100(a)Core75T4competitorRNAsimmediateearlyisolatedfrom E.coilcells501" (green)Delayed early255' (red)late17' (blue)afterinfectionwithT4100(b)HoloenzymeH3-RNAsof3genes75competetoT4DNAwith50competitor25immediateEarly2505007!Delayed earlyCompetitorRNAadded(μg)
immediate early Delayed early late immediate Early Delayed early
100(a)Core75Core enzyme (No g)immediate early50has no specificityDelayed early25late100Holoenzyme(with α)(b)Holoenzyme75is highly specificforimmediate early and50delayed early genes of25immediate Earlytranscription5007!Delayed early250CompetitorRNAadded(μg)
Holoenzyme (with σ ) is highly specific for immediate early and delayed early genes of transcription Core enzyme (No σ) has no specificity immediate early Delayed early late immediate Early Delayed early
100H3-T7DNA+HoloE/CoreEeaeHoloenzyme(DNA-Eat37°c)ExcessunlabeledT7DNA10competitor中HoloE/CoreEdissociatedCorefromDNA-E,thenrebind/Filtered mixtures through2040600NitrocelluloseTime (min)37°cg stimulates tight bindingMonitor H3-T7 DNA/E retainedbetweenRNApolymeraseandtemplate strand of promoter
Sigma stimulates tight binding between RNA polymerase and promoter Excess unlabeled T7 DNA competitor Filtered mixtures through Nitrocellulose 37 37oc
14C-ATP measured RNAWhyY32P-ATPelongationand y32P-GTPooo(ebe used?g could appearto stimulateelongation by stimulationinitiation and providing[y32P]ATP2more chainsfor core Etoelongation!!14C-ATP ?IGTF stimulateinitiation of0.501.0CoreE+o (μg/ml)transcription
γ-P32ATP or γ-P32GTP ?! σ stimulate initiation of transcription Core E +σ (μg/ml) 14C-ATP measured RNA elongation σ could appear to stimulate elongation by stimulation initiation and providing more chains for core E to 14C-ATP ? elongation!! Why γ32P-ATP and γ32P-GTP be used?
o factor can be reused
σfactor can be reused