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 as sembly of an initiation complex comprising the TATA-binding protein (T BP), a 90-kDa polypeptide (TFIIIB90), and a 70-kDA polypeptide (TFIIIB 70). TFIIIB70 interacts with TBP, a unique pol III subunit, C34, and t he 131-kDa subunit of the pol III-specific complex, TFIIIC. TFIIIB70 w as expressed in Escherichia coli and purified to homogeneity. The spec ific transcription activity of rTFIIIB(70) is 22-58% that of the nativ e yeast and in vitro synthesized factor. However, only a small fractio n (0.07-0.32%) of the TFIIIB70 from these sources results in the synth esis of full-length RNA. The data suggest that TFIIIB70 function may b e limited by an unfavorable recruitment equilibrium into the preinitia tion complex. Quantitative DNase I ''footprint'' titrations of yeast T BP to the adenovirus major late promoter were conducted at a series of constant TFIIIB70 concentrations. A value of -0.7 +/- 0.2 kcal/mol wa s determined for the cooperative free energy of formation of the TBP . TFIIIB70. DNA complex at concentrations of TFIIIB70 sufficient to par tition all of the binding cooperatively to the TBP binding isotherm. A K-d of 44 +/- 23 nM characterizes the TFIIIB70 concentration dependen ce of the TBP . TFIIIB70 cooperativity. The relationship delta log K/d elta log (TFIIIB70) is consistent with the linkage of a single molecul e of TFIIIB70 with the TBP-promoter binding reaction.