Chapter 6 The mechanism of transcription in bacteria

Luc Luc Luc Luc -509 Luc -406 Luc -324 Luc Relative Light Units (RLU) Luc pGL2-Basic vector Analysis of the cag-8 gene promoter activity Ji et al. BBRC 2006 luciferase

lacZ GFP Chap 5

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

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

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

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

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

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

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