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MOLECULAR BIOLOGY Ch 6. Prokaryotic Transcription: Initiation, Elongation & Termination. SDS PAGE. RNA Polymerase Holoenzyme. holoenzyme. sigma. core. -. '. . . +. Viral Transcription: immediate early genes, delayed early genes, late genes Sigma Factor : - PowerPoint PPT Presentation
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Prokaryotic Transcription:
Initiation, Elongation & Termination
MOLECULAR BIOLOGY
Ch 6
holoenzymecore
sigma
+
-
'
SDS PAGE
RNA PolymeraseHoloenzyme
'
α
α
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
Hybridization-Competition Experiment
Sigma stimulates tight binding between RNA polymerase and promoter:
Experiment:
- labeled DNA + core or holo. - added unlabeled DNA - filtered the mixture
Transcriptional INITIATION
ElectronMicroscopy
on2-D
Crystals
Sigma aids in DNA melting in promoter region
- creates tighter binding of RNA Pol to promoter region
Tight binding of RNA Pol to promoter is Temp dependent
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 bondformation)
-35TTGACa
-10TAtAaT
- up mutations: strongest - down mutations: weaker- in/dels between boxes: deleterious
Consensus Sequence of Promoter Regions
α subunit: - recognizes UP element - C-term. end binds to UP element
UP Element in strongest promoters
- seen upstream to E. coli’s 7 rRNA genes - stimulates transcription 30X
- binding sites for activators (TAP-Fis)- between -60 & -150
Fis sites act as enhancers
4 Homologous Regions of Sigma Factor
Region 1: 70 & 43
between 1&2: 245 a.a. deletion in 43
- cannot loosen binding between nonpromoter region and RNA Pol (needs factor)
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)
helix-turn-helix motif
ß subunit: - phosphodiester bond formation - determinant of rifampicin &
streptolydigin - sensitivity or resistance
- weak bonding at melted DNA zone (active site) and downstream binding
Transcriptional ELONGATION
RNA Polymerase
(streptolydigin: stops elongation of transcription)
RNA Polymeraseß’ subunit:
-most + charged of all subunits
-zinc finger motif
-strong binding downstream of active site
Transcriptional ELONGATIONZinc Fingers
Zinc Fingers within Major Groove of DNA
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)
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 A3’ 5’
RNA made
5’-AUACUUGACGUACAAGUAU-3’
10 structure 20 structure
DNA sequences (palindrome) signal termination
TERMINATION of Prok. Transcription
A=UU=AA=UC=GU=AU=AG=C
AC G U
A
5’ 3’
1) Rho- (Rho independent):- template strand: 16-20 bases upstream of term. pt has an inverted repeat followed by poly A’s
Types of Prok. Transcription Termination Mechanisms
2) Rho+ (Rho dependent):- template strand with inverted repeat but no poly A’s following- Rho protein decreases net rate of transcription
Types of Termination Mechanisms
- Rho binds to RNA releases transcript from DNA template
- Rho has no effect on initiation
Rho-Dependent
Termination
of Prok.Transcription