Chapter 6 The Transcription Apparatus of Prokaryotes

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Chapter 6The Transcription Apparatus of
Prokaryotes
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RNA Polymerase Structure

• The subunit content of an RNA polymerase
  holoenzyme is ?, ?, 2?, ? and ?.
• ? 160 kD ? 150 kD ? 40 kD ?70 70 kD ?
  10 kD
• 3 regions of conservation -35, -10 and the
  length of spacer 17 bp ? 1 bp

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Fig. 6.1
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Sigma as a Specificity Factor

• The E. coli enzyme is composed of a core, which
  contains the basic transcription machinery, and a
  s factor, which directs the core to transcribe
  specific genes.

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Promoters

• The polymerase binding sites, including the
  transcription initiation sites, are called
  promoters.

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Core polymerase
RNase-resistance
holoenzyme
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Binding of RNA polymerase to Promoters

• 3H-labeled T7 DNA to bind to E. coli core
  polymerase (blue) or holoenzyme (red).
• Next they added an excess of unlabeled T7 DNA so
  that any polymerase that dissociated from the
  labeled DNA would be likely to re-bind to
  unlabeled DNA
• Filter the mixtures through NC at various times
  to monitor the dissociation.

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T ½ 30- 60 hrs
T ½ less than 1 min
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• High temperature promotes DNA melting (strand
  separation), this finding is consistent with the
  notion that tight binding involves local melting
  of the DNA.

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More stable
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Polymerase/Promoter Binding

• Holoenzyme binds DNA loosely at first
• Complex loosely bound at promoter closed
  promoter complex, dsDNA in closed form
• Holoenzyme melts DNA at promoter forming open
  promoter complex - polymerase tightly bound

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Summary

• The sigma-factor allows initiation of
  transcription by causing the RNA polymerase
  holoenzyme to bind tightly to a promoter.
• This tight binding depends on local melting of
  the DNA to form an open complex and is only in
  the presence of sigma.

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Promoter Structure

• Prokaryotic promoters contain two regions
  centered at 10 and 35 base pairs upstream of
  the transcription start site. In general, the
  more closely regions within a promoter resemble
  these consensus sequences, the stronger that
  promoter will be.

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enhancer
30 X increase of activation
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Transcription Initiation

• Carpousis allowed E. coli RNA polymerase to
  synthesize 32P-labeled RNA in vitro using a DNA
  containing lac UV5 promoter, heparin to bind any
  free RNA polymerase
• Heparin negatively charged polysaccharide that
  competes with DNA in binding tightly to free RNA
  polymerase
• Abortive transcripts would be up to 9-10 nt in
  size.

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Lane 1 no DNA lane 2, ATP only lane 3-7 ATP
with concentrations of CTP, GTP, and UTP
increasing by two-fold in each lane.
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• Because the heparin in the assay prevented free
  polymerase from re-associating with the DNA, this
  result implied that the polymerase was making
  many small, abortive transcripts without ever
  leaving the promoter.
• The abortive transcripts up to 9 to 10 nt in size.

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Fig. 6.9
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The Functions of sigma

• ? stimulates initiation, but not elongation, of
  transcription.
• ? can be re-used by different core polymerases,
  and the core, not ?, governs rifampicin
  sensitivity or resistance.
• Rifampicin blocks prokaryotic transcription
  initiation but not elongation.

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The incorporation of the 14CATP measured bulk
RNA synthesis the incorporation of the ?-32P
nucleotide measured initiation
Even though sigma seems to stimulate both
initiation and elongation, it was due to an
indirect effect of enhanced initiation
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Further experiment

• ? stimulates initiation, but not elongation, of
  transcription was further demonstrated by the use
  of
• Rifampicin( blocks prokaryotic transcription
  initiation but not elongation). They held the
  number of RNA chains constant and then use
  ultracentrifugation to measure the length of the
  RNA in the presence and absence of sigma.

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• Experiment demonstrate that sigma can be
  recycled.
• The key was to run the transcription reaction at
  low ionic strength, which prevent RNA polymerase
  core from dissociating from the DNA template at
  the end of a gene.

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The number of RNA chain Constant by allowing a
certain amount of initiation to occur and then
blocking any further initiation by rifampicin
then add rifampicin-resistant core polymerase
- rifampicin
rifampicin
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Reuse of s

• During initiation s can be recycled for
  additional use in a process called the s cycle
• Core enzyme can release s which then associates
  with another core enzyme

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Sigma may not associate from core During
Elongation

• Fluorescence resonance energy transfer (FRET)
  two fluorescent molecules close to each other
  will engage in transfer of resonance energy, and
  the efficiency of this energy transfer will
  decrease rapidly as the two molecules move apart.

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Summary

• The sigma factor changes its relationship to the
  core polymerase during elongation, but it may not
  dissociate from the core. Instead it may just
  shift position and become more loosely bound to
  the core.

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Local DNA melting at the promoter

• When A is base-paired with T, the N1 nitrogen of
  A is hidden in the middle of the double helix and
  is protected from methylation
• S1 nuclease can cut the DNA at each of the
  unformed base pairs because these are local
  single-stranded regions.

