A study of the RNA polymerase III transcription specificity factor IIIB(70) from Saccharomyces cerevisiae

Abstract

In yeast, RNA polymerase III (pol III) transcription in vitro requires three factors (TFIIIA, TFIIIB, TFIIIC). Depending on the template, TFIIIA and/or TFIIIC are required to direct TFIIIB binding upstream of the transcriptional start site. Once bound, TFIIIB, as the only essential initiation factor, can recruit pol III for multiple rounds of transcription. TFIIIB is comprised of three subunits, a 70 kDa polypeptide (TFIIIB{dollar}\sb{lcub}70{rcub}),{dollar} a 90 kDa polypeptide (TFIIIB{dollar}\sb{lcub}90{rcub}),{dollar} and the TATA-binding protein (TBP). Employing a genetic strategy, I cloned PCF4, the gene encoding TFIIIB{dollar}\sb{lcub}70{rcub}.{dollar} TFIIIB{dollar}\sb{lcub}70{rcub}{dollar} is homologous to TFIIB, a pol II transcription factor that binds to TBP and mediates the recruitment of pol II. Similarly, TFIIIB{dollar}\sb{lcub}70{rcub}{dollar} interacts directly with TBP, TFIIIC{dollar}\sb{lcub}131{rcub}{dollar} and a unique pol III subunit. Therefore, TFIIIB{dollar}\sb{lcub}70{rcub}{dollar} can be thought of as a "polymerase specificity factor" by its ability to mediate interactions among the universal transcription factor, TBP and pol III-specific components.;Quantitative DNase 1 "footprint" titrations of yeast TBP to (i) the high affinity TATA box of the adenovirus major late promoter (AdMLP TATA), (ii) the lower affinity yeast U6 sRNA TATA box (yU6 TATA), (iii) the AdMLP TATA sequence placed in the yU6 context (yU6 hybrid TATA) and (iv) a low affinity nonconsensus TATA box were conducted in the presence and absence of pure recombinant TFIIIB{dollar}\sb{lcub}70{rcub}.{dollar} TFIIIB{dollar}\sb{lcub}70{rcub}{dollar} cooperatively interacts with TBP, increasing the equilibrium association constants of TBP for these sites 3, 17, 5 and 12-fold respectively. The data suggest that TATA flanking sequences play a role in TBP binding as well as in TBP-TFIIIB{dollar}\sb{lcub}70{rcub}{dollar} cooperativity. The observed cooperativity on the AdMLP TATA was found to be salt dependent with complete loss of cooperativity at low salt. The two lower affinity TATAs (yU6 TATA and nonconsensus TATA) display higher degrees of cooperativity than the higher affinity AdMLP and yU6 hybrid TATAs. Since most pol III genes contain low affinity sites for TBP binding, the cooperative binding of TFIIIB{dollar}\sb{lcub}70{rcub}{dollar} and TBP to DNA may represent an important driving force in the assembly of pol III-specific transcription complexes.