TATA-BOX DNA-BINDING ACTIVITY AND SUBUNIT COMPOSITION OF RNA-POLYMERASE-III TRANSCRIPTION FACTOR-IIIB FROM XENOPUS-LAEVIS

The RNA polymerase III transcription initiation factor TFIIIB contains the TATA-box-binding protein (TBP) and polymerase III-specific TBP-as sociated factors (TAFs). Previous studies have shown that DNA oligonuc leotides containing the consensus TATA-box sequence inhibit polymerase III transcription, implying that the DNA binding domain of TBP is exp osed in TFIIIB. We have investigated the TATA-box DNA binding activity of Xenopus TFIIIB, using transcription inhibition assays and a gel mo bility shift assay. Gel shift competition assays with mutant and nonsp ecific DNAs demonstrate the specificity of the TFIIIB-TATA box DNA com plex. The apparent dissociation constant for this protein-DNA interact ion is similar to 0.4 nM, similar to the affinity of yeast TBP for the same sequence. TFIIIB transcriptional activity and TATA-box binding a ctivity cofractionate during a series of four ion-exchange chromatogra phic steps, and reconstituted transcription reactions demonstrate that the TATA-box DNA-protein complex contains TFIIIB TAF activity. Polype ptides with apparent molecular masses of 75 and 92 kDa are associated with TBP in this complex. These polypeptides were renatured after elut ion from sodium dodecyl sulfate-gels and tested individually and in co mbination for TFIIIB TAF activity. Recombinant TBP along with protein fractions containing the 75- and 92-kDa polypeptides were sufficient t o reconstitute TFIIIB transcriptional activity and DNA binding activit y, suggesting that Xenopus TFIIIB is composed of TBP along with these polypeptides.