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Cloning and characterization of an evolutionarily divergent DNA-binding subunit of mammalian TFIIIC.

Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, New York 10021.

ABSTRACT

Transcription factor IIIC (TFIIIC) is required for the assembly of a preinitiation 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 VAI RNA gene and contains five polypeptides ranging in size from 63 to 220 kDa; the largest of these directly contacts DNA. Here we describe the cloning of cDNAs 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 evolutionary divergence for RNA polymerase III factors. Antibodies raised against the N terminus of recombinant human TFIIIC alpha specifically inhibit binding of natural TFIIIC to DNA. Furthermore, immunodepletion assays indicate that TFIIIC alpha is absolutely required for RNA polymerase III transcription of 5S RNA, tRNA, and VAI RNA genes but not for the 7SK RNA and U6 small nuclear RNA genes. Transcription from the tRNA and VAI RNA genes in TFIIIC-depleted nuclear extracts can be restored by addition of purified TFIIIC. In contrast, restoration of 5S RNA gene transcription requires readdition of both TFIIIC and TFIIIA, indicating a promoter-independent interaction between these factors. Immunoprecipitation experiments demonstrate a tight association of all five polypeptides previously identified in the TFIIIC2 fraction, confirming the multisubunit structure of the human factor.

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