An immunoaffinity purified Schizosaccharomyces pombe TBP-containing complex directs correct initiation of the S.pombe rRNA gene promoter.
The multi-protein complex SL1, containing TBP, which is essential for RNA polymerase I catalyzed transcription, has been analyzed in fission yeast. It was immunopurified based on association of component subunits with epitope-tagged TBP. To enable this analysis, a strain of Schizosaccharomyces pombe was created where the only functional TBP coding sequences were those of FLAG-TBP. RNA polymerase I transcription components were fractionated from this strain and the TBP-associated polypeptides were subsequently immunopurified together with the epitope- tagged TBP. An assessment of the activity of this candidate SL1 complex was undertaken cross-species. This fission yeast TBP-containing complex displays two activities in redirecting transcriptional initiation of an S. pombe rDNA gene promoter cross-species in Saccharomyces cerevisiae transcription reactions: it both blocks an incorrect transcriptional start site at +7 and directs initiation at the correct site for S. pombe rRNA synthesis. This complex is essential for accurate initiation of the S.pombe rRNA gene: rRNA synthesis is reconstituted when this S.pombe TBP-containing complex is combined with a S.pombe fraction immunodepleted of TBP.
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Department of Chemistry, New York University, New York, NY 10003, USA.
Guo
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D001483:Base Sequence; D002846:Chromatography, Affinity; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular; D004275:DNA, Ribosomal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010455:Peptides; D035463:Pol1 Transcription Initiation Complex Proteins; D011401:Promoter Regions, Genetic; D012318:RNA Polymerase I / Q000378:metabolism; D012331:RNA, Fungal / Q000096:biosynthesis / Q000235:genetics; D012335:RNA, Ribosomal / Q000096:biosynthesis / Q000235:genetics*; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism*; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*; D019473:Transcription Factors, TFII / Q000378:metabolism; D014158:Transcription, Genetic
D004275:DNA, Ribosomal; D004268:DNA-Binding Proteins; D009842:Oligopeptides; D010455:Peptides; D035463:Pol1 Transcription Initiation Complex Proteins; D012331:RNA, Fungal; D012335:RNA, Ribosomal; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D019473:Transcription Factors, TFII; C063660:transcription initiation factor TIF-IB; C085706:FLAG peptide; D012318:RNA Polymerase I