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Slide 1
Chapter 8 Chapter 8 Major Shifts in Prokaryotic
Transcription
Slide 2
Modification of the Host RNA Polymerase Transcription of phage
SPO1 genes in infected B. subtilis cells proceeds according to a
temporal program in which early genes are transcribed first, then
middle genes, and finally late genes. This switching is directed by
a set of phage-encoded factors that associated with the host core
RNA polymerase and change its specificity from early to middle to
late.
Slide 3
RNA polymerase changes specificity gp28: (1) diverts the hosts
polymerase from transcribing host (2) switches from early to middle
phage transcription gene gp33 and gp34: The switch from middle to
late transcription occurs in much the same way, except that two
polypeptides team up to bind to the polymerase core and change its
specificity.
Slide 4
Fig. 8.1
Slide 5
Genetic evidence: Mutants of gp28, gp34 or 33 prevent
early-to-middle, middle-to-late switch Biochemical data: Pero
measured polymerase specificity by transcribing SP01 DNA in vitro
with core (a), enzyme B (b) or enzyme C (c), in the presence of [ 3
H]UTP to label the RNA product. Next, they hybridized the labeled
RNA to SP01 DNA in the presence of the following competitors, early
SP01 RNA (green); middle RNA (blue); and late RNA (red). Look for
the competition for the products:
Slide 6
Control of Transcription During Sporulation B. subtilis can
exist indefinitely in the vegetative, as long as conditions are
appropriate for growth. Under starvation conditions, this organism
forms endospores, that can survive for years until favorable
conditions return Sporulation is a fundamental change
Slide 7
Control of Transcription During Sporulation When the bacterium
B. subtilis sporulates, a whole new set of sporulation-specific
genes is turned on, and many, but not all, vegetative genes are
turned off. This switch takes place largely at the transcription
level. It is accomplished by several new factors that displace the
vegetative factor from the core RNA polymerase.
Slide 8
Slide 9
More than one new sigma factors are involved in sporulation At
least three sigma 29 (sigma E), sigma 30 (sigma H), and sigma 32
(sigma C) in addition to sigma 43 (sigma A) are involved.
Slide 10
The DNA region contains two promoters: a vegetative and a
sporulation
Slide 11
In vitro transcription: Plasmid p213 + labeled nt+ Sigma E or
sigma A, then hybridized the labeled RNA to southern blot
containing EcoRI-HincII fragments of the plasmid Sigma E has some
ability to recognize vegetative promoters
Slide 12
spoIID: well-characterize Sporulation gene. Rong prepared a
restriction fragment containing the spoIID promoter and transcribed
it in vitro with B. subtillis core RNA polymerase plus sigma E (
middle lane) or sigma B plus sigma C. Only the enzyme containing
sigma E made the proper transcript.
Slide 13
Genes with Multiple Promoters Some prokaryotic genes must be
transcribed under conditions where two different factors are
active. These genes contain two different promoters. This ensures
their expression no matter which factor is present and allows for
different control under different conditions.
Slide 14
Spo VG: transcribed by E B and E E. The last purification step
was DNA- cellulose column chromatography. The polymerase activity
in each fraction (red). The insert shows the results of a run-off
transcription assay using a DNA with two SpoVG promoters.
Slide 15
Fig. 8.7
Slide 16
Purified sigma factors B and E by gel electrophoresis and
tested them with core polymerase by the same run-off transcription
assay.
Slide 17
Fig. 8.8
Slide 18
Fig. 8.9
Slide 19
The E. coli Heat Shock Genes When cells experience an increase
in temperature, or a variety of other environmental insults, they
mount a defense called the heat shock response. Molecular
chaperones, proteases are produced. At least 17 new heat shock
transcripts begins when at higher temperature (42 o C). This shift
of transcription required -32 ( H).
Slide 20
Slide 21
Infection of E. coli by Phage Infection of E. coli by Phage
Phage can replicate in either of two ways: lytic and
lysogenic.
Slide 22
A bacterium harboring the integrated phage DNA is called a
lysogen The integrated DNA is called a prophage
Slide 23
Slide 24
Cro gene product blocks the transcription of repressor CI N:
antiterminator Extension of transcripts controlled by the same
promoters. Q: antiterminator
Slide 25
Lytic reproduction of Phage Lytic reproduction of Phage The
immediate early/delayed early/late transcriptional switching in the
lytic cycle of phage is controlled by antiterminators.
