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Site-specific fluorescence dynamics in an RNA ‘thermometer’ reveals the role of ribosome binding in its temperature-sensitive switch function

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 replacin...

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Autores principales: Narayan, Satya, Kombrabail, Mamta H., Das, Sudipta, Singh, Himanshu, Chary, Kandala V. R., Rao, Basuthkar J., Krishnamoorthy, Guruswamy
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2015
Materias:
RNA
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4288164/
https://www.ncbi.nlm.nih.gov/pubmed/25477380
http://dx.doi.org/10.1093/nar/gku1264
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author Narayan, Satya
Kombrabail, Mamta H.
Das, Sudipta
Singh, Himanshu
Chary, Kandala V. R.
Rao, Basuthkar J.
Krishnamoorthy, Guruswamy
author_facet Narayan, Satya
Kombrabail, Mamta H.
Das, Sudipta
Singh, Himanshu
Chary, Kandala V. R.
Rao, Basuthkar J.
Krishnamoorthy, Guruswamy
author_sort Narayan, Satya
collection PubMed
description 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.
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spelling pubmed-42881642015-02-19 Site-specific fluorescence dynamics in an RNA ‘thermometer’ reveals the role of ribosome binding in its temperature-sensitive switch function Narayan, Satya Kombrabail, Mamta H. Das, Sudipta Singh, Himanshu Chary, Kandala V. R. Rao, Basuthkar J. Krishnamoorthy, Guruswamy Nucleic Acids Res RNA 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. Oxford University Press 2015-01-09 2014-12-03 /pmc/articles/PMC4288164/ /pubmed/25477380 http://dx.doi.org/10.1093/nar/gku1264 Text en © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle RNA
Narayan, Satya
Kombrabail, Mamta H.
Das, Sudipta
Singh, Himanshu
Chary, Kandala V. R.
Rao, Basuthkar J.
Krishnamoorthy, Guruswamy
Site-specific fluorescence dynamics in an RNA ‘thermometer’ reveals the role of ribosome binding in its temperature-sensitive switch function
title Site-specific fluorescence dynamics in an RNA ‘thermometer’ reveals the role of ribosome binding in its temperature-sensitive switch function
title_full Site-specific fluorescence dynamics in an RNA ‘thermometer’ reveals the role of ribosome binding in its temperature-sensitive switch function
title_fullStr Site-specific fluorescence dynamics in an RNA ‘thermometer’ reveals the role of ribosome binding in its temperature-sensitive switch function
title_full_unstemmed Site-specific fluorescence dynamics in an RNA ‘thermometer’ reveals the role of ribosome binding in its temperature-sensitive switch function
title_short Site-specific fluorescence dynamics in an RNA ‘thermometer’ reveals the role of ribosome binding in its temperature-sensitive switch function
title_sort site-specific fluorescence dynamics in an rna ‘thermometer’ reveals the role of ribosome binding in its temperature-sensitive switch function
topic RNA
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4288164/
https://www.ncbi.nlm.nih.gov/pubmed/25477380
http://dx.doi.org/10.1093/nar/gku1264
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