title: TFB1 or TFB2 is sufficient for Thermococcus kodakaraensis viability and for basal transcription in vitro.
abstract: Archaeal RNA polymerases (RNAPs) are most similar to eukaryotic RNAP II (Pol II) but require the support of only two archaeal general transcription factors, TBP (TATA-box binding protein) and TFB (archaeal homologue of the eukaryotic general transcription factor TFIIB) to initiate basal transcription. However, many archaeal genomes encode more than one TFB and/or TBP leading to the hypothesis that different TFB/TBP combinations may be employed to direct initiation from different promoters in Archaea. As a first test of this hypothesis, we have determined the ability of RNAP purified from Thermococcus kodakaraensis (T.k.) to initiate transcription from a variety of T.k. promoters in vitro when provided with T.k. TBP and either TFB1 or TFB2, the two TFBs encoded in the T.k. genome. With every promoter active in vitro, transcription initiation occurred with either TFB1 or TFB2 although the optimum salt concentration for initiation was generally higher for TFB2 (approximately 250 mM K(+)) than for TFB1 (approximately 200 mM K(+)). Consistent with this functional redundancy in vitro, T.k. strains have been constructed with the TFB1- (tfb1; TK1280) or TFB2- (tfb2; TK2287) encoding gene deleted. These mutants exhibit no detectable growth defects under laboratory conditions. Domain swapping between TFB1 and TFB2 has identified a central region that contributes to the salt sensitivity of TFB activity, and deleting residues predicted to form the tip of the B-finger region of TFB2 had no detectable effects on promoter recognition or transcription initiation but did eliminate the production of very short (< or =5 nt) abortive transcripts.
authors.affiliation: Department of Microbiology, Ohio State University, Columbus, OH 43210, USA.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Santangelo
authors.forename: L'ubomíra
authors.initials: L
authors.lastname: Cubonová
authors.forename: Cindy L
authors.initials: CL
authors.lastname: James
authors.forename: John N
authors.initials: JN
authors.lastname: Reeve
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics / Q000502:physiology*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019848:Gene Expression Regulation, Archaeal; D050296:Microbial Viability; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D019712:Thermococcus / Q000235:genetics / Q000502:physiology*; D015533:Transcriptional Activation
chemical_list: D019843:Archaeal Proteins; D012321:DNA-Directed RNA Polymerases