Transcription

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Transcription

• General
• Prokaryotic
• Mechanism
• Apparatus
• Regulation
• Eukaryotic
• Mechanism
• Apparatus
• Regulation

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Transcription similar to replication but
different

• RNA polymerases use ribonucleotides (NTPs) and
  Uracil instead of Thymine.
• Produces single-stranded molecules that are
  complementary to one strand of the DNA template
  (or non-coding strand).
• Only part of the genome is transcribed
  functional genome or transcriptome

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Transcription Basic Enzymology

• DNA-dependent RNA Polymerases
• RNA Pols polymerize in the 5 to 3 direction
  (rNTP added only to the 3 end)
• 3 OH of chain reacts with the a phosphate of
  incoming rNTP, liberating pyrophosphate
• Added ribonucleotide follows Watson-Crick
  pairing rules, determined by template strand
• RNA polymerases dont need a primer, but do need
  DS DNA

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E. coli RNA polymerase

• - 2 copies
• b - 1 copy
• b - 1 copy
• s - 1 copy

Core
holoenzyme
There are multiple forms of s (sigma).
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Transcription in 4 Steps

• Binding
• Initiation
• Elongation
• Termination

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1. Binding

• Occurs at promoters
• (-10, -35) or s70 most common type
• Recognized by s70 subunit
• E. coli has at least 2 other promoter types
• Recognized by other s factors

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Figure 6.8
A Prokaryotic Promoter
Capital letters denote bases found in those
positions 50 of promoters examined. Small
letters
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Consensus TTGACA 17/- 1bp TATAAT
-35 -10
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-10
-35
Side view
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Looking up from below the DNA.
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The very strong rRNA promoter has an extra
upstream element, UP.
Recognized by the a subunit, strengthens binding
of Polymerase to the promoter.
Fig. 6.9
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2. Initiation

• Polymerase bound tightly to promoter, Closed
  Complex
• Polymerase unwinds DNA (-10 to 3), Open
  Complex
• Starts synthesizing RNA

Fig. 6.7
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3. Elongation

• After 10 nucleotides have been added, 5 end
  ribonucleotide unpairs from template.
• The s subunit dissociates from core.
• The size of RNA-DNA hybrid (10 bp) maintained
  during elongation.
• Sigma recycles to new polymerase molecules.

Fig. 6.14
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4. Termination

• Two types of termination events in E. coli
• Rho independent
• Rho dependent
• Rho independent also called intrinsic
• Triggered by an Inverted Repeat (IR) that occurs
  downstream of Rho independent genes
• IR followed by an A-rich region

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IR in DNA produces a stem-loop in RNA.
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????????begins to ?????
Stem-loop formation competes with the RNA-DNA
hybrid (Open Complex). Causing DNA helix to
reform (Closed complex).
????????????? and Destabilizes hybrid (RNA
Pull-out)
???????????
Fig. 6.51
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Rho-dependent termination

• Rho is a RNA helicase, and an ATPase
• Travels along RNA chasing polymerase
• When polymerase stalls at an IR (no A-T rich
  region), Rho dissociates the RNA-DNA hybrid
• DNA helix closes

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Rho in action
Rho is a hexamer helicase, like DnaB except
prefers RNA.
Rho binds transcripts at stretches of 100 nt
free of 2nd structure and rich in cytosines.
Can unwind RNA-DNA hybrids.
Fig. 6.56
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Closer look at the Prokaryotic RNA Polymerase
Sigma factors

• There are 7 sigma factors in E. coli differ in
  their affinity for the Core enzyme.
• They dont all recognize the same promoters
  either.
• Sigma factors are themselves regulated, e.g.,
• ?32 becomes activated by heat shock (42C).
• Displaces ?70 from the core.
• Directs the RNAP to heat-shock promoters.

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Conserved regions of sigma factors
2.1 - Core binding 2.3 - DNA melting 2.4 -
(-10) recognition
4.1 - Activators bind 4.2 - (-35) recognition
Polymerase core has non-specific affinity for DNA
that is reduced by ? factors, thereby promoting
specific binding.
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Figure 6.5
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Function of s

• Provides promoter specificity
• Increases affinity of RNA Pol for promoter
• Allows transcription initiation
• Stimulates transcription initiation,
• not elongation

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What are the functions of the other subunits of
the E. coli RNAP?
Identifying the active site.
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Drugs that inhibit E. coli RNAP

• Rifampicin - blocks at initiation (Open Complex
  forms), rifampicin resistance mutations mapped to
  the b subunit.
• Streptolydigin- inhibits elongation stage,
  resistance mutations also map to b subunit.
• What do initiation and elongation have in common?
  Phosphodiester bond formation. Suggests ß subunit
  is key.

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Fig. 6.33
Direct evidence for ß active site by Affinity
Labeling.
(1) Reagent in (a) resembles ATP, but it reacts
with NH2 groups when binds to active site. (2)
Then add radioactive a -32P-UTP, which is added
(i.e., polymerized) onto reagent I.
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(3) Separate enzyme subunits by
SDS-polyacrylamide gel electrophoresis, and see
which ones are radioactive. Only the b subunit
is highly radioactive. b subunit must have
active site.
Fig. 6.33
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E. coli RNA polymerase (redux)

• Functions of other subunits
• a - binds the UP element found upstream of very
  strong promoters (rRNA), and activators.
• b - active site of Pol, also binds nascent RNA,
  RNA-DNA hybrid, and DS DNA in front of the
  bubble.
• b also binds nascent RNA, RNA-DNA hybrid, and
  DS DNA in front of the bubble.

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Nudler et al., 1998, Science 281, pg. 425
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RNAP Backsliding and Editing

• If wrong nucleotide incorporated, elongation can
  become arrested.
• Backsliding now competes with elongation
• Pol backs up, extruding some of nascent RNA
• Gre proteins activate RNAP core to cleave small
  piece that has wrong nucleotide.
• Pol starts elongating again.

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RNAP core
Square is the next NTP to be added. Green
nascent RNA that will be cleaved off Red
older RNA