Dissociable Rpb4-Rpb7 subassembly of rna polymerase II binds to single-strand nucleic acid and mediates a post-recruitment step in transcription initiation.
The Rpb4 and Rpb7 subunits of yeast RNA polymerase II form a heterodimeric complex essential for promoter-directed transcription initiation in a reconstituted system. Results of template competition experiments indicate that the Rpb4-Rpb7 complex is not required for stable recruitment of polymerase to active preinitiation complexes, suggesting that Rpb4-Rpb7 mediates an essential step subsequent to promoter binding. Sequence and structure-based alignments revealed a possible OB-fold single-strand nucleic acid-binding motif in Rpb7. Purified Rpb4-Rpb7 complex exhibited both single-strand DNA- and RNA-binding activities, and a small deletion in the putative OB-fold nucleic acid-binding surface of Rpb7 abolished binding activity without affecting the stability of the Rpb4-Rpb7 complex or its ability to associate with polymerase. The same mutation destroyed the transcription activity of the Rpb4-Rpb7 complex. A separate deletion elsewhere in the OB-fold motif of Rpb7 also blocked transcription but did not affect nucleic acid binding, suggesting that the OB-fold of Rpb7 mediates both DNA-protein and protein-protein interactions required for productive initiation.
Orlicky
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Banting and Best Department of Medical Research and Department of Medical Genetics and Microbiology, C. H. Best Institute, University of Toronto, Ontario M5G 1L6, Canada.
Tran
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Sayre
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Edwards
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AM
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D002460:Cell Line; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology; D017353:Gene Deletion; D007313:Insecta; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012319:RNA Polymerase II / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D016944:Sulfolobus / Q000201:enzymology; D014158:Transcription, Genetic
D004277:DNA, Single-Stranded; D029701:Saccharomyces cerevisiae Proteins; D012319:RNA Polymerase II; C423309:RPB4 protein, S cerevisiae; C470553:RPB7 protein, S cerevisiae