CLONING AND CHARACTERIZATION OF AN EVOLUTIONARILY DIVERGENT DNA-BINDING SUBUNIT OF MAMMALIAN TFIIIC

Transcription factor IIIC (TFIIIC) is required for the assembly of a p reinitiation complex on 5S RNA, tRNA, and adenovirus VA RNA genes and contains two separable components, TFIIIC1 and TFIIIC2. TFIIIC2 binds to the 3' end of the internal control region of the VA, RNA gene and c ontains five polypeptides ranging in size from 63 to 220 kDa; the larg est of these directly contacts DNA. Here we describe the cloning of cD NAs encoding all (rat) or part (human) of the 220-kDa subunit (TFIIIC alpha). Surprisingly, TFIIIC alpha has no homology to any of the yeast TFIIIC subunits already cloned, suggesting a significant degree of ev olutionary divergence for RNA polymerase III factors. Antibodies raise d against the N terminus of recombinant human TFIIIC alpha specificall y inhibit binding of natural TFIIIC to DNA. Furthermore, immunodepleti on assays indicate that TFIIIC alpha is absolutely required for RNA po lymerase III transcription of 5S RNA, tRNA, and VA, RNA genes but not for the 7SK RNA and U6 small nuclear RNA genes. Transcription from the tRNA and VA,RNA genes in TFIIIC-depleted nuclear extracts can be rest ored by addition of purified TFIIIC. In contrast, restoration of 5S RN A gene transcription requires readdition of both TFIIIC and TFIIIA, in dicating a promoter-independent interaction between these factors. Imm unoprecipitation experiments demonstrate a tight association of all fi ve polypeptides previously identified in the TFIIIC2 fraction, confirm ing the multisubunit structure of the human factor.