title: Binding and release of the 6S transcriptional control RNA.
abstract: 6S RNA is an important noncoding RNA that regulates eubacterial transcription. In Escherichia coli this RNA binds to the sigma(70) RNA polymerase holoenzyme and is released by the synthesis of a short product RNA. In order to determine how binding and release are controlled by the 6S RNA sequence, we used in vitro selection to screen a high diversity library containing approximately 4 x 10(12) sequences for functional 6S RNA variants. Residues critical for binding were found to be located in a "-35" region upstream of the 6S RNA transcription bubble mimic structure. Mutating these phylogenetically conserved residues invariably led to decreases in binding and removing them abolished binding, implicating these nucleotides in a biologically important interaction with the Esigma(70) complex. Interestingly, mutation of phylogenetically conserved "-10" residues that were also upstream of the site of pRNA synthesis was found to influence 6S RNA release rates in addition to modulating -35 binding. These results indicate how 6S RNA -35 binding to sigma(70) RNA polymerase holoenzyme can regulate expression from "strong" and "weak" -35 DNA promoters and suggest that 6S RNA release rates have been fine tuned over evolutionary time so as to correctly regulate cellular levels of transcription.
authors.affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.
authors.forename: Lindsay
authors.initials: L
authors.lastname: Shephard
authors.forename: Neil
authors.initials: N
authors.lastname: Dobson
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Unrau
mesh_terms: D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D016384:Consensus Sequence; D017124:Conserved Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D048168:Escherichia coli K12 / Q000235:genetics* / Q000378:metabolism*; D019143:Evolution, Molecular; D015723:Gene Library; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D010802:Phylogeny; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D022661:RNA, Untranslated; D017384:Sequence Deletion; D012808:Sigma Factor / Q000378:metabolism
chemical_list: C411065:6S RNA; D012329:RNA, Bacterial; D022661:RNA, Untranslated; D012808:Sigma Factor; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases