The mechanism of Transcription in Prokaryotes

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Chapter 6

• The mechanism of Transcription in Prokaryotes

Reporter Xulirong
200722040153
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Outline

• RNA Polymerase Structure
• Promoters
• Transcription Initiation
• Elongation
• Termination of Transcription

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1.RNA Polymerase Structure
As early as 1960-1961, RNA polymerases were
discovered in animals, plants and bacteria.
And the bacteria enzyme was the
first to be studied in great detail.
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1.RNA Polymerase Structure
SDS-PAGE on the E.coli RNA polymerase ß 150kD ß
160kD s 70kD a 40kD
Fig 6.1 Separation of the subunits of E.coli RNA
polymerase
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1.RNA Polymerase Structure
Structure Of E.coli RNA Polymerase
Structure
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• Holoenzyme ß,ß,s,a2
• Core enzyme ß,ß,a2

1.RNA Polymerase Structure
Table 6.1 Ability of core enzyme and holoenzyme
to transcribe DNAs
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Compare the two enzymes

• s restored specificity to the nonspecific core
• How to verify that?
• Hybridization competition (Bautz and coworkers)

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Hybridization competition(2 steps)
RNase
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Fig 6.3 s confers specificity for the T4
immediate early genes
1.RNA Polymerase Structure
Green immediate early Red delayed
early Blue late
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Table 6.2 Viral transcription phases
1.RNA Polymerase Structure
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1.RNA Polymerase Structure

• The holoenzyme is highly specific for the
  immediate early genes, while the core enzyme has
  no specificity.
• The core enzyme transcribes both DNA strands.
  
• How to prove?

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• (1)hybridizing the labeled product of the
  holoenzyme or the core enzyme to authentic T4
  phage RNA
• (2)checked for RNase resistance.

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Fig 6.4 Testing for asymmetric or symmetric
transcription in vitro
1.RNA Polymerase Structure
If the RNA is made asymmetrically, it will be not
able to hybridize to the in vivo RNA and become
sensitive to RNase.
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• s direct the core to transcribe specific genes.
• In fact ,it was named for this character, and in
  Greek letters it stands for specificity.

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2. Promoters
Fig 6.8 a prokaryotic promoter

• Core promoter elements-10 box and -35 box
• The spacing between the promoter elements is also
  important.
• Mutations that destroy matches with the consensus
  sequence tend to be down mutations more like up
  mutations.

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Some very strong promoters have an
additional element further upstream called an UP
element. eg. rrn genesFis sites enhancers
2. Promoters
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Fig 6.9 The rrnB P1 promoter
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Binding of RNA Polymerase to Promoters
2. Promoters
Holoenzyme binds much more tightly to the T7 DNA
than does the core enzyme.
Fig 6.5 s stimulates tight binding between RNA
polymerase and promoter
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2. Promoters
In a separate experiment, they switched the
procedure around, and revealed that holoenzyme
also had loose binding sites on the DNA.
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2. Promoters

• Holoenzyme finds two kinds of binding sites on
  DNA tight binding sites (promoter) and loose
  binding sites (everywhere)

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Fig 6.6 The effect of temperature on the
dissociation of the polymerase-T7 DNA complex
A striking enhancement of tight binding at
elevated temperature was found.
2. Promoters
Because high temperature promotes DNA melting.
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3. Transcription Initiation
Until 1980, It was thought that transcription
initiation ended when RNA polymerase formed the
first phosphodiester bond. Wrong!
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3. Transcription Initiation
Carpousis and Gralla found that the RNA
polymerase was making many small, abortive
transcripts without ever leaving the promoter .
Heparin(???) a negatively charged
polysaccharide that competes with DNA in binding
tightly to free RNA polymerase .
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(No Transcript)
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The Function of s
3. Transcription Initiation

• s selects genes for transcription by causing
  tight binding between RNA polymerase and
  promoters.
• s can dissociate from core after sponsoring
  polymerase-promoter binding, which indicates that
  it can be reused again and again.

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3. Transcription Initiation
Fig 6.14 The s cycle
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s may not dissociate from core during elongation

• Richard noted in2001
• (1) the evidence favoring the scycle model relies
  on harsh separation techniques, such as
  electrophoresis or chromatography.

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s may not dissociate from core during elongation

• (2)previous work failed to distinguish between
  active and inactive RNA polymerases.

