title: Nhp6 is a transcriptional initiation fidelity factor for RNA polymerase III transcription in vitro and in vivo.
abstract: The binding of the RNA polymerase III (pol III) transcription factor TFIIIC to the box A intragenic promoter element of tRNA genes specifies the placement of TFIIIB on upstream-lying DNA. In turn, TFIIIB recruits pol III to the promoter and specifies transcription initiating 17-19 base pairs upstream of box A. The resolution of the pol III transcription apparatus into recombinant TFIIIB, highly purified TFIIIC, and pol III is accompanied by a loss of precision in specifying where transcription initiation occurs due to heterogeneous placement of TFIIIB. In this paper we show that Nhp6a, an abundant high mobility group B (HMGB) family, non-sequence-specific DNA-binding protein in Saccharomyces cerevisiae restores transcriptional initiation fidelity to this highly purified in vitro system. Restoration of initiation fidelity requires the presence of Nhp6a prior to TFIIIB-DNA complex formation. Chemical nuclease footprinting of TFIIIC- and TFIIIB-TFIIIC-DNA complexes reveals that Nhp6a markedly alters the TFIIIC footprint over box A and reduces the size of the TFIIIB footprint on upstream DNA sequence. Analyses of unprocessed tRNAs from yeast lacking Nhp6a and its closely related paralogue Nhp6b demonstrate that Nhp6 is required for transcriptional initiation fidelity of some but not all tRNA genes, in vivo.
authors.affiliation: Division of Biological Sciences and Center for Molecular Genetics, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0634, USA. gak@biomail.ucsd.edu
authors.forename: George A
authors.initials: GA
authors.lastname: Kassavetis
authors.forename: David F
authors.initials: DF
authors.lastname: Steiner
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D015966:Gene Expression Regulation, Fungal; D024001:HMGN Proteins; D066298:In Vitro Techniques; D007438:Introns; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism / Q000502:physiology*; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012320:RNA Polymerase III / Q000737:chemistry*; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism / Q000502:physiology*; D013696:Temperature; D035361:Transcription Factor TFIIIB / Q000737:chemistry*; D019474:Transcription Factors, TFIII / Q000737:chemistry*; D014158:Transcription, Genetic; D000098832:Transcription Factor TFIIIC
chemical_list: D004268:DNA-Binding Proteins; D024001:HMGN Proteins; C086471:NHP6A protein, S cerevisiae; C086472:NHP6B protein, S cerevisiae; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D035361:Transcription Factor TFIIIB; D019474:Transcription Factors, TFIII; D000098832:Transcription Factor TFIIIC; D004247:DNA; D012343:RNA, Transfer; D012320:RNA Polymerase III