAP subunit and one copy of CTD.2. The AR1-CTD interaction facilitates the binding of CTD to the DNA downstream of CAP.3. Possibly, interaction between same copy of CTD and the bound at the –35 element.4. The interaction between the second CTD and CAP is unclear.

The result: increasing the affinity of RNAP for promoter DNA, resulting in an increase in the binding constant KB, for the formation of the RNAP-promoter closed complex

Transcription activation by CAP at class I CAP-dependent promoters (cont.)

(-103, -93, -83, or –72)

Transcription activation: Possibly, the second copy of CTD may interact with the DNA downstream of CAP, and may interact with the bound at the –35 element.

Results: increasing the affinity of RNAP for promoter DNA, resulting in an increase in the binding constant KB, for the formation of the RNAP-promoter closed complex

Transcription activation by CAP at class II CAP-dependent promoters (cont.)

(-42)

Transcription activation:1. Interaction between the AR1 of the upstream CAP subunit and one copy of CTD

(either CTDI or CTDII, but preferentially CTDI). The AR1-CTD interaction facilitates the binding of CTD to the DNA upstream of CAP.

Results: increase in the binding constant KB, for the formation of the RNAP-promoter closed complex

• Interaction between the AR2 of the downstream CAP subunit and NTDI.

Result: increase the rate constant, kf, for isomerization of closed complex to open complex.

Transcription activation by CAP at class III CAP-dependent promoters

(-103 or –93) (-62)

Transcription activation:

Each CAP dimer functions through a class I mechanism with AR1 of the downstream subunit of each CAP dimer interacting with one copy of CTD

Results: synergistic transcription activation

Transcription activation by CAP at class III CAP-dependent promoters (cont.)

(-103, -93, or -83) (-42)

Transcription activation:

The upstream CAP dimer functions by a class I mechanism, with AR1 of the downstream subunit interacting with one copy of CTD; the downstream CAP dimer functions by a class II mechanism, with AR1 and AR2 interacting with the other copy of CTD and NTD, respectively.

Results: synergistic transcription activation

(a) Glucose present (cAMP low); no lactose;

lacIPi P O lacYlacZ lacA

Repressormonomer

Repressortetramer

mRNAX

(b) Glucose present (cAMP low); lactose present

Repressormonomer

Repressortetramer

mRNA

Inducer

High level of mRNAX

Inactiverepressor

High

(c) No glucose (cAMP high); lactose present

cAMPCAP

Glucose effect on the E. coli lac operon

lacIPi P O lacYlacZ lacA

Repressormonomer

Repressortetramer

mRNAX

lacIPi P O lacYlacZ lacA

Repressormonomer

Repressortetramer

mRNAX

No lactose inside the cells!(inducer exclusion)!

(a) Glucose present (cAMP low); no lactose;

lacIPi P O lacYlacZ lacA

Repressormonomer

Repressortetramer

mRNAX

(b) Glucose present (cAMP low); lactose present

Repressormonomer

Repressortetramer

mRNA

Inducer

High level of mRNAX

Inactiverepressor

High

(c) No glucose (cAMP high); lactose present

cAMPCAP

Glucose effect on the E. coli lac operon

lacIPi P O lacYlacZ lacA

Repressormonomer

Repressortetramer

mRNAX

lacIPi P O lacYlacZ lacA

Repressormonomer

Repressortetramer

mRNAX

No lactose inside the cells!(inducer exclusion)!

Inducer exclusion: How does it work?

1. Uptake of glucose dephosphorylates enzyme IIglc.

2. Dephosphorylated enzyme IIglc binds to and inhibits lactose permease.

3. Inhibition of lactose permease prevents lactose from entering the cell.

4. Hence, the term inducer exclusion.

Questions about positive regulationof the lac operon?

Dual positive and negative control of transcription initiation:

the E. coli ara operon

The E. coli L-arabinose operon

+

+

AraC exists in two states

P1 P2

Arabinose

Arabinose

ActivatorAntiactivator

AraC acts as a positive or negative regulator based on its conformational state and binding affinity for

various sites in the two promoter regions.

AraC encodes the regulatorAraO1 and AraO2 encode operatorsCAP is a CAP binding siteAraI is an additional regulatory regionAraBAD are the structural genes

If AraC concentration becomes too high, AraC will also bind to AraO1 and repress its own expression.

No arabinose

+ arabinose

In the absence of arabinose, the P1 form of AraC binds AraO2 and AraI to prevent any P2 form from binding and activating expression--this is anti-activation, not repression!

In the presence of arabinsose, AraC shifts to the P2 form and bindsAraI and acts to activate transcription.

Therefore AraC is an Activator, Repressor and Anti-activator!!

