Download RNA polymerase changes specificity

Chapter 8
Major Shifts in
Prokaryotic
Transcription
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 σ factors that
associated with the host core RNA
polymerase and change its specificity from
early to middle to late.
RNA polymerase changes specificity
• gp28: (1) diverts the host’s 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.
Fig. 8.1
• 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 [3H]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:
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
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 newσ factors
that displace the vegetativeσ factor from the
core RNA polymerase.
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.
The DNA region contains two promoters: a vegetative and a
sporulation
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
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.
Genes with Multiple Promoters
• Some prokaryotic genes must be transcribed
under conditions where two differentσ
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.
Spo VG: transcribed
by EB and E E.
The last purification
step was DNAcellulose 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.
Fig. 8.7
Purified sigma
factors B and E
by gel
electrophoresis
and tested them
with core
polymerase by
the same run-off
transcription
assay.
Fig. 8.8
Fig. 8.9
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 oC).
• This shift of transcription required -32 (H).
Infection of E. coli by Phage 
• Phage  can replicate in either of two ways:
lytic and lysogenic.
A bacterium harboring the
integrated phage DNA is
called a lysogen
The integrated DNA is
called a prophage
Cro gene product blocks the
transcription of  repressor CI
N: antiterminator
Extension of transcripts
controlled by the same
promoters. Q: antiterminator
Lytic reproduction of Phage 
• The immediate early/delayed early/late
transcriptional switching in the lytic cycle
of phage  is controlled by antiterminators.
N utilization site
NusA
N: function by
restricting the
pause time at the
terminator
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.
Highly conserved among Nut sites
Help to stabilize the
antitermination complex
contains an inverted repeat
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
Highly conserved among Nut sites
Help to stabilize the
antitermination complex
contains an inverted repeat
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.
Fig. 8.15
Fig. 8.17
Qut: Q utilization site
Q binds directly to qut site not to the transcript
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 PRE.
Fig. 8.18
Delayed early transcription from PR
gives cII mRNA that is transcribed
to CII (purple), which allows RNA
polymerase (blue and red) to bind
to PRE and transcribe the cI gene
Autoregulation of cI Gene During
Lysogeny
• The promoter that is used to maintain
lysogeny is PRM.
• It comes into play after transcription from
PRE makes possible that burst of repressor
synthesis that establishes lysogeny.
• This repressor binds to OR1 and OR2
cooperatively, but leave OR3 open. RNA
polymerase binds to PRM,, in a way that
contacts the repressor bound to OR2.
Fig. 8.19
Run-off transcription (this construct does not contain OL,
therefore, need to use very high concentration of repressor)
High levels of repressor can repress transcription from PRM, may involve interaction
of repressor dimers bound to OR1, OR2 and OR3, with repressor dimers bound to
OL1, OL2 and OL3 via DNA looping.
RNA polymerase-repressor
Interaction
• Intergenic suppressor mutation studies show
that the crucial interaction between
repressor and RNA polymerase involves
region 4 of the σ subunit of the polymerase.
Fig. 8.23
Fig. 8.24
Fig. 8.25
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.
Fig. 8.26
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.
Fig. 8.27
Chapter 9
DNA – Protein
Interactions in
Prokaryotes
Helix 2 of the motif (red) lies in the major groove of its DNA target
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
9-51
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 proteinDNA interactions
• Changing these amino acids can change specificity of
the repressor
9-52
Probing Binding Specificity by SiteDirected Mutagenesis
• Key amino acids in
recognition helices of 2
repressors are proposed
• These amino acids are
largely different
between the two
repressors
9-53
The helix-turn-helix
motif of the upper
monomer (red and
blue) is inserted into
the major groove of
the DNA)
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.
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 halfsites
– 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
9-57
Fig. 9.6
General structural features
Interactions With Bases
9-60
Amino Acid/DNA Backbone Interactions
• Hydrogen bond at Gln
33 maximizes
electrostatic attraction
between positively
charged amino end of ahelix and negatively
charged DNA
• The attraction works to
stabilize the bond
9-61
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.
Hydrogen bonds
are represented by
dashed lines, the
van der Waals
interaction between
the Gln 29 side
chain and the 5methyl group of the
thymine paired to
adenine 3 is
represented by
concentric arcs
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.
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
9-65
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.
Fig. 9.13
The trp repressor requires tryptophan to force the
recognition helices of the repressor dimer into the
proper position for interacting with the trp operator.
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 DNA’s base
sequence.
Hydrogen Bonding Capabilities of the
Four Different Base Pairs
• The four different base
pairs present four
different hydrogenbonding profiles to
amino acids
approaching either
major or minor groove
9-71
The Importance of Multimeric DNABinding 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
9-72
9.4 DNA-Binding Proteins: Action at a
Distance
• There are numerous examples in which DNAbinding proteins can influence interactions at
remote sites in DNA
• This phenomenon is common in eukaryotes
• It can also occur in several prokaryotes
9-73
The gal Operon
• The E. coli gal operon has
two distinct operators, 97 bp
apart
– One located adjacent to the
gal promoter
• External operator, OE
– Other is located within first
structural gene, galE
• 2 separated operators -both
bind to repressors that
interact by looping out the
intervening DNA
9-74
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
9-75
Fig. 9.17
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
9-77
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
9-78
Fig. 9.18
Fig. 9.19
Fig. 9.20