Sarkosyl block of transcription reinitiation by RNA polymerase II as visualized by the colliding polymerases reinitiation assay

Abstract

There are Indications that different concentrations of Sarkosyl can block transcription Initiation by RNA polymerase II In vitro at different functional steps [Hawley and Roeder (1985) J. Blot. Chem. 260, 8163-8172]. Consequently, this reagent could be a very useful tool for mechanistic studies. So far, however, evidence for the selectivity of Sarkosyl effects on RNA polymerase II transcription has been only indirect. To directly investigate the effect of Sarkosyl on transcription Initiation and reinitiation by RNA polymerase II, we employed the reinitiation assay based on utilization of templates containing G-free cassettes (colliding polymerases reinitiation assay, or CoPRA). These experiments showed unambiguously that, under the appropriate conditions, Sarkosyl can be used to block transcription reinitiation by RNA polymerase II while allowing a first round of initiations from preassembled Initiation complexes. This Inhibition Is not due to a disruption of the Sll-dependent elongation of the reinitiated transcripts, and the levels of Sarkosyl that prevent transcription reinitiation coincide with the levels that block preinitlatlon complex assembly. However, Sarkosyl addition to transcription reactions reconstituted with partially purified transcription factors was found to have several undesirable side effects. The usefulness and limitations of the Sarkosyl-based and CoPRA assays for measurements of transcription reinitiation are discussed. © 1994 Oxford University Press.