The regulatory regions of the PC and PBAD promoters

The domain structure of one subunit of the dimeric AraC protein

The PC and PBAD Regions in the presence or absence of arabinose

+ L-arabinose

Hypothetical model of the activation of the PBAD promoter

1. PBAD – class II promoter2. Possible interactions: between the CTD of RNAP

and the CAP protein and AraC protein and DNA

1. Find mutations that render the regulation uninducible or constitutive.

2. Decide by performing a complementation test if the mutants are dominant or recessive.

3. If they are recessive, decide if the system is regulated by repression or by activation. A recessive mutated activator has most likely lost function: the system will become uninducible. A recessive mutated repressor has also lost function, but now the system will show constitutive expression.

4. Decide if the elements of the system act in cis or in trans to each other: are they proteins or DNA binding sites?

5. Construct a model.

Strategies for Understanding Regulation

Questions about ara regulation?

A. Transcriptional control1. Transcription initiation a) Positive b) Negative2. Transcription termination Attenuation

B. Translational control1. Positive2. Negative

C. Post-translational control--Proteolysis

Regulatory mechanisms used to control gene expression

DNAPromoteroperator

Attenuator Stop signal

RNA polymeraseRegulatory proteins aa-tRNAs RNA polymerase

Transcription

Transcriptional control(a) Transcription initiation: positive/negative(b) Transcription termination: attenuation/anti-termination

Regulation of gene expression

mRNA

Degradation

Translational controlTranslation initiation: positive/negative

Ribosomebindingsite

Stop signal

Regulatory proteinsAntisense RNAs

Translation

Protein

Post-translational control(e.g., proteolysis)

RNAP

Transcription termination players:Termination sequence

RNA polymeraseand sometimes the Rho ( factor

A B C D

Promoter Operon of 4 genes Terminator

X

Rho-independentterminator

Rho-independentterminator

Rho-dependentterminator

Two major types of Terminator Sequences1. Rho-independent2. Rho-dependent

Attenuation: Premature termination of transcription of operons for amino acid biosynthesis

(trp, his, leu, etc.)

Relies on coupled transcription and translation and RNA secondary structure

P/O trpDtrpE trpC

mRNA

Tryptophanrepressor

mRNA

L trpAtrpBP/O trpR

1

23

4

The trp leader mRNA encodes the LEADER PEPTIDE

MetLysAlaIlePheValLeuLysGlyTrpTrpArgThrSer5’-AUGAAAGCAAUUUUCGUACUGAAAGGUUGGUGGCGCACUUCC U CCCAUAGACUAACGAAAUGCGUACCACUUAUGUGACGGGCAAAGA GCCCGCCUAAUGAGCGGGCUUUUUUUUGAACAAAAUUAGAGA-3’

Organization of Tryptophane Biosynthesis Genes

End product of the pathway

1 2 3 4

mRNA forms secondary structures

Adapted from http://www.andrew.cmu.edu/user/berget/Education/attenuation/atten.html

3 and 4 form a Rho-independent

terminator

Two possible alternative structures can form2 is complementary to 1 and 33 is complementary to 2 and 4

2 and 3 form the Pre-emptor, which prevents

Terminator formation

Pre-emptor

Tryptophan absent Tryptophan present

UGGUGGCGCACUUCCU

UGGUGGCGCACUUCCU

RegulatoryOperon Leader Peptide Sequence Amino Acid(s) his Met-Thr-Arg-Val-Gln-Phe-Lys-His-His-His-His -His-His-His-Pro-Asp His pheA Met-Lys-His-Ile-Pro-Phe-Phe-Phe-Ala-Phe-Phe -Phe-Thr-Phe-Pro Phe leu Met-Ser-His-Ile-Val-Arg-Phe-Thr-Gly-Leu-Leu -Leu-Leu-Asn-Ala-Phe-Ile-Val-Agr-Gly-Agr-Pro -Val-Gly-Gly-Ile-Gln-His Leu thr Met-Lys-Agr-Ile-Ser-Thr-Thr-Ile-Thr-Thr-Thr -Ile-Thr-Ile-Thr-Thr-Gly-Asn-Gly-Ala-Gly Thr, Ile

ilv Met-Thr-Ala-Leu-Leu-Arg-Val-Ile-Ser-Leu-Val -Val-Ile-Ser-Val-Val-Val-Ile-Ile-Ile-Pro-Pro -Cys-Gly-Ala-Ala-Leu-Gly-Arg-Gly-Lys-Ala Leu, Val, Ile

Biosynthetic Operons Regulated by Attenuation

Attenuation can also occur at the level of Protein-RNA interaction:

Regulation of the trp operon in Bacillus

Model of trp transcriptional

control

Binding of activated TRAP

in the leaderpeptide

results in the formation of a

terminatorstructure

Take home message:

Transcription of genes to produce mRNAcan be controlled at the level of

initiation and/or termination

STOP