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Lane RS shows the results when both RNA
polymerase ( R) and S1 nuclease (S) were used.
On binding to a promoter, RNA polymerase causes
the melting of at least 10 bp,
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Structure of Sigma

• Sigma 70 family There are four conserved regions
  in sigma 70 family proteins.
• The best evidence for the functions of these
  regions shows that sub-regions 2.4 and 4.2 are
  involved in promoter 10 box and 35 box
  recognition.

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• Region 1 found only in the primary sigmas (
  sigma 70 and 43)
• Region 2 most highly conserved sigma region,
  2.4 -10 box binding
• Region 3 helix-turn-helix DNA binding domain
• Region 4 4.2 -35 box binding

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Fig. 6.20
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Fig. 6.21
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?P lacking the tac promoter
? In this experiment contains only the region 4,
not region 2.
Because pTac DNA competes much better than ?P
DNA, they concluded that the fusion protein
with region 4 can bind to the tac promoter.
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The role of the ?-subunit in UP element
recognition

• The RNA polymerase ?-subunit has an independently
  folded C-terminal domain that can recognize and
  bind to a promoters UP element. This allows very
  tight binding between polymerase and promoter.
• a subunit response to activator, repressor,
  elongation factor and transcription factors

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?-235 polymerase missing 94 C-terminal amino
acid of the ? subunit
-88 wild type promoter SUB irrelevant sequence
instead -41 deletion UP
In vitro transcription. What is the conclusion
you get from this experiment?
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The bold brackets indicate the footprints in the
UP element caused by the ?-subunit, and the thin
bracket indicates the footprint caused by the
holoenzyme.
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Elongation
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Core Polymerase Functions in Elongation

• The role of ß in phosphodiester bond formation
  The core subunitß binds nucleotides at the active
  site of the RNA polymerase where phosphodiester
  bonds are formed. Rifampicin can block initiation
  by preventing the formation of that first bond.
• The core subunit ßcan bind weakly to DNA by
  itself in vitro. In fact, both ß andßbind to DNA
  as indicated by different experiments.

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The affinity-labeling reactions First, add
reagent I to RNA polymerase. The reagent binds
covalently to amino groups at the active site.
Next, add radioactive UTP, which forms a
phosphodiester bond (blue) with the enzyme-bound
reagent I. This reaction should occur only at the
active site, so only that site becomes
radioactively labeled.
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Labeled the active site as mentioned above, then
separate the polymerase subunits to identify the
subunits that compose the active site
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Hydrophobic interaction ? and ?
Electrostatic interaction , ?
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Termination of Transcription

• Rho-independent Termination inverted repeats and
  Hairpins, a string of Ts in the nontemplate strand

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rU-dA have a melting temperature 20 degree lower
than rU-rA or rA-dT pairs
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An assay for attenuation

• If attenuation works, and transcription
  terminates at the attenuator, a short 140-nt
  transcript should be the result.
• When change the string of eight Ts in the
  nontemplate strand, creating a trp a1419 mutant,
  attenuation was weakened.
• This result is consistent with the weak rU-dA
  pairs are important in termination.

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TTTTGAA trp a1419, attenuation weakened IMP
inosine monophosphate, a GMP analogue, weaken
base-pairing in the hairpin, IC weaker than GC
pair
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The essence of a bacterial terminator is twofold

• 1. Base-pairing of something to the transcript to
  destabilize the RNA-DNA hybrid
• 2. Something that causes transcription to pause
• A normal intrinsic terminator satisfies the first
  condition by causing a hairpin to form in the
  transcript, and the second by causing a string of
  Us to be incorporated just downstream of the
  hairpin.

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• Rho-dependent Termination consist of an inverted
  repeat, which can cause a hairpin to form in the
  transcript, but no string of Ts.
• Rho affects chain elongation, but not initiation.
  
• Rho causes production of shorter transcripts.
• Rho is an RNA helicase, composed of 6 identical
  subunits, each subunit has an RNA binding domain
  and ATPase domain
• Rho releases the RNA product from the DNA
  template.

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Chapter 7Operons Fine Control ofProkaryotic
Transcription
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The lac Operon

• Lactose metabolism in E.coli is carried out by
  two enzymes, with possible involement by a third.
  The genes for all three enzymes are clustered
  together and transcribed together from one
  promoter, yielding a polycistronic message.

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• The lac Operon It contains three structural
  genes genes that code for proteins
  ?-galactosidase (lacZ), galactoside permease
  (lacY), and galactoside transacetylase (lacA).
• They all are transcribed together on one messager
  RNA, called a polycistronic message, starting
  from a single promoter.

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• Negative Control of the lac Operon
• Repressor-operator Interactions
• Lac repressor binds to lac operator was
  demonstrated by filter-binding assay.

