A mutation in the second largest subunit of TFIIIC increases a rate-limiting step in transcription by RNA polymerase III

Gerald Rameau, Karen Puglia, Alex Crowe, Indra Sethy, Ian M. Willis

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Abstract

In previous studies, we have shown that the PCF1-1 mutation of Saccharomyces cerevisiae suppresses the negative effect of a tRNA gene A block promoter mutation in vivo and increases the transcription of a variety of RNA polymerase III genes in vitro. Here, we report that PCF1 encodes the second largest subunit of transcription factor IIIC (TFIIIC) and that the PCF1-1 mutation causes an amino acid substitution in a novel protein structural motif, a tetratricopeptide repeat, in this subunit. In agreement with the nature of the mutation, in vitro transcription studies with crude extracts indicate that PCF1-1 facilitates the rate-limiting step in transcription, namely, the recruitment of TFIIIB to the template. Additionally, biochemical fractionation of wild-type and mutant cell extracts shows that PCF1-1 increases the amount of the 70-kDa TFIIIB subunit detectable by Western (immunoblot) analysis in purified TFIIIB fractions and the transcription activity of a TFIIIB" fraction containing the 90-kDa subunit of this factor. We suggest that the effect of PCF1-1 on TFIIIB activity in vitro is a consequence of its increased rate of recruitment in vivo.

Original languageEnglish (US)
Pages (from-to)822-830
Number of pages9
JournalMolecular and Cellular Biology
Volume14
Issue number1
StatePublished - 1994

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Transcription Factor TFIIIB
RNA Polymerase III
Mutation
Amino Acid Motifs
Amino Acid Substitution
Transfer RNA
Cell Extracts
Complex Mixtures
Genes
Saccharomyces cerevisiae
Western Blotting
transcription factor TFIIIC
In Vitro Techniques

ASJC Scopus subject areas

  • Cell Biology
  • Genetics
  • Molecular Biology

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A mutation in the second largest subunit of TFIIIC increases a rate-limiting step in transcription by RNA polymerase III. / Rameau, Gerald; Puglia, Karen; Crowe, Alex; Sethy, Indra; Willis, Ian M.

In: Molecular and Cellular Biology, Vol. 14, No. 1, 1994, p. 822-830.

Research output: Contribution to journalArticle

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