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Nanopore molecular trajectories of a eukaryotic reverse transcriptase reveal a long-range RNA structure sensing mechanism

Eukaryotic reverse transcriptases (RTs) can have essential or deleterious roles in normal human physiology and disease. Compared to well-studied helicases, it remains unclear how RTs overcome the ubiquitous RNA structural barriers during reverse transcription. Herein, we describe the development of...

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Autores principales: Shaw, Alan, Craig, Jonathan M., Amiri, Hossein, Kim, Jeonghoon, Upton, Heather E., Pimentel, Sydney C., Huang, Jesse R., Marqusee, Susan, Collins, Kathleen, Gundlach, Jens H., Bustamante, Carlos J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10104057/
https://www.ncbi.nlm.nih.gov/pubmed/37066208
http://dx.doi.org/10.1101/2023.04.05.535757
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author Shaw, Alan
Craig, Jonathan M.
Amiri, Hossein
Kim, Jeonghoon
Upton, Heather E.
Pimentel, Sydney C.
Huang, Jesse R.
Marqusee, Susan
Collins, Kathleen
Gundlach, Jens H.
Bustamante, Carlos J.
author_facet Shaw, Alan
Craig, Jonathan M.
Amiri, Hossein
Kim, Jeonghoon
Upton, Heather E.
Pimentel, Sydney C.
Huang, Jesse R.
Marqusee, Susan
Collins, Kathleen
Gundlach, Jens H.
Bustamante, Carlos J.
author_sort Shaw, Alan
collection PubMed
description Eukaryotic reverse transcriptases (RTs) can have essential or deleterious roles in normal human physiology and disease. Compared to well-studied helicases, it remains unclear how RTs overcome the ubiquitous RNA structural barriers during reverse transcription. Herein, we describe the development of a Mycobacterium smegmatis porin A (MspA) nanopore technique to sequence RNA to quantify the single-molecule kinetics of an RT from Bombyx mori with single-nucleotide resolution. By establishing a quadromer map that correlates RNA sequence and MspA ion current, we were able to quantify the RT’s dwell time at every single nucleotide step along its RNA template. By challenging the enzyme with various RNA structures, we found that during cDNA synthesis the RT can sense and actively destabilize RNA structures 11–12 nt downstream of its front boundary. The ability to sequence single molecules of RNA with nanopores paves the way to investigate the single-nucleotide activity of other processive RNA translocases.
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spelling pubmed-101040572023-04-15 Nanopore molecular trajectories of a eukaryotic reverse transcriptase reveal a long-range RNA structure sensing mechanism Shaw, Alan Craig, Jonathan M. Amiri, Hossein Kim, Jeonghoon Upton, Heather E. Pimentel, Sydney C. Huang, Jesse R. Marqusee, Susan Collins, Kathleen Gundlach, Jens H. Bustamante, Carlos J. bioRxiv Article Eukaryotic reverse transcriptases (RTs) can have essential or deleterious roles in normal human physiology and disease. Compared to well-studied helicases, it remains unclear how RTs overcome the ubiquitous RNA structural barriers during reverse transcription. Herein, we describe the development of a Mycobacterium smegmatis porin A (MspA) nanopore technique to sequence RNA to quantify the single-molecule kinetics of an RT from Bombyx mori with single-nucleotide resolution. By establishing a quadromer map that correlates RNA sequence and MspA ion current, we were able to quantify the RT’s dwell time at every single nucleotide step along its RNA template. By challenging the enzyme with various RNA structures, we found that during cDNA synthesis the RT can sense and actively destabilize RNA structures 11–12 nt downstream of its front boundary. The ability to sequence single molecules of RNA with nanopores paves the way to investigate the single-nucleotide activity of other processive RNA translocases. Cold Spring Harbor Laboratory 2023-11-01 /pmc/articles/PMC10104057/ /pubmed/37066208 http://dx.doi.org/10.1101/2023.04.05.535757 Text en https://creativecommons.org/licenses/by-nc-nd/4.0/This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (https://creativecommons.org/licenses/by-nc-nd/4.0/) , which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
spellingShingle Article
Shaw, Alan
Craig, Jonathan M.
Amiri, Hossein
Kim, Jeonghoon
Upton, Heather E.
Pimentel, Sydney C.
Huang, Jesse R.
Marqusee, Susan
Collins, Kathleen
Gundlach, Jens H.
Bustamante, Carlos J.
Nanopore molecular trajectories of a eukaryotic reverse transcriptase reveal a long-range RNA structure sensing mechanism
title Nanopore molecular trajectories of a eukaryotic reverse transcriptase reveal a long-range RNA structure sensing mechanism
title_full Nanopore molecular trajectories of a eukaryotic reverse transcriptase reveal a long-range RNA structure sensing mechanism
title_fullStr Nanopore molecular trajectories of a eukaryotic reverse transcriptase reveal a long-range RNA structure sensing mechanism
title_full_unstemmed Nanopore molecular trajectories of a eukaryotic reverse transcriptase reveal a long-range RNA structure sensing mechanism
title_short Nanopore molecular trajectories of a eukaryotic reverse transcriptase reveal a long-range RNA structure sensing mechanism
title_sort nanopore molecular trajectories of a eukaryotic reverse transcriptase reveal a long-range rna structure sensing mechanism
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10104057/
https://www.ncbi.nlm.nih.gov/pubmed/37066208
http://dx.doi.org/10.1101/2023.04.05.535757
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