title: The G+C-rich discriminator region of the tyrT promoter antagonises the formation of stable preinitiation complexes.
abstract: RNA polymerase forms a highly stable preinitiation complex at many prokaryotic promoters in the absence of ribonucleotides. These are often characterised by the longevity of the DNA strand-separated (open) complex in the presence of heparin. In contrast, such complexes are notoriously unstable at the promoters for rRNA and tRNA under similar conditions. The high G+C content within the DNA-melting region of these promoters has been implicated in this seemingly anomalous behaviour. Here, we used rapid-pulse UV laser photo-footprinting to monitor the transient structural intermediates formed at the Escherichia coli tyrT promoter. Promoter derivatives with A+T for G/C base substitutions within the G+C-rich discriminator region (-7 to -1) augmented the stability of complexes on both linear fragments and supercoiled plasmid DNA. Analysis of the lifetime of the preinitiation complexes as a function of the discriminator sequence reveals a direct relationship between the A+T content of the DNA-melting region and the stability of the ensuing complex. Our results are consistent with the premise that a G/C block to DNA-untwisting and/or DNA-melting operates to prevent the formation of the stable isomers that are implicated in most other transcription initiation pathways.
authors.affiliation: Unité de Physicochimie des Macromolécules Biologiques, Institut Pasteur, CNRS URA 1773, Paris, 75724, France.
authors.forename: I K
authors.initials: IK
authors.lastname: Pemberton
authors.forename: G
authors.initials: G
authors.lastname: Muskhelishvili
authors.forename: A A
authors.initials: AA
authors.lastname: Travers
authors.forename: M
authors.initials: M
authors.lastname: Buckle
mesh_terms: D001482:Base Composition / Q000235:genetics*; D020029:Base Pairing / Q000235:genetics; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004278:DNA, Superhelical / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D006207:Half-Life; D007700:Kinetics; D007834:Lasers; D009154:Mutation / Q000235:genetics; D009691:Nucleic Acid Denaturation / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D012365:RNA, Transfer, Tyr / Q000235:genetics*; D013816:Thermodynamics; D014158:Transcription, Genetic / Q000235:genetics*
chemical_list: D004269:DNA, Bacterial; D004278:DNA, Superhelical; D012365:RNA, Transfer, Tyr; D012321:DNA-Directed RNA Polymerases