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DNA

Enzymes in the central dogma. Cellular enzymes. (Mostly) RNA virus enzymes. DNA. DNA polymerase. Reverse transcriptase. RNA polymerase. RNA-dependent RNA polymerase. RNA. Ribosome. Protein. The process of transcription. 3’. 5’. DNA template. RNA. 5’. 3’.

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DNA

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  1. Enzymes in the central dogma Cellular enzymes (Mostly) RNA virus enzymes DNA DNA polymerase Reverse transcriptase RNA polymerase RNA-dependent RNA polymerase RNA Ribosome Protein

  2. The process of transcription 3’ 5’ DNA template RNA 5’ 3’

  3. The three steps of transcription: initiation, elongation and termination RNA polymerase Non-template strand DNA Template strand 5’ RNA 3’ Fig. 3.14 5’

  4. E. coli promoter Fig. 6.6

  5. The following DNA contains a promoter. What is the sequence of the RNA made? - Clicker Question - 5’-AGTACGTACTTGACATAGATGCGCGCTCGATGTATAATGCGCCACCAGAGTGATCGA-3’ 3’-TCATGCATGAACTGTATCTACGCGCGAGCTACATATTACGCGGTGGTCTCACTAGCT-5’ -10 -35 A. 5’-OHUCUCACUAGCUppp-3’ B. 5’-pppUCUCACUAGCUOH-3’ C. 5’-pAGAGUGAUCGAp-3’ D. 5’-pppAGAGUGAUCGAOH-3’ E. 5’-pppAGAGUGAUCGAp-3’

  6. E. coli RNA polymerase ‘    ‘    Core (2’)  + Holo-enzyme (2’) 

  7. ‘     Sigma factor is needed for promoter binding (2’) ‘    (2’) Fig. 6.3

  8. Experiment to test whether the RNA polymerase melts the region around the transcription start site DMS: Chemical that methylates unpaired As 32P Nuclease Autorad Nuclease S1: Nuclease that cleaves only single stranded DNA Technique: see DNase/DMS footprinting (Weaver Ch. 5, p. 116-119) Fig. 6.16

  9. Evidence that the RNA polymerase melts the region around the transcription start site RNA polymerase: + + - - Nuclease S1: - + + - Melted region Fig. 6.17

  10. In the shown experiment, the lane labeled R-S+ contains no RNA polymerase, but is still treated with Nuclease S1. Why is this control experiment important? It tells you that: A. Nuclease S1 cleaves ssDNA. B. Nuclease S1 does not cleave the DNA when RNA polymerase was not there. C. RNA polymerase does not cleave dsDNA. D. RNA polymerase is active in transcription. - Clicker Question -

  11. Evidence that the sigma subunit is reused Incorporation of 32P-ATP/GTP (2’) 2) 1) Add holoenzyme (2’) Fig. 6.11

  12. Evidence that sigma stays bound during transcription elongation Fig. 6.13b Fig. 6.14b

  13. The steps of transcription initiation in bacteria ? Fig. 6.9

  14. The alpha subunit of RNA polymerase can bind upstream (UP) elements in strong promoters Fig. 6.26

  15. Nucleotides cross-link to the RNA polymerase  subunit SDS-PAGE/ autorad Fig. 6.29 Fig. 6.30

  16. Both  and ’ subunits interact with DNA during transcription cross-linker ‘  32P   * DNA SDS-PAGE/ autorad Fig. 6.33

  17. Model of the transition from closed (RPc) to open (RPo) promoter complex     ’ ’ Fig. 6.43a

  18. - Clicker Question - An E. coli strain has acquired a lethal mutation in the promoter -10 box of an essential gene. Researchers subjected the strain to a mutagen, and selected a secondary mutation (i.e not in the same gene), which restored growth. Which of the following genes is most likely to carry the secondary mutation? A. RNA polymerase a subunit. B. RNA polymerase b subunit. C. RNA polymerase b’ subunit. D. RNA polymerase s subunit. E. RNA polymerase e subunit.

  19. - Clicker Question - You have isolated an antibiotic which kills E. coli cells by interacting with RNA polymerase. You discover that the antibiotic causes low production of ribosomal RNA but does not affect most mRNAs. Which of the following RNA polymerase subunits is most likely to interact with the drug? A. RNA polymerase a subunit. B. RNA polymerase b subunit. C. RNA polymerase b’ subunit. D. RNA polymerase s subunit. E. RNA polymerase e subunit.

  20. Model for Rho-independent (simple) transcription termination in bacteria RNA 3’end of simple terminator: 5’ 3’ UUUUUU Fig. 6.46

  21. Some terminators depend on the protein Rho DNA:RNA Free RNA Heavy Heavy Light Light DNA/RNA separated in CsCl density gradients Fig. 6.50

  22. Model for Rho-dependent transcription termination in bacteria Fig. 6.51

  23. - Clicker Question - Pa gene a Pb gene b Gene b is immediately downstream of gene a in E. coli and has its own promoter and a Rho-dependent terminator. An E. coli strain has acquired a mutation that causes the appearance of an RNA containing both genes a and b, but no sequence downstream of gene b. In which part of the genome is the mutation most likely to be? A. In the promoter for gene a. B. In the promoter for gene b. C. At the end of gene a. D. The RNA polymerase s subunit gene. E. The Rho factor gene.

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