A cryptic DNA binding domain at the COOH terminus of TFIIIB70 affects formation, stability, and function of preinitiation complexes

Abstract

TFIIIC-dependent assembly of yeast TFIIIB on class III genes unmasks a high avidity of TFIIIB for DNA. TFIIIB contains TATA-binding protein (TBP), TFIIIB90/B', and TFIIIB70/Brf1, which is homologous to TFIIB. Using limited proteolysis, we have found that the COOH terminus of TFIIIB70 (residues 510- 596) forms a protease-resistant domain that binds DNA tightly as seen by Southwestern, DNase I footprinting, and gel shift assays. Consistent with a role for this DNA binding activity, preinitiation complexes were formed less efficiently with truncated TFIIIB70 lacking the COOH-terminal domain and displayed an increased sensitivity to heparin. B' (TFIIIB70 + TBP)·TFIIIC·DNA complexes were also particularly unstable. In addition, TFIIIB·TFIIIC·DNA complexes containing truncated TFIIIB70 were impaired in promoting transcription initiation.