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

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

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

Electron Microscopy on 2-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 bond formation)

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

 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 Polymerase ß’ subunit: -most + charged of all subunits -zinc finger motif -strong binding downstream of active site Transcriptional ELONGATION Zinc 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 A 3’ 5’ RNA made 5’-AUACUUGACGUACAAGUAU-3’ 1 0 structure 2 0 structure DNA sequences (palindrome) signal termination TERMINATION of Prok. Transcription

1) Rho- (Rho independent): - template strand: 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