title: Site-specific fluorescence dynamics in an RNA 'thermometer' reveals the role of ribosome binding in its temperature-sensitive switch function.
abstract: RNA thermometers control the translation of several heat shock and virulence genes by their temperature-sensitive structural transitions. Changes in the structure and dynamics of MiniROSE RNA, which regulates translation in the temperature range of 20-45°C, were studied by site specifically replacing seven adenine residues with the fluorescent analog, 2-aminopurine (2-AP), one at a time. Dynamic fluorescence observables of 2-AP-labeled RNAs were compared in their free versus ribosome-bound states for the first time. Noticeably, position dependence of fluorescence observables, which was prominent at 20°C, was persistent even at 45ºC, suggesting the persistence of structural integrity up to 45ºC. Interestingly, position-dependent dispersion of fluorescence lifetime and quenching constant at 45°C was ablated in ribosome-bound state, when compared to those at 20°C, underscoring loss of structural integrity at 45°C, in ribosome-bound RNA. Significant increase in the value of mean lifetime for 2-AP corresponding to Shine-Dalgarno sequences, when the temperature was raised from 20 to 45°C, to values seen in the presence of urea at 45°C was a strong indicator of melting of the 3D structure of MiniROSE RNA at 45°C, only when it was ribosome bound. Taken all together, we propose a model where we invoke that ribosome binding of the RNA thermometer critically regulates temperature sensing functions in MiniROSE RNA.
authors.affiliation: Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai 400005, India.
authors.forename: Satya
authors.initials: S
authors.lastname: Narayan
authors.affiliation: Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai 400005, India.
authors.forename: Mamta H
authors.initials: MH
authors.lastname: Kombrabail
authors.affiliation: Tata Institute of Fundamental Research, Center for Interdisciplinary Sciences, Hyderabad 500075, India.
authors.forename: Sudipta
authors.initials: S
authors.lastname: Das
authors.affiliation: Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai 400005, India.
authors.forename: Himanshu
authors.initials: H
authors.lastname: Singh
authors.affiliation: Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai 400005, India Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai 400005, India.
authors.forename: Kandala V R
authors.initials: KV
authors.lastname: Chary
authors.affiliation: Department of Chemistry, Indian Institute of Technology, Kanpur 208016, India.
authors.forename: Basuthkar J
authors.initials: BJ
authors.lastname: Rao
authors.affiliation: Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai 400005, India gk@tifr.res.in.
authors.forename: Guruswamy
authors.initials: G
authors.lastname: Krishnamoorthy
mesh_terms: D015075:2-Aminopurine / Q000737:chemistry; D005453:Fluorescence; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D058928:Riboswitch; D013696:Temperature
chemical_list: D058928:Riboswitch; D015075:2-Aminopurine; D012313:RNA