Interactions between the tetratricopeptide repeats-containing transcription factor TFIIIC131 and its ligand, TFIIIB70: Evidence for a conformational change in the complex

Robyn D. Moir, Karen V. Puglia, Ian M. Willis

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Abstract

In the transcription of tRNA and 5 S genes by RNA polymerase III, recruitment of the transcription factor (TF)IIIB is mediated by the promoter-bound assembly factor TFIIIC. A critical limiting step in this process is the interaction between the tetratricopeptide repeat (TPR)-containing subunit of TFIIIC (TFIIIC131) and the TFIIB-related factor Brf1p/TFIIIB70. To facilitate biochemical studies of this interaction, we expressed a fragment of TFIIIC131, TFIIIC131-(1-580), that includes the minimal TFIIIB70 interaction domain defined by two-hybrid studies together with adjacent sequences, up to the end of TPR9, implicated in the assembly reaction. TFIIIC131-(1-580) interacts with TFIIIB70 in solution and inhibits the formation of TFIIIB70·TFIIIC·DNA complexes. In a coupled equilibrium binding assay, the formation of TFIIIC131-(1-580)·TFIIIB70 complexes was adequately described by a single-site binding model and yielded an apparent equilibrium dissociation constant of 334 ± 23 nM. CD spectroscopy and limited proteolysis experiments defined a well structured and largely protease-resistant core in TFIIIC131-(1-580) comprising part of the hydrophilic amino terminus, TPR1-5, the intervening non-TPR region, and TPR6-8. CD spectra showed that trifluoroethanol induced significant α-helical structure in TFIIIC131-(1-580). A more modest monovalent ion-dependent CD difference was observed in mixtures of TFIIIC131-(1-580) and TFIIIB70, suggesting that formation of the binary complex may proceed with the acquisition of α-helicity.

Original languageEnglish (US)
Pages (from-to)26591-26598
Number of pages8
JournalJournal of Biological Chemistry
Volume275
Issue number34
DOIs
Publication statusPublished - Aug 25 2000

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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