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

Chen Zhao,Fei Liu,Anna Marie Pyle

doi:10.1261/rna.063479.117

Chen Zhao, Fei Liu + Show 1 more

Open Access

https://doi.org/10.1261/rna.063479.117

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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.

Highlights

Long RNA molecules control numerous aspects of gene expression, such as mRNAs, regulatory RNAs, viral genomes, components of the machinery for translation, RNA processing, and many other processes (Mercer et al 2009; Kung et al 2013)
Efficient copying of a highly structured viral genome In order to assess the relative processivity and reactivity of MarathonRT on a large, biologically relevant template, we examined cDNA synthesis from the genome of hepatitis C virus (HCV)
To evaluate relative cDNA synthesis efficiency by the MarathonRT, we extended reverse-transcription from six different sites in the genome (Fig. 2A,C), resulting in cDNA fragments that range from 4.9 to 9.5 kb in length (Fig. 2C)

Summary

INTRODUCTION

Long RNA molecules control numerous aspects of gene expression, such as mRNAs, regulatory RNAs, viral genomes, components of the machinery for translation, RNA processing, and many other processes (Mercer et al 2009; Kung et al 2013). Many studies have noted the unusually high minal extension (RT0) and a specific set of insertions be- processivity of group II intron maturases (Fig. 1A; Mohr tween the seven conserved sequence motifs that are found et al 2013) and related non-LTR retrotransposon RTs in all RTs (RT1–7) A thermally stable group palm subdomains, includes the catalytic center, and mediates II intron maturase (known as TGIRT) has been successfully polymerase fidelity and processivity (Fig. 1A; Zimmerly et al used for cDNA library construction (Mohr et al 2013; 2001; Blocker et al 2005; Zhao and Pyle 2017). RT domain, we designed mutations that improve the properties of the RT, thereby demonstrating that this family of RTs can be further optimized and engineered to create a new generation of powerful enzyme tools for meeting the needs of cutting-edge RNA science

RESULTS

DISCUSSION

MATERIALS AND METHODS