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 Kd of 44 +/- 23 nM characterizes the TFIIIB70 concentration dependence of the TBP.TFIIIB70 cooperativity. The relationship deltalog K/deltalog (TFIIIB70) is consistent with the linkage of a single molecule of TFIIIB70 with the TBP-promoter binding reaction.
Librizzi
M D
MD
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA. illis@aecom.yu.edu
Moir
R D
RD
Brenowitz
M
M
Willis
I M
IM
D004268:DNA-Binding Proteins / Q000378:metabolism*; D046911:Macromolecular Substances; D011401:Promoter Regions, Genetic; D012320:RNA Polymerase III / Q000378:metabolism*; D012367:RNA, Viral / Q000096:biosynthesis; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D035381:TATA-Binding Protein Associated Factors; D035181:TATA-Box Binding Protein; D035361:Transcription Factor TFIIIB; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic
C441028:BRF1 protein, human; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D012367:RNA, Viral; D011994:Recombinant Proteins; D035381:TATA-Binding Protein Associated Factors; D035181:TATA-Box Binding Protein; D035361:Transcription Factor TFIIIB; D014157:Transcription Factors; D012320:RNA Polymerase III