INTERACTIONS BETWEEN YEAST TFIIIB COMPONENTS

Yeast transcription factor TFIIIB is a multicomponent factor comprised of the TATA-binding protein TBP and of associated factors TFIIIB70 an d B''. Epitope-tagged or histidine-tagged TFIIIB70 could be quantitati vely removed from TFIIIB by affinity chromatography. TBP and B'' (appa rent mass 160-200 kDa) could be easily separated by gel filtration or ion-exchange chromatography. While only weak interactions were detecte d between TBP and B'', direct binding of [S-35]-labeled TBP to membran e-bound TFIIIB70 could be demonstrated in absence of DNA. On tRNA gene s, there was no basal level of transcription in the complete absence o f TBP. The two characterized TFIIIB components (recombinant rTFIIIB70 and rTBP) and a fraction cochromatographing with B'' activity were fou nd to be required for TFIIIC-independent transcription of the TATA-con taining Us RNA gene in vitro. Therefore, beside the TFIIIC-dependent a ssembly process, each TFIIIB component must have an essential role in DNA binding or RNA polymerase recruitment.