Transcription overview
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Transcription overview. DNA is the template for transcription. RNA chain growth is 5 ’ to 3 ’. Prokaryotic promoters. Prokaryotic promoters. The carboxy terminal domain (CTD) of a binds to the UP element of a promoter. Overview of transcription initiation.

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Transcription overview

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Transcription overview

Transcription overview


Transcription overview

DNA is the template for transcription


Transcription overview

RNA chain growth is 5’ to 3’


Transcription overview

Prokaryotic promoters


Transcription overview

Prokaryotic promoters


Transcription overview

The carboxy terminal domain (CTD) of a binds

to the UP element of a promoter.


Transcription overview

Overview of transcription initiation


Transcription overview

Included: E.coli RNA pol; a strong promoter; and heparin

Lane 1: no DNA

Lane 2 ATP only

Lanes 3-7: increasing concentrations of other three NTPs


Transcription overview

s Stimulates Transcription Initiation

Expt 1: Added [g32P]ATP, or [g32P] GTP to label first position of RNA (initiation), OR used [14C] ATP to label all C’s in the mRNA (elongation) and asked if s stimulated incorporation of the labels.

Result: s appeared to stimulate both initiation and elongation.


Transcription overview

Expt 2: Allowed a certain amount of initiation to occur, and then added rifampicin to inhibit any further initiation. By ultracentrifugation, looked at lengths of mRNAs produced with and without s.

Result: The presence/absence of s made no difference to the mRNA lengths, which would have NOT have been the case if s stimulated elongation.

Conclusion: s stimulates initiation but not elongation of transcription.


Transcription overview

Re-use of s

- rifampicin, core from rifampicin-resistant strain

Experiment done at low ionic strength. Label is gamma 32P labelled NTPs to measure inititation only.


Transcription overview

Re-use of s

- rifampicin, core from rifampicin-resistant strain

Experiment done at low ionic strength. Lable is gamma 32P labelled NTPs to measure inititation only.

+rifampicin, core from rifampicin-resistant strain


Transcription overview

Classic View of the s Cycle

Caveat: Evidence for above model might be artifactual; due to harsh separation conditions of initiation vs. elongation versions of RNA Pol

Recent evidence:s may stay associated with RNA Pol even during elongation. One model is that the s cycle is really just a shifting of position of s so that it becomes more loosely associated with RNA

Pol.

The Stochastic Model


Transcription overview

Local DNA Melting at the Promoter

•RNA Pol-promoter complexes are more stable at elevated temps. suggesting that local melting occurs upon tight of RNA Pol to DNA

•E. coli RNA Pol binding to T7 gene promoters caused a hyperchromic shift in the A260 of DNA: indicative of DNA strand separation

•Gamper and Hearst expt. indicates that RNA Pol melts a short region of DNA (171 bp) form a transcription bubble that moves with the polymerase as it transcribes DNA. This exposes the template as RNA Pol moves along it.


Figure 6 19

Figure 6.19


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