Prokaryotic Transcription: Initiation, Elongation & Termination

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

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

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

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

13. UP Element in strongest promoters - seen upstream to E. coli’s 7 rRNA genes - stimulates transcription 30X  subunit: - recognizes UP element - C-term. end binds to UP element

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

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. 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)

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

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. 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’

23. 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 A’s

24. Types of Termination Mechanisms • 2) Rho+ (Rho dependent): • - template strand with inverted repeat but • no poly A’s following • - Rho protein decreases net rate of transcription

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