title: Competitive folding of anti-terminator/terminator hairpins monitored by single molecule FRET.
abstract: The control of transcription termination by RNA-binding proteins that modulate RNA-structures is an important regulatory mechanism in bacteria. LicT and SacY from Bacillus subtilis prevent the premature arrest of transcription by binding to an anti-terminator RNA hairpin that overlaps an intrinsic terminator located in the 5'-mRNA leader region of the gene to be regulated. In order to investigate the molecular determinants of this anti-termination/termination balance, we have developed a fluorescence-based nucleic acids system that mimics the competition between the LicT or SacY anti-terminator targets and the overlapping terminators. Using Förster Resonance Energy Transfer on single diffusing RNA hairpins, we could monitor directly their opening or closing state, and thus investigate the effects on this equilibrium of the binding of anti-termination proteins or terminator-mimicking oligonucleotides. We show that the anti-terminator hairpins adopt spontaneously a closed structure and that their structural dynamics is mainly governed by the length of their basal stem. The induced stability of the anti-terminator hairpins determines both the affinity and specificity of the anti-termination protein binding. Finally, we show that stabilization of the anti-terminator hairpin, by an extended basal stem or anti-termination protein binding can efficiently counteract the competing effect of the terminator-mimic.
authors.affiliation: CNRS UMR5048, Centre de Biochimie Structurale, 29 rue de Navacelles, 34090 Montpellier, France; INSERM U1054, 34090 Montpellier, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Clerte
authors.forename: Nathalie
authors.initials: N
authors.lastname: Declerck
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Margeat
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics; D001426:Bacterial Proteins / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D059370:RNA Folding; D012329:RNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D055672:Static Electricity; D013728:Terminator Regions, Genetic; D014157:Transcription Factors / Q000378:metabolism*; D061806:Transcription Termination, Genetic
chemical_list: D001426:Bacterial Proteins; C098955:LicT protein, Bacteria; D012329:RNA, Bacterial; D016601:RNA-Binding Proteins; C076894:SacY protein, Bacillus subtilis; D014157:Transcription Factors; C064872:antiterminator proteins, Bacteria