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