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 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 K_d 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.