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s may not dissociate from core during elongation

• How to test the hypothesis?
• FRET technique
• (fluorescence resonance energy transfer)

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FRET techniques rationale

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

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Fluorescence donor Green Accepter red FRET
efficiency purple line FRET decrease s
dissociate Otherwise it does not
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• FRET analysis of score association after
  promoter clearance

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FRET analysis

• There could be a s cycle, but the s would
  cycle between being tightly bound to core in the
  initiating state and loosely bound in the
  elongating state.

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3. Transcription Initiation
Structure and function of s

• Each bacterium has a primary s-factor that
  transcribes its vegetative genes-those required
  for everyday growth.
• 4 Regions of s

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3. Transcription Initiation
Fig 6.21 Homologous regions in various E.coli and
B.subtilis s factors
The conservation of sequence in these regions
suggests that they are important in the function
of s.
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Region 1
3. Transcription Initiation
Its role appears to be to prevent s from
binding by itself to DNA. This is important
because s binding to promoters could inhibit
holoenzyme binding and thereby inhibit
transcription.
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Region 2
3. Transcription Initiation

• Most highly conserved
• It can be divided into 4 parts 2.1-2.4
• 2.1 necessary and sufficient for core binding
• 2.4 recognize the -10 box

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3. Transcription Initiation
Region 3
Involved in both core and DNA binding
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Region 4
3. Transcription Initiation

• It plays a key role in promoter recognition.
• 4.2 govern binding to the -35 box.

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The Role of a-Subunit in UP Element Recognition
3. Transcription Initiation
It is demonstrated by foot printing assay.
C-terminal domain is required for UP binding
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4. Elongation
After initiation, the core continues to
elongate the RNA, ß- and ß-subunits are involved
in phosphodiester bond formation.
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Core Polymerase Function in Elongation
4. Elongation
s can determine the specificity of
initiation, however the core polymerase contains
the RNA synthesizing machinery, so the core is
the central player in elongation.
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Evidence for the role of ß
(1) add Reagent I to RNA polymerase (bind at the
active site) (2)a labeled nucleotide UTP
that would form a phosphodiester bond with the
complex
4. Elongation
Affinity labeling RNA polymerase at active site
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• Reagent I an ATP analog, when added to
  polymerase, it went to the active site as an ATP
  would normally do.
• Pitfall affinity reagent might bind to other
  amino groups on the enzyme surface.

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Evidence for the role of ß
4. Elongation
Then they dissociated the labeled enzyme
and subjected the subunits to SDS-PAGE. (Fig 6.33
at P157)
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Evidence for the role of ß

• The result shows that the ß-subunit is the
  only core subunit labeled, suggesting that it is
  at or very near the site where phosphodiester
  bond formation occurs.

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The roles of ß and ß in DNA binding
4. Elongation
The interaction between the RNA transcript and
the DNA is not the most important contributor to
the stability of the ternary DNA-RNA-protein
complex (salt-sensitive, weak). Indeed, the
strong binding site is a vital part of a sliding
clamp that holds the polymerase tightly on the
DNA.
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Structure of the Elongation Complex
4. Elongation
Vitro studies have suggested that processivity of
transcription depends on an RNA-DNA hybrid at
least 9 bp long.
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A model for the participation of sregion 1.1
informing the open promoter complex
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The switch from closed to open promoter complex
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5.Termination of Transcription

• When the RNA polymerase reaches a terminator at
  the end of a gene it falls off the template,
  releasing the RNA.
• 2 kinds of terminators
• Intrinsic terminators
• ?-dependent terminators

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5.Termination of Transcription

• Each terminators contain two common elements
• (1)an inverted repeat
• (2)a T-rich region in the non template strand
  followed the inverted repeat .

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(a) The polymerase paused at a sting of rU-dA bp
(c) The RNA and pol dissociate completely from
the DNA
A model for intrinsic termination
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?-Dependent Termination
5. Termination of Transcription

• ? was firstly discovered as a protein that cause
  an depression of transcription. The depression is
  the result of termination.
• ?, Hexamer, each has ATPase activity and RNA-DNA
  helicase activity

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A model of ?-dependent termination

• ? binds to the ? loading site (upstream of the
  termination site on the transcript, 60-100 nt
  long)

5. Termination of Transcription
!
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• Thank you!