Prokaryotic Transcription:

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MOLECULAR BIOLOGY Ch 6

• Prokaryotic Transcription
• Initiation,
• Elongation Termination

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SDS PAGE
RNA Polymerase Holoenzyme
holoenzyme
sigma
core
-
?'
?
?

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Viral Transcription immediate early genes,
delayed early genes, late genes Sigma Factor -
directs the core to transcribe specific
genes hybridization-competition experiment -
holoenzyme specific for immed. early genes -
core enzyme transcripts compete w/ all genes
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Hybridization-Competition Experiment
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Transcriptional INITIATION
Sigma stimulates tight binding between RNA
polymerase and promoter Experiment
- labeled DNA core or holo. - added
unlabeled DNA - filtered the mixture
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Electron Microscopy on 2-D Crystals
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Sigma aids in DNA melting in promoter region
- creates tighter binding of RNA Pol to promoter
region
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Tight binding of RNA Pol to promoter is Temp
dependent
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Sigma factor can be recycled (reused)

• exper
• holoenzyme DNA
• wait 10 min
• (initiation ceased)
• add rifampicin-resistant
• core enzyme rifampicin

(rifampicin normally prevents first
phosphodiester bond formation)
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Consensus Sequence of Promoter Regions
-35 TTGACa
-10 TAtAaT
- up mutations strongest - down mutations
weaker - in/dels between boxes deleterious
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UP Element in strongest promoters - seen
upstream to E. colis 7 rRNA genes -
stimulates transcription 30X
? subunit - recognizes UP element - C-term.
end binds to UP element
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Fis sites act as enhancers
- binding sites for activators (TAP-Fis) -
between -60 -150
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4 Homologous Regions of Sigma Factor
Region 1 ?70 ?43 between 12 245 a.a.
deletion in ?43 - cannot loosen binding between
nonpromoter region and RNA Pol (needs ?
factor)
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Region 2 highest homology (2.1) hydrophobic,
binds to core (2.4) binds to -10 box (has a
helix) Region 3 ? (helix - turn - helix)
Region 4 (4.2) binds to -35 box
(helix-turn-helix)
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helix-turn-helix motif
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Transcriptional ELONGATION
RNA Polymerase
ß subunit - phosphodiester bond formation -
determinant of rifampicin streptolydigin -
sensitivity or resistance - weak bonding at
melted DNA zone (active site) and
downstream binding
(streptolydigin stops elongation of
transcription)
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Transcriptional ELONGATION
Zinc Fingers

• RNA Polymerase
• ß subunit
• most charged of all subunits
• zinc finger motif
• strong binding downstream
• of active site

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Zinc Fingers within Major Groove of DNA
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Models of Transcription Elongation
- more than likely RNA Pol moves in straight
line w/ topisomerases relieving supercoils -
RNA/DNA hybrid forms for 20 bases - 40 nt/sec
(prok)
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TERMINATION of Prok. Transcription
DNA sequences (palindrome) signal termination
5 3 A T A C T T G A C G T A C A A G
T A T T A T G A A C T G C A T G T T C A T
A 3 5
RNA made
10 structure
20 structure
5-AUACUUGACGUACAAGUAU-3
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Types of Prok. Transcription Termination
Mechanisms
1) Rho- (Rho independent) - template strand
16-20 bases upstream of term. pt has an
inverted repeat followed by poly As
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Types of Termination Mechanisms
2) Rho (Rho dependent) - template strand with
inverted repeat but no poly As following -
Rho protein decreases net rate of transcription
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Rho-Dependent Termination of Prok.Transcription

• Rho binds to RNA
• releases transcript
• from DNA template
• Rho has no effect on
• initiation