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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...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Cold Spring Harbor Laboratory
2023
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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. |
format | Online Article Text |
id | pubmed-10104057 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Cold Spring Harbor Laboratory |
record_format | MEDLINE/PubMed |
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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