Expression and purification of the RNA polymerase III transcription specificity factor IIIB70 from Saccharomyces cerevisiae and its cooperative binding with TATA-binding protein

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Abstract

Transcription by RNA polymerase III (pol III) in yeast requires the assembly of an initiation complex comprising the TATA-binding protein (TBP), a 90-kDa polypeptide (TFIIIB90), and a 70-kDa polypeptide (TFIIIB70). TFIIIB70 interacts with TBP, a unique pol III subunit, C34, and the 131-kDa subunit of the pol III-specific complex, TFIIIC. TFIIIB70 was expressed in Escherichia coli and purified to homogeneity. The specific transcription activity of rTFIIIB70 is 22-58% that of the native yeast and in vitro synthesized factor. However, only a small fraction (0.07-0.32%) of the TFIIIB70 from these sources results in the synthesis of full-length RNA. The data suggest that TFIIIB70 function may be limited by an unfavorable recruitment equilibrium into the preinitiation complex. Quantitative DNase I "footprint" titrations of yeast TBP to the adenovirus major late promoter were conducted at a series of constant TFIIIB70 concentrations. A value of -0.7 ± 0.2 kcal/mol was determined for the cooperative free energy of formation of the TBP-TFIIIB70-DNA complex at concentrations of TFIIIB70 sufficient to partition all of the binding cooperativity to the TBP binding isotherm. A Kd of 44 ± 23 nM characterizes the TFIIIB70 concentration dependence of the TBP-TFIIIB70 cooperativity. The relationship δlog K/δlog (TFIIIB70) is consistent with the linkage of a single molecule of TFIIIB70 with the TBP-promoter binding reaction.

Original languageEnglish (US)
Pages (from-to)32695-32701
Number of pages7
JournalJournal of Biological Chemistry
Volume271
Issue number51
DOIs
StatePublished - Jan 1 1996

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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