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Coupling between conformational dynamics and catalytic function at the active site of the lead-dependent ribozyme

In common with other self-cleaving RNAs, the lead-dependent ribozyme (leadzyme) undergoes dynamic fluctuations to a chemically activated conformation. We explored the connection between conformational dynamics and self-cleavage function in the leadzyme using a combination of NMR spin-relaxation anal...

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Autores principales: White, Neil A., Sumita, Minako, Marquez, Victor E., Hoogstraten, Charles G.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6191710/
https://www.ncbi.nlm.nih.gov/pubmed/30111534
http://dx.doi.org/10.1261/rna.067579.118
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author White, Neil A.
Sumita, Minako
Marquez, Victor E.
Hoogstraten, Charles G.
author_facet White, Neil A.
Sumita, Minako
Marquez, Victor E.
Hoogstraten, Charles G.
author_sort White, Neil A.
collection PubMed
description In common with other self-cleaving RNAs, the lead-dependent ribozyme (leadzyme) undergoes dynamic fluctuations to a chemically activated conformation. We explored the connection between conformational dynamics and self-cleavage function in the leadzyme using a combination of NMR spin-relaxation analysis of ribose groups and conformational restriction via chemical modification. The functional studies were performed with a North-methanocarbacytidine modification that prevents fluctuations to C2′-endo conformations while maintaining an intact 2′-hydroxyl nucleophile. Spin-relaxation data demonstrate that the active-site Cyt-6 undergoes conformational exchange attributed to sampling of a minor C2′-endo state with an exchange lifetime on the order of microseconds to tens of microseconds. A conformationally restricted species in which the fluctuations to the minor species are interrupted shows a drastic decrease in self-cleavage activity. Taken together, these data indicate that dynamic sampling of a minor species at the active site of this ribozyme, and likely of related naturally occurring motifs, is strongly coupled to catalytic function. The combination of NMR dynamics analysis with functional probing via conformational restriction is a general methodology for dissecting dynamics-function relationships in RNA.
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spelling pubmed-61917102019-11-01 Coupling between conformational dynamics and catalytic function at the active site of the lead-dependent ribozyme White, Neil A. Sumita, Minako Marquez, Victor E. Hoogstraten, Charles G. RNA Article In common with other self-cleaving RNAs, the lead-dependent ribozyme (leadzyme) undergoes dynamic fluctuations to a chemically activated conformation. We explored the connection between conformational dynamics and self-cleavage function in the leadzyme using a combination of NMR spin-relaxation analysis of ribose groups and conformational restriction via chemical modification. The functional studies were performed with a North-methanocarbacytidine modification that prevents fluctuations to C2′-endo conformations while maintaining an intact 2′-hydroxyl nucleophile. Spin-relaxation data demonstrate that the active-site Cyt-6 undergoes conformational exchange attributed to sampling of a minor C2′-endo state with an exchange lifetime on the order of microseconds to tens of microseconds. A conformationally restricted species in which the fluctuations to the minor species are interrupted shows a drastic decrease in self-cleavage activity. Taken together, these data indicate that dynamic sampling of a minor species at the active site of this ribozyme, and likely of related naturally occurring motifs, is strongly coupled to catalytic function. The combination of NMR dynamics analysis with functional probing via conformational restriction is a general methodology for dissecting dynamics-function relationships in RNA. Cold Spring Harbor Laboratory Press 2018-11 /pmc/articles/PMC6191710/ /pubmed/30111534 http://dx.doi.org/10.1261/rna.067579.118 Text en © 2018 White et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society http://creativecommons.org/licenses/by-nc/4.0/ This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
spellingShingle Article
White, Neil A.
Sumita, Minako
Marquez, Victor E.
Hoogstraten, Charles G.
Coupling between conformational dynamics and catalytic function at the active site of the lead-dependent ribozyme
title Coupling between conformational dynamics and catalytic function at the active site of the lead-dependent ribozyme
title_full Coupling between conformational dynamics and catalytic function at the active site of the lead-dependent ribozyme
title_fullStr Coupling between conformational dynamics and catalytic function at the active site of the lead-dependent ribozyme
title_full_unstemmed Coupling between conformational dynamics and catalytic function at the active site of the lead-dependent ribozyme
title_short Coupling between conformational dynamics and catalytic function at the active site of the lead-dependent ribozyme
title_sort coupling between conformational dynamics and catalytic function at the active site of the lead-dependent ribozyme
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6191710/
https://www.ncbi.nlm.nih.gov/pubmed/30111534
http://dx.doi.org/10.1261/rna.067579.118
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