Conditional expression of RNA polymerase II in mammalian cells. Deletion of the carboxyl-terminal domain of the large subunit affects early steps in transcription

Abstract

The carboxyl-terminal domain (CTD) of the large subunit of mammalian RNA polymerase II contains 52 repeats of a heptapeptide that is the target of a variety of kinases. The hyperphosphorylated CTD recruits important factors for mRNA capping, splicing, and 3'-processing. The role of the CTD for the transcription process in vivo, however, is not yet clear. We have conditionally expressed an alpha-amanitin-resistant large subunit with an almost entirely deleted CTD (LS*Delta5) in B-cells. These cells have a defect in global transcription of cellular genes in the presence of alpha-amanitin. Moreover, pol II harboring LS*Delta5 failed to transcribe up to the promoter-proximal pause sites in the hsp70A and c-fos gene promoters. The results indicate that the CTD is already required for steps that occur before promoter-proximal pausing and maturation of mRNA.