Chapter 8 Major Shifts in Prokaryotic Transcription

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Chapter 8Major Shifts inProkaryotic
Transcription
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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 s factors that associated with the
  host core RNA polymerase and change its
  specificity from early to middle to late.

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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.

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Fig. 8.1
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• 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 3HUTP 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

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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

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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 news factors that
  displace the vegetatives factor from the core RNA
  polymerase.

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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.

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The DNA region contains two promoters a
vegetative and a sporulation
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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
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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.
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Genes with Multiple Promoters

• Some prokaryotic genes must be transcribed under
  conditions where two differents 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.

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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.
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Fig. 8.7
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Purified sigma factors B and E by gel
electrophoresis and tested them with core
polymerase by the same run-off transcription
assay.
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Fig. 8.8
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Fig. 8.9
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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).

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Infection of E. coli by Phage ?

• Phage ? can replicate in either of two ways
  lytic and lysogenic.

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A bacterium harboring the integrated phage DNA is
called a lysogen The integrated DNA is called a
prophage
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Cro gene product blocks the transcription of ?
repressor CI N antiterminator
Extension of transcripts controlled by the same
promoters. Q antiterminator
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Lytic reproduction of Phage ?

• The immediate early/delayed early/late
  transcriptional switching in the lytic cycle of
  phage ? is controlled by antiterminators.

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N utilization site
NusA
N function by restricting the pause time at the
terminator
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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.

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Highly conserved among Nut sites
Help to stabilize the antitermination complex
contains an inverted repeat
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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

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Highly conserved among Nut sites
Help to stabilize the antitermination complex
contains an inverted repeat
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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.
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Fig. 8.15
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Fig. 8.17
Qut Q utilization site Q binds directly to qut
site not to the transcript
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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.

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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
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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.

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Fig. 8.19
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Run-off transcription (this construct does not
contain OL, therefore, need to use very high
concentration of repressor)
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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.
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RNA polymerase-repressor Interaction

• Intergenic suppressor mutation studies show that
  the crucial interaction between repressor and RNA
  polymerase involves region 4 of the s subunit of
  the polymerase.

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Fig. 8.23
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Fig. 8.24
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Fig. 8.25
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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.

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Fig. 8.26
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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.

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Fig. 8.27
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Chapter 9DNA Protein Interactions in
Prokaryotes
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Helix 2 of the motif (red) lies in the major
groove of its DNA target
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The l 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

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Binding Specificity of Repressor-DNA Interaction
Site

• Repressors of l-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

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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

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The helix-turn-helix motif of the upper monomer
(red and blue) is inserted into the major groove
of the DNA)
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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.
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High-Resolution Analysis of l 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

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Fig. 9.6
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General structural features
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Interactions With Bases
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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

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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.
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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
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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.
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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

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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.
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Fig. 9.13
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The trp repressor requires tryptophan to force
the recognition helices of the repressor dimer
into the proper position for interacting with the
trp operator.
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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.

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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

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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

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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

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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

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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

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Fig. 9.17
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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

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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

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Fig. 9.18
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Fig. 9.19
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Fig. 9.20