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

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.

Biochemical properties of bacterial reverse transcriptase-related (rvt) gene products: multimerization, protein priming, and nucleotide preference

“…8B). Overall, our results are consistent with the view that the catalytic center is in the closed conformation provided by the greatly extended loop 2a (Zhao, et al 2017) and is largely inaccessible to external substrates, but is capable of adding dNTP onto suitable hydroxyl groups in the vicinity of the catalytic center (either short oligos captured during enzyme preparation, or accessible hydroxyls in the protein itself).…”

Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

“…Unlike retroviral RTs, which evolved to help retroviruses evade host defenses by introducing and propagating mutational variations (Hu and Hughes 2012), group II intron RTs evolved to function in retrohoming, a retrotranposition mechanism that requires faithful synthesis of a full-length cDNA of a long, highly structured group II intron RNA (Lambowitz and Belfort 2015). Their beneficial properties for RNA-seq include high fidelity, processivity, and strand displacement activity, along with a proficient templateswitching activity that is minimally dependent upon base pairing and enables the seamless attachment of RNA-seq adapters to target RNAs without RNA tailing or ligation (Mohr et al 2013;Zhao et al 2018). Thermostable group II intron RTs (TGIRTs) from bacterial thermophiles combine these beneficial properties with the ability to function at high temperatures (60-65 ο C), which help melt out stable RNA secondary structures that can impede reverse transcription (Mohr et al 2013).…”

Beyond mass spectrometry, the next step in proteomics

“…Another shortcoming is that reverse transcription to DNA is required. Most commercial reverse transcriptases are of limited processivity, which precludes synthesis of especially long or complex structured mRNA (93). However, recent developments in nanopore sequencing have allowed for direct sequencing of RNA molecules (94)(95)(96).…”