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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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The carboxy terminal domain (CTD) of a binds

to the UP element of a promoter.



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


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.


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.


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.


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


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


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.



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