Conserved functional domains of the RNA polymerase III general transcription factor BRF.
In Saccharomyces cerevisiae, two components of the RNA polymerase III (Pol III) general transcription factor TFIIIB are the TATA-binding protein (TBP) and the B-related factor (BRF), so called because its amino-terminal half is homologous to the Pol II transcription factor IIB (TFIIB). We have cloned BRF genes from the yeasts Kluyveromyces lactis and Candida albicans. Despite the large evolutionary distance between these species and S. cerevisiae, the BRF proteins are conserved highly. Although the homology is most pronounced in the amino-terminal half, conserved regions also exist in the carboxy-terminal half that is unique to BRF. By assaying for interactions between BRF and other Pol III transcription factors, we show that it is able to bind to the 135-kD subunit of TFIIIC and also to TBP. Surprisingly, in addition to binding the TFIIB-homologous amino-terminal portion of BRF, TBP also interacts strongly with the carboxy-terminal half. Deleting two conserved regions in the BRF carboxy-terminal region abrogates this interaction. Furthermore, TBP mutations that selectively inhibit Pol III transcription in vivo impair interactions between TBP and the BRF carboxy-terminal domain. Finally, we demonstrate that BRF but not TFIIB binds the Pol III subunit C34 and we define a region of C34 necessary for this interaction. These observations provide insights into the roles performed by BRF in Pol III transcription complex assembly.
Khoo
B
B
Wellcome/CRC Institute, Cambridge University, UK.
Brophy
B
B
Jackson
S P
SP
D000595:Amino Acid Sequence; D001665:Binding Sites; D005075:Biological Evolution; D002176:Candida albicans / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers; D004926:Escherichia coli; D005800:Genes, Fungal; D007716:Kluyveromyces / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012320:RNA Polymerase III / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D035361:Transcription Factor TFIIIB; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
C090956:BRF1 protein, S cerevisiae; D017931:DNA Primers; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D035361:Transcription Factor TFIIIB; D014157:Transcription Factors; D012320:RNA Polymerase III