Abstract
We have used Sarkosyl to study the events comprising specific transcription initiation in vitro by HeLa RNA polymerase II. On the basis of different sensitivities to the Sarkosyl concentration, we have defined three functional steps in initiation at the adenovirus major late promoter: 1. a template commitment step that occurs in the presence of 0.015% Sarkosyl; 2. a nucleotide-independent conversion of the committed complex to a "rapid start complex" capable of initiating an RNA chain, a step blocked by 0.015% Sarkosyl; and 3. a step that requires nucleoside triphosphates, converts the rapid start complex to a stably initiated complex, and is sensitive to Sarkosyl concentrations greater than 0.05%. The subsequent elongation of the initiated RNA chain is resistant to Sarkosyl, except that Sarkosyl causes pausing or premature termination at a specific site about 186 nucleotides downstream of the major late cap site. Using this assay, we have further characterized these steps and the resulting intermediate complexes.
Highlights
On the basis of different sensitiv- moters i n vitro but must be supplemented by other proteins ities to the Sarkosyl concentration, we have defined present in crude extracts[5].Chromatographic fractionation three functional steps in initiation at the adenovirus procedures have separated HeLa extracts active for specific major late promoter: 1. a template commitment step RNA polymerase I1 transcription into at least five fractions that occurs in the presence of 0.015%Sarkosyl; 2. a nucleotide-independent conversion of the committed complex to a “rapidstart complex”capableof initiating an RNA chain, a step blocked by 0.015%Sarkosyl;and 3. a step that requires nucleoside triphosphates, converts the rapid start complex to a stably initiated complex, and is sensitive to Sarkosyl concentrations >0.05%
The ammonium sulfate precipitation stepw, hile increasing themax- Effect of Sarkosyl on Steps inInitiation-Our transcription imum amount of transcription observed, did not appear toaffect the system was reconstituted with transcription factors that had pattern of titration significantly
The P-11 breakthrough (0.1 M KCI) fraction contains the required factor TFIIA. Because this fractionalso contains most of the nucleotides present in the nuclear extract, we used TFIIA that had been purified by two additional chromatographic steps
Summary
The ammonium sulfate precipitation stepw, hile increasing themax- Effect of Sarkosyl on Steps inInitiation-Our transcription imum amount of transcription observed, did not appear toaffect the system was reconstituted with transcription factors that had pattern of titration significantly. After the column was washed with BC100, TFIIAactivity was stepelutedwith BC250 (0.25 M KCI) This the protein fractions in the absence of nucleotides; and 2) a second incubation after addition of nucleotides to allow initiation and productionof a 536-nucleotide run-off transcript. A slightly higher concentration of Sarkosyl (0.015%) almost completely inhibitedtranscription when present during the preincubation(lane 5 ) but did not inhibit initiation of the RNA chain when added following the preinpromoter). (We show below that under our standard conditions, transcription initiation iscomplete 30 s after nucleotide addition.) A higher concentration of Sarkosyl (0.25%) prevented initiation not (usually 25 p l ) of transcription stop mix: 10 mM EDTA, 0.1 M sodium only when present during the preincubation and when acetate, pH 5.5, 0.5% sodium dodecyl sulfate, 1 mg/ml yeast RNA. C1 protein factors (data not shown) but did not require nucleotides, suggesting that the resulting intermediate was a partially or completely assembled,but not initiated, transcription complex.
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