Cargando…
An ultraprocessive, accurate reverse transcriptase encoded by a metazoan group II intron
Group II introns and non-LTR retrotransposons encode a phylogenetically related family of highly processive reverse transcriptases (RTs) that are essential for mobility and persistence of these retroelements. Recent crystallographic studies on members of this RT family have revealed that they are st...
Autores principales: | , , |
---|---|
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/PMC5769746/ https://www.ncbi.nlm.nih.gov/pubmed/29109157 http://dx.doi.org/10.1261/rna.063479.117 |
_version_ | 1783292952346361856 |
---|---|
author | Zhao, Chen Liu, Fei Pyle, Anna Marie |
author_facet | Zhao, Chen Liu, Fei Pyle, Anna Marie |
author_sort | Zhao, Chen |
collection | PubMed |
description | Group II introns and non-LTR retrotransposons encode a phylogenetically related family of highly processive reverse transcriptases (RTs) that are essential for mobility and persistence of these retroelements. Recent crystallographic studies on members of this RT family have revealed that they are structurally distinct from the retroviral RTs that are typically used in biotechnology. However, quantitative, structure-guided analysis of processivity, efficiency, and accuracy of this alternate RT family has been lacking. Here, we characterize the processivity of a group II intron maturase RT from Eubacterium rectale (E.r.), for which high-resolution structural information is available. We find that the E.r. maturase RT (MarathonRT) efficiently copies transcripts at least 10 kb in length and displays superior intrinsic RT processivity compared to commercial enzymes such as Superscript IV (SSIV). The elevated processivity of MarathonRT is at least partly mediated by a loop structure in the finger subdomain that acts as a steric guard (the α-loop). Additionally, we find that a positively charged secondary RNA binding site on the surface of the RT diminishes the primer utilization efficiency of the enzyme, and that reengineering of this surface enhances capabilities of the MarathonRT. Finally, using single-molecule sequencing, we show that the error frequency of MarathonRT is comparable to that of other high-performance RTs, such as SSIV, which were tested in parallel. Our results provide a structural framework for understanding the enhanced processivity of retroelement RTs, and they demonstrate the potential for engineering a powerful new generation of RT tools for application in biotechnology and research. |
format | Online Article Text |
id | pubmed-5769746 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Cold Spring Harbor Laboratory Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-57697462018-02-01 An ultraprocessive, accurate reverse transcriptase encoded by a metazoan group II intron Zhao, Chen Liu, Fei Pyle, Anna Marie RNA Article Group II introns and non-LTR retrotransposons encode a phylogenetically related family of highly processive reverse transcriptases (RTs) that are essential for mobility and persistence of these retroelements. Recent crystallographic studies on members of this RT family have revealed that they are structurally distinct from the retroviral RTs that are typically used in biotechnology. However, quantitative, structure-guided analysis of processivity, efficiency, and accuracy of this alternate RT family has been lacking. Here, we characterize the processivity of a group II intron maturase RT from Eubacterium rectale (E.r.), for which high-resolution structural information is available. We find that the E.r. maturase RT (MarathonRT) efficiently copies transcripts at least 10 kb in length and displays superior intrinsic RT processivity compared to commercial enzymes such as Superscript IV (SSIV). The elevated processivity of MarathonRT is at least partly mediated by a loop structure in the finger subdomain that acts as a steric guard (the α-loop). Additionally, we find that a positively charged secondary RNA binding site on the surface of the RT diminishes the primer utilization efficiency of the enzyme, and that reengineering of this surface enhances capabilities of the MarathonRT. Finally, using single-molecule sequencing, we show that the error frequency of MarathonRT is comparable to that of other high-performance RTs, such as SSIV, which were tested in parallel. Our results provide a structural framework for understanding the enhanced processivity of retroelement RTs, and they demonstrate the potential for engineering a powerful new generation of RT tools for application in biotechnology and research. Cold Spring Harbor Laboratory Press 2018-02 /pmc/articles/PMC5769746/ /pubmed/29109157 http://dx.doi.org/10.1261/rna.063479.117 Text en © 2018 Zhao et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society http://creativecommons.org/licenses/by/4.0/ This article, published in RNA, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Zhao, Chen Liu, Fei Pyle, Anna Marie An ultraprocessive, accurate reverse transcriptase encoded by a metazoan group II intron |
title | An ultraprocessive, accurate reverse transcriptase encoded by a metazoan group II intron |
title_full | An ultraprocessive, accurate reverse transcriptase encoded by a metazoan group II intron |
title_fullStr | An ultraprocessive, accurate reverse transcriptase encoded by a metazoan group II intron |
title_full_unstemmed | An ultraprocessive, accurate reverse transcriptase encoded by a metazoan group II intron |
title_short | An ultraprocessive, accurate reverse transcriptase encoded by a metazoan group II intron |
title_sort | ultraprocessive, accurate reverse transcriptase encoded by a metazoan group ii intron |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5769746/ https://www.ncbi.nlm.nih.gov/pubmed/29109157 http://dx.doi.org/10.1261/rna.063479.117 |
work_keys_str_mv | AT zhaochen anultraprocessiveaccuratereversetranscriptaseencodedbyametazoangroupiiintron AT liufei anultraprocessiveaccuratereversetranscriptaseencodedbyametazoangroupiiintron AT pyleannamarie anultraprocessiveaccuratereversetranscriptaseencodedbyametazoangroupiiintron AT zhaochen ultraprocessiveaccuratereversetranscriptaseencodedbyametazoangroupiiintron AT liufei ultraprocessiveaccuratereversetranscriptaseencodedbyametazoangroupiiintron AT pyleannamarie ultraprocessiveaccuratereversetranscriptaseencodedbyametazoangroupiiintron |