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Prokaryotic Transcription: Initiation, Elongation & Termination MOLECULAR BIOLOGY Ch 6.

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Presentation on theme: "Prokaryotic Transcription: Initiation, Elongation & Termination MOLECULAR BIOLOGY Ch 6."— Presentation transcript:

1 Prokaryotic Transcription: Initiation, Elongation & Termination MOLECULAR BIOLOGY Ch 6


3 holoenzyme core sigma + - ''   SDS PAGE RNA Polymerase Holoenzyme

4 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

5 Hybridization-Competition Experiment

6 Sigma stimulates tight binding between RNA polymerase and promoter: Experiment: - labeled DNA + core or holo. - added unlabeled DNA - filtered the mixture Transcriptional INITIATION


8 Electron Microscopy on 2-D Crystals

9 Sigma aids in DNA melting in promoter region - creates tighter binding of RNA Pol to promoter region

10 Tight binding of RNA Pol to promoter is Temp dependent

11 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)

12 -35 TTGACa -10 TAtAaT - up mutations: strongest - down mutations: weaker - in/dels between boxes: deleterious Consensus Sequence of Promoter Regions

13  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

14 - binding sites for activators (TAP-Fis) - between -60 & -150 Fis sites act as enhancers

15 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)

16 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)

17 helix-turn-helix motif

18 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)

19 RNA Polymerase ß’ subunit: -most + charged of all subunits -zinc finger motif -strong binding downstream of active site Transcriptional ELONGATION Zinc Fingers

20 Zinc Fingers within Major Groove of DNA

21 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)

22 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 5’-AUACUUGACGUACAAGUAU-3’ 1 0 structure 2 0 structure DNA sequences (palindrome) signal termination TERMINATION of Prok. Transcription

23 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

24 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

25 - Rho binds to RNA releases transcript from DNA template - Rho has no effect on initiation Rho-Dependent Termination of Prok.Transcription

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