Abstract
Initiation of mRNA synthesis by RNA polymerase II occurs by a multistep pathway that involves the polymerase along with several auxiliary factors. Here I describe an analysis of the kinetics and mechanism of action of Drosophila RNA polymerase II by using the detergent Sarkosyl to inhibit different steps in the pathway leading to transcription initiation. The template DNA and Drosophila transcription factors assemble into a partially Sarkosyl-resistant initiation complex with a kinetically first order time course characterized by a t1/2 of about 3 min, and subsequent conversion of the initiation complex into the elongating complex occurs rapidly (less than 5 s). During the formation of the first two phosphodiester bonds, there is a stepwise transition from the initiation complex to the 0.25% Sarkosyl-resistant elongating polymerase. In contrast to the mammalian RNA polymerase II systems, the Drosophila extracts possess the ability to carry out many rounds (approximately 10) of transcription in vitro. Finally, the transcription factors are committed to the template DNA as an initiation complex, but then no longer appear to remain committed to the DNA after initiation of transcription. This finding suggests that there is complete assembly and disassembly of the transcription complex during each round of transcription by RNA polymerase II.
