title: Aptamers to the sigma factor mimic promoter recognition and inhibit transcription initiation by bacterial RNA polymerase.
abstract: Promoter recognition by bacterial RNA polymerase (RNAP) is a multi-step process involving multiple protein-DNA interactions and several structural and kinetic intermediates which remain only partially characterized. We used single-stranded DNA aptamers containing specific promoter motifs to probe the interactions of the Thermus aquaticus RNAP σ(A) subunit with the -10 promoter element in the absence of other parts of the promoter complex. The aptamer binding decreased intrinsic fluorescence of the σ subunit, likely as a result of interactions between the -10 element and conserved tryptophan residues of the σ DNA-binding region 2. By monitoring these changes, we demonstrated that DNA binding proceeds through a single rate-limiting step resulting in formation of very stable complexes. Deletion of the N-terminal domain of the σ(A) subunit increased the rate of aptamer binding while replacement of this domain with an unrelated N-terminal region 1.1 from the Escherichia coli σ(70) subunit restored the original kinetics of σ-aptamer interactions. The results demonstrate that the key step in promoter recognition can be modelled in a simple σ-aptamer system and reveal that highly divergent N-terminal domains similarly modulate the DNA-binding properties of the σ subunit. The aptamers efficiently suppressed promoter-dependent transcription initiation by the holoenzyme of RNA polymerase, suggesting that they may be used for development of novel transcription inhibitors.
authors.affiliation: Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia.
authors.forename: Nataliya
authors.initials: N
authors.lastname: Miropolskaya
authors.affiliation: Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia. Electronic address: akulb@img.ras.ru.
authors.forename: Andrey
authors.initials: A
authors.lastname: Kulbachinskiy
mesh_terms: D052157:Aptamers, Nucleotide / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012808:Sigma Factor / Q000235:genetics*; D015533:Transcriptional Activation / Q000235:genetics*
chemical_list: D052157:Aptamers, Nucleotide; D001426:Bacterial Proteins; D012808:Sigma Factor; D012321:DNA-Directed RNA Polymerases
keywords: Aptamer; Promoter; RNA polymerase; Transcription initiation; σ subunit