1 / 22

Transcription overview

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.

maura
Download Presentation

Transcription overview

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Transcription overview

  2. DNA is the template for transcription

  3. RNA chain growth is 5’ to 3’

  4. Prokaryotic promoters

  5. Prokaryotic promoters

  6. The carboxy terminal domain (CTD) of a binds to the UP element of a promoter.

  7. Overview of transcription initiation

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. Figure 6.19

More Related