1 / 10

Chapter 6 The mechanism of transcription in bacteria

Chapter 6 The mechanism of transcription in bacteria. +1. -2000. -1500. -1000. -500. +117. Luc. -2175. Luc. -1620. Luc. -1144. Luc. -626. Luc. -509. Luc. -406. Luc. -324. Luc. -204. Luc. Luc. pGL2-Basic vector. 0. 20000. 40000. 60000. 80000.

cahil
Download Presentation

Chapter 6 The mechanism of transcription in bacteria

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. Chapter 6 The mechanism of transcription in bacteria

  2. +1 -2000 -1500 -1000 -500 +117 Luc -2175 Luc -1620 Luc -1144 Luc -626 Luc -509 Luc -406 Luc -324 Luc -204 Luc Luc pGL2-Basic vector 0 20000 40000 60000 80000 Relative Light Units (RLU) Analysis of the cag-8 gene promoter activity luciferase Ji et al. BBRC 2006

  3. Chap 5 GFP lacZ

  4. Chap 5 Gel Mobility Shift Assay DNase I footprinting Knock-out mouse ; Gene targeting Thymidine kinase Neomycinr ES cell Knock-down; RNAi = si RNA

  5. E. coli RNA polymerase; SDS-PAGE Holoenzyme, core enzyme phosphocellulose σ ; Specificity factor Asymmmetric transcription 30% 100% RNase-resistant Holoenzyme • Core enzyme; • Basal activity • no strand specificity

  6. Binding of RNA Polymerase to Promoters • How tightly does core enzyme vs. holoenzyme bind DNA? • nitrocellulose filter binding assay chap 5, p.116 • Holoenzyme binds filters tightly • Core enzyme binding is more transient Unlabeled DNA ; competitor

  7. Promoter sequence; Consensus sequence -10 box, -35 box -10 box; Pribnow box Cf. Euk; TATA box at -28 σ-cycle

  8. β and β’ subunits ; DNA binding Affinity labeling Reagent I; ATP analog salt sensitive site; ionic bond Salt insensitive site; hydrophobic interaction Active site Phosphodiester bond formation; β subunit

  9. Stereo views of RNA polymerase core enzyme Open crab claw Model 1; Avoid twisting Wrapping of RNA around DNA Model 2; Avert wraping negative supertwist ahead positive supertwist behind

  10. Termination Hairpin & T-string ρ-dependent Inverted repeat; palindrome ρ-independent Inverted repeat; palindrome Hairpin but no T-string ρ helicase ; dissociate the DNA/ RNA hybrid

More Related