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...

Descripción completa

Detalles Bibliográficos
Autores principales: Zhao, Chen, Liu, Fei, Pyle, Anna Marie
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