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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 :

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prokaryotic transcription initiation elongation termination

MOLECULAR BIOLOGY

Ch 6

Prokaryotic Transcription:

Initiation,

Elongation & Termination

slide3

SDS PAGE

RNA Polymerase

Holoenzyme

holoenzyme

sigma

core

-

'

+

slide4

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
slide6

Transcriptional INITIATION

Sigma stimulates tight binding between RNA polymerase and promoter:

Experiment:

- labeled DNA + core or holo.

- added unlabeled DNA

- filtered the mixture

slide8

Electron

Microscopy

on

2-D

Crystals

slide9

Sigma aids in DNA melting in promoter region

- creates tighter binding of RNA Pol to promoter region

slide11

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)

slide12

Consensus Sequence of Promoter Regions

-35

TTGACa

-10

TAtAaT

- up mutations: strongest

- down mutations: weaker

- in/dels between boxes: deleterious

slide13

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

slide14

Fis sites act as enhancers

  • - binding sites for activators (TAP-Fis)
      • - between -60 & -150
slide15

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)
slide16

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)

slide18

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)

slide19

Transcriptional ELONGATION

Zinc Fingers

  • RNA Polymerase
  • ß’ subunit:
  • most + charged of all subunits
  • zinc finger motif
  • strong binding downstream
  • of active site
slide21

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)
slide22

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’

slide23

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
slide24

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
slide25

Rho-Dependent

Termination

of Prok.Transcription

  • Rho binds to RNA
  • releases transcript
  • from DNA template
  • Rho has no effect on
  • initiation