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RNA. 2018 Feb;24(2):183-195. doi: 10.1261/rna.063479.117. Epub 2017 Nov 6.

An ultraprocessive, accurate reverse transcriptase encoded by a metazoan group II intron.

Zhao C1, Liu F2,3, Pyle AM2,3,4.

Author information

1
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
2
Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA.
3
Howard Hughes Medical Institute, Chevy Chase, Maryland 20815, USA.
4
Department of Chemistry, Yale University, New Haven, Connecticut 06520, USA.

Abstract

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.

KEYWORDS:

RNA-seq; group II intron; intron-encoded protein; maturase; processivity; reverse transcriptase

PMID:
29109157
PMCID:
PMC5769746
DOI:
10.1261/rna.063479.117
[Indexed for MEDLINE]
Free PMC Article

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