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The repressor is an allosteric protein one in
which the binding of one molecular to the protein
changes the shape of a remote site on the protein
and alter its interaction with a second molecule.
Inducer 1st molecule operator 2nd molecule
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Constitutive mutants had a defect in the gene
(lacI)
Constitutive mutant
Because it is dominant only with respect to genes
on the same DNA
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Because the mutant repressor will bind to
operators even in the presence of inducer or of
WT repressor
Constitutive and dominant
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The mechanism of Repression

• RNA polymerase can bind to the lac promoter in
  the presence of the repressor. The function of
  the repressor appears to inhibit the transition
  from the non-productive synthesis of the abortive
  transcripts to real, processive transcription.

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Assaying the binding between lac operator and lac
repressor

• Cohen and colleagues labeled lacO-containing DNA
  with 32P and added increasing amounts of lac
  repressor
• They assayed binding between repressor and
  operators by measuring the radioactivity attached
  to NC.
• Only labeled DNA bound to repressor would attach
  to NC.
• IPTG prevents repressor-operator binding.

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Mutant O with low affinity
Wild type operator
Nonsense DNA
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1. Incubation of a DNA fragment containing the lac
   promoter with (lanes 2 and 3) or without (lane 1)
   lac repressor (LacR).
2. After repressor-operator binding had occurred,
   they added RNA polymerase. After 20 minutes for
   OC to form, they added heparin and all components
   except CTP.

3. Finally, after 5 more minutes, they ?-32P CTP
alone or with the inducer IPTG then wait for 10
minutes for RNA synthesis. The result showed that
transcription occurred even when repressor bound
to the DNA before polymerase could, repressor did
not prevent polymerase from binding and forming
an open promoter complexes. ( but the condition
is nonphysiological conditions, too much proteins)
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Effect of lac repressor on dissociation of RNA
polymerase from the lac promoter

• Record made complexes between RNA polymerase and
  DNA containing the lac promoter-operator region
• They allowed the complexes to synthesize abortive
  transcripts in the presence of a UTP analog
  fluorescently labeled.
• As the polymerase incorporates UMP from this
  analog into transcripts, the labeled
  pyrophosphate released increases in fluorescence
  intensity.

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( condition likely in vivo)
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• The latest evidence supports the repressor, by
  binding to the operator, blocks access by the
  polymerase to the adjacent promoter.

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Effects of mutations in the three lac operators

• WT or mutant lac operon on ? phage
• Infect and lysogenize E. coli
• Assay for ?-galactosidase in the presence or
  absence of IPTG

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IPTG/-IPTG
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Positive Control of the lac Operon

• It is mediated by a factor called catabolite
  activator protein (CAP) in conjunction with
  cyclic AMP, to stimulate transcription.
• Sensed the lack of glucose, increase of cAMP.
• CAP is dimeric and binds to 22 bp operator
  sequences, accelerates the initiation of
  transcription at these promoters.

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Once the first phosphodiester bond forms, the
polymerase is resistant to rifampicin inhibition
until it re-initiates.
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CAP binding sites in the lac, gal and ara operons
all contain the sequence TGTGA Lac operon has
remarkably weak promoter , -35 box
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Mechanism of CAP Action

• The CAP-cAMP complex stimulates transcription of
  the lac operon by binding to an activator site
  adjacent to the promoter and helping RNA
  polymerase to bind to the promoter. This closed
  complex then converts to an open promoter
  complex. CAP-cAMP causes recruitment through
  protein-protein interactions, by bending the DNA,
  or by a combination of these phenomena.

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Binding of CAP-cAMP to the activator site does
cause the DNA to bend

• When a piece of DNA is bent, it migrates more
  slowly during electrophoresis.
• The closer the bend is to the middle of the DNA,
  the more slowly the DNA electrophoreses.
• Actual electrophoresis results with CAP-cAMP and
  DNA fragments containing the lac promoter at
  various points in the fragment, dependent on
  which restriction enzyme was used to cut the DNA.

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Fig. 7.19
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Fig. 7.20
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Tryptophans Role in Negative Control of the trp
Operon

• The trp Operon contains the genes for the enzymes
  that E. coli needs to make the amino acid
  tryptophan.
• The trp operon responds to a repressor that
  includes a corepressor, tryptophan, which signals
  the cell that it has made enough of this amino
  acid. The corepressor binds to the aporepressor,
  changing its conformation so it can bind to the
  trp operator, thereby repressing the operon.

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Fig. 7.28
5 structural genes
High conc. of tryptophan is a signal to turn off
the operon
Trp repressor
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Control of the trp Operon by Attenuation

• Because of the weak repression of the trp operon,
  another extra control called attenuation exists.
• Attenuation imposes an extra level of control on
  an operon, over and above the repressor-operator
  system. It operates by causing premature
  termination of transcription of the operon when
  the operons products are abundant.

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Figure 7.30 Two Structures available to the
leader-attenuator transcript.
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Riboswitches

• Is a region in the 5-UTR of an mRNA that
  contains two modules an aptamer that can bind a
  ligand, and an expression plateform whose change
  in conformation can cause a change in expression
  of the gene.
• FMN can bind to an aptamer in a riboswitch called
  the RFN element in the 5-UTR of the ribD mRNA.
• Upon binding FMN, the base pairing in the
  riboswitch changes to create a terminator that
  attenuates transcription.

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Fig. 7.34
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Fig. 7.35