transcription overview
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
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 binds to the UP element of a promoter.
Overview of transcription initiation
Included: E.coli RNA pol; a strong promoter; and heparinLane 1: no DNALane 2 ATP onlyLanes 3-7: increasing concentrations of other three NTPs
Stimulates Transcription Initiation
Expt 1: Added [32P]ATP, or [32P] GTP to label first position of RNA (initiation), OR used [14C] ATP to label all C’s in the mRNA (elongation) and asked if stimulated incorporation of the labels.
Result: 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 .
Result: The presence/absence of made no difference to the mRNA lengths, which would have NOT have been the case if stimulated elongation.
Conclusion: stimulates initiation but not elongation of transcription.
Re-use of - 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
+rifampicin, core from rifampicin-resistant strain
- rifampicin, core from rifampicin-resistant strain
Experiment done at low ionic strength. Lable is gamma 32P labelled NTPs to measure inititation only.
Classic View of the Cycle
Caveat: Evidence for above model might be artifactual; due to harsh separation conditions of initiation vs. elongation versions of RNA Pol
Recent evidence: may stay associated with RNA Pol even during elongation. One model is that the cycle is really just a shifting of position of so that it becomes more loosely associated with RNAPol.
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 (171 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