Biochemical analysis of transcription termination by RNA polymerase III from yeast Saccharomyces cerevisiae.

Abstract

Eukaryotic RNA polymerase III (pol III) transcribes short noncoding RNA genes such as those encoding tRNAs, 5S rRNA, U6 snRNA, and a few others. As compared to its pol II counterpart, Pol III has several advantages, including the relative simplicity, stability, and more direct connectivity of its transcription machinery. Only two transcription factor complexes, TFIIIB and TFIIIC, are required to faithfully initiate and direct multiple rounds of transcription by pol III. Moreover, in contrast to an intricate multipartite mechanism of pol II termination, pol III termination is extremely simple, responsive to a monopartite signal (oligo T stretch on the nontemplate DNA strand) and mediated by a stably associated termination subcomplex of three integral subunits (Arimbasseri et al. Transcription 4(6), 2013). This makes pol III a valuable model for dissecting intrinsic molecular mechanisms of eukaryotic transcription termination. In this chapter, we provide protocols we adapted to study the biochemistry of transcription termination by S. cerevisiae pol III.