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

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 rTFIIIB(70) 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 cooperatively to the TBP binding isotherm. A K-d of 44 +/- 23 nM characterizes the TFIIIB70 concentration dependence of the TBP . TFIIIB70 cooperativity. The relationship delta log K/delta log (TFIIIB70) is consistent with the linkage of a single molecule of TFIIIB70 with the TBP-promoter binding reaction.