Separation and partial characterization of three functional steps in transcription initiation by human RNA polymerase II

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.