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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.

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Transcription overview

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  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

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