Slide 26
N utilization site NusA N: function by restricting the pause
time at the terminator
Slide 27
Antitermination Five proteins (N, NusA, NusB, NusG and S10)
collaborate in antitermination at the immediate early terminators.
Antitermination in the late region requires Q, which binds to the
Q-binding region of the qut site as RNA polymerase is stalled just
downstream of the late promoter.
Slide 28
Highly conserved among Nut sites Help to stabilize the
antitermination complex contains an inverted repeat
Slide 29
NusA, NusB, NusG, ribosomal S10 proteins interfere with
antitermination Gel mobility shift assay: binding between N and RNA
fragment containing box B NusA+ N bound to the complex: Fig.
8.16
Slide 30
Highly conserved among Nut sites Help to stabilize the
antitermination complex contains an inverted repeat
Slide 31
Nus A and S10 bind to RNA polymerase, and N and Nus B bind to
the box B and box A regions of the nut site in the growing
transcript.
Slide 32
Fig. 8.15
Slide 33
Fig. 8.17 Qut: Q utilization site Q binds directly to qut site
not to the transcript
Slide 34
Establishing Lysogeny Phage establishes lysogeny by causing
production of enough repressor to bind to the early operators and
prevent further early RNA synthesis. The promoter used for
establishment of lysogeny is P RE.
Slide 35
Fig. 8.18 Delayed early transcription from P R gives cII mRNA
that is transcribed to CII (purple), which allows RNA polymerase
(blue and red) to bind to P RE and transcribe the cI gene
Slide 36
Autoregulation of cI Gene During Lysogeny The promoter that is
used to maintain lysogeny is P RM. It comes into play after
transcription from P RE makes possible that burst of repressor
synthesis that establishes lysogeny. This repressor binds to O R 1
and O R 2 cooperatively, but leave O R 3 open. RNA polymerase binds
to P RM,, in a way that contacts the repressor bound to O R 2.
Slide 37
Fig. 8.19
Slide 38
Slide 39
Run-off transcription (this construct does not contain O L,
therefore, need to use very high concentration of repressor)
Slide 40
High levels of repressor can repress transcription from P RM,
may involve interaction of repressor dimers bound to O R 1, O R 2
and O R 3, with repressor dimers bound to O L 1, O L 2 and O L 3
via DNA looping.
Slide 41
RNA polymerase-repressor Interaction Intergenic suppressor
mutation studies show that the crucial interaction between
repressor and RNA polymerase involves region 4 of the subunit of
the polymerase.
Slide 42
Fig. 8.23
Slide 43
Fig. 8.24
Slide 44
Fig. 8.25
Slide 45
Determining the fate of a Infection: lysis or lysogeny Depends
on the outcome of a race between the products of the cI and cro
genes. The winner of the race is further determined by the CII
concentration, which is determined by the cellular protease
concentration, which is in turn determined by environmental factors
such as the richness of the medium.
Slide 46
Fig. 8.26
Slide 47
Lysogen Induction When a lysogen suffers DNA damage, it induces
the SOS response. The initial event in this response is the
appearance of a coprotease activity in the RecA protein. This
causes the repressors to cut themselves in half, removing them from
the operators and inducing the lytic cycle. In this way, progeny
phages can escape the potentially lethal damage that is occurring
in their host.
Slide 48
Fig. 8.27
Slide 49
Chapter 9 Chapter 9 DNA Protein Interactions in
Prokaryotes
Slide 50
Helix 2 of the motif (red) lies in the major groove of its DNA
target
Slide 51
9-51 The Family of Repressors Repressors have recognition
helices that lie in the major groove of appropriate operator
Specificity of this binding depends on amino acids in the
recognition helices
Slide 52
9-52 Binding Specificity of Repressor-DNA Interaction Site
Repressors of -like phage have recognition helices that fit
sideways into the major groove of the operator DNA Certain amino
acids on the DNA side of the recognition helix make specific
contact with bases in the operator These contacts determine the
specificity of protein- DNA interactions Changing these amino acids
can change specificity of the repressor
Slide 53
9-53 Probing Binding Specificity by Site- Directed Mutagenesis
Key amino acids in recognition helices of 2 repressors are proposed
These amino acids are largely different between the two
repressors
Slide 54
Slide 55
The helix-turn-helix motif of the upper monomer (red and blue)
is inserted into the major groove of the DNA)
Slide 56
The repressor of the lambda-like phages have recognition
helices that fit sideways into the major groove of the operator
DNA. Certain amino acids on the DNA side of the recognition helix
make specific contact with bases in the operator, and these
contacts determine the specificity of the protein-DNA interaction.
