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

Giorgio Lagna, Robert Kovelman, Jun Sukegawa, Robert G. Roeder

Research output: Contribution to journalArticlepeer-review

44 Citations (Scopus)

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 VA(I) 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 (TFIIICα). Surprisingly, TFIIICα 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 TFIIICα specifically inhibit binding of natural TFIIIC to DNA. Furthermore, immunodepletion assays indicate that TFIIICα is absolutely required for RNA polymerase III transcription of 5S RNA, tRNA, and VA(I) RNA genes but not for the 7SK RNA and U6 small nuclear RNA genes. Transcription from the tRNA and VA1 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.

Original languageEnglish
Pages (from-to)3053-3064
Number of pages12
JournalMolecular and cellular biology
Volume14
Issue number5
DOIs
Publication statusPublished - 1994 May
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

Fingerprint

Dive into the research topics of 'Cloning and characterization of an evolutionarily divergent DNA-binding subunit of mammalian TFIIIC'. Together they form a unique fingerprint.

Cite this