Identification of an autonomously initiating RNA polymerase III holoenzyme containing a novel factor that is selectively inactivated during protein synthesis inhibition.

Abstract

Transcription by RNA polymerase III (Pol III) requires multiple general initiation factors that, in isolated form, assemble onto the promoter in an ordered fashion. Here, it is shown that all components required for transcription of the VA1 and tRNA genes, including TFIIIB, TFIIIC, and RNA Pol III, can be coimmunopurified from a HeLa cell line that constantly expresses a FLAG epitope-tagged subunit of human RNA Pol III. This finding of an RNA Pol III "holoenzyme" suggests similarities between transcription initiation by RNA Pol II and RNA Pol III and has led to the identification of a novel general initiation factor (TDF, translation dependent factor) that is present within the holoenzyme. TDF is selectively inactivated during protein synthesis inhibition by cycloheximide and at a late stage of adenovirus infection, thus accounting for the loss of RNA Pol III-mediated transcription of the tRNA and VA RNA genes under these conditions. On the basis of these observations, possible mechanisms for the global regulation of transcription by RNA Pol III and for disassembly of RNA Pol III initiation complexes are proposed.