TFIIIC1 Acts through a Downstream Region To Stabilize TFIIIC2 Binding to RNA Polymerase III Promoters

Abstract

An in vitro system reconstituted with highly purified RNA polymerase III, TFIIIC2, and TFIIIB has been used to identify two chromatographically distinct human RNA polymerase III transcription factors, TFIIIC1 and TFIIIC1', which are functionally equivalent to the previously defined TFIIIC1 (S. T. Yoshinaga, P. A. Boulanger, and A. J. Berk, Proc. Natl. Acad. Sci. USA 84:3585-3589, 1987). Interactions between TFIIIC2, TFIIIC1 (or TFIIIC1'), and the VA1 and tRNA1(Met) templates have been investigated by DNase I footprint analysis. Homogeneous TFIIIC2 alone shows only a weak footprint over the B-box region of the VA1 and tRNA1(Met) templates, whereas TFIIIC1 (or TFIIIC1') alone shows both a strong interaction over the downstream termination region and a very weak interaction near the A-box region. Importantly, when both factors are present simultaneously, TFIIIC1 (or TFIIIC1') dramatically enhances the level of TFIIIC2 binding and extends the footprint to a region that includes the A box. The downstream termination region is essential for this cooperative interaction between TFIIIC2 and TFIIIC1 (or TFIIIC1') on the VA1 and tRNA1(Met) templates and plays a role in the overall accuracy and efficiency of RNA polymerase III transcription.