Changing these amino acids can change the specificity of the
repressor.
Slide 57
9-57 High-Resolution Analysis of Repressor-Operator
Interactions General Structural Features Recognition helices of
each repressor monomer nestle into the DNA major grooves in the 2
half- sites Helices approach each other to hold the two monomers
together in the repressor dimer DNA is similar in shape to B-form
DNA Bending of DNA at the two ends of the DNA fragment as it curves
around the repressor dimer
Slide 58
Fig. 9.6
Slide 59
General structural features
Slide 60
9-60 Interactions With Bases
Slide 61
9-61 Amino Acid/DNA Backbone Interactions Hydrogen bond at Gln
33 maximizes electrostatic attraction between positively charged
amino end of a- helix and negatively charged DNA The attraction
works to stabilize the bond
Slide 62
The most important contacts occur in the major groove, where
amino acids make hydrogen bonds with DNA bases and with the DNA
backbone. Some of these hydrogen bonds are stabilized by
hydrogen-bond Networks involving two amino acids and two or more
sites on the DNA.
Slide 63
Hydrogen bonds are represented by dashed lines, the van der
Waals interaction between the Gln 29 side chain and the 5- methyl
group of the thymine paired to adenine 3 is represented by
concentric arcs
Slide 64
This implies hydrogen bonding between the protein and DNA at
these sites. This analysis also shows probable hydrogen bonding
between three glutamine residues in the recognition helix and three
base pairs in the repressor. It also reveals a potential van der
Waals contact between one of these glutamines and a base in the
operator.
Slide 65
9-65 The Role of Tryptophan The trp repressor requires
tryptophan to force the recognition helices of the repressor dimer
into proper position for interacting with the trp operator
Slide 66
DNA deviates significantly from its normal regular shape. It
bends somewhat to accommodate the necessary base/amino acid
contacts. The central part of the helix is wound extra
tightly.
Slide 67
Slide 68
Fig. 9.13
Slide 69
The trp repressor requires tryptophan to force the recognition
helices of the repressor dimer into the proper position for
interacting with the trp operator.
Slide 70
General considerations on Protein-DNA interactions Specificity
of binding between a protein and a specific stretch of DNA: 1.
Specific interactions between bases and amino acids 2. the ability
of the DNA to assume a certain shape, which also depends on the
DNAs base sequence.
Slide 71
9-71 Hydrogen Bonding Capabilities of the Four Different Base
Pairs The four different base pairs present four different
hydrogen- bonding profiles to amino acids approaching either major
or minor groove
Slide 72
9-72 The Importance of Multimeric DNA- Binding Proteins Target
sites for DNA-binding proteins are usually symmetric or repeated
Most DNA-binding proteins are dimers that greatly enhances binding
between DNA and protein as the 2 protein subunits bind
cooperatively
Slide 73
9-73 9.4 DNA-Binding Proteins: Action at a Distance There are
numerous examples in which DNA- binding proteins can influence
interactions at remote sites in DNA This phenomenon is common in
eukaryotes It can also occur in several prokaryotes
Slide 74
9-74 The gal Operon The E. coli gal operon has two distinct
operators, 97 bp apart One located adjacent to the gal promoter
External operator, O E Other is located within first structural
gene, galE 2 separated operators -both bind to repressors that
interact by looping out the intervening DNA
Slide 75
9-75 Effect of DNA Looping on DNase Susceptibility Operators
separated by Integral number of double-helical turns can loop out
DNA to allow cooperative binding Nonintegral number of turns
requires proteins to bind to opposite faces of DNA and no
cooperative binding
Slide 76
Fig. 9.17
Slide 77
9-77 Enhancers Enhancers are nonpromoter DNA elements that bind
protein factors and stimulate transcription Can act at a distance
Originally found in eukaryotes Recently found in prokaryotes
Slide 78
9-78 Prokaryotic Genes Can Use Enhancers E. coli glnA gene is
an example of a prokaryotic gene depending on an enhancer for its
transcription Enhancer binds the NtrC protein interacting wit
polymerase bound to the promoter at least 70 bp away Hydrolysis of
ATP by NtrC allows formation of an open promoter complex The two
proteins interact by looping out the DNA Phage T4 late enhancer is
mobile, part of the phage DNA-replication apparatus