Structure of a Thermostable Group II Intron Reverse Transcriptase with Template-Primer and Its Functional and Evolutionary Implications

Abstract

SUMMARY Bacterial group II intron reverse transcriptases (RTs) function in both intron mobility and RNA splicing and are evolutionary predecessors of retrotransposon, telomerase, and retroviral RTs, as well as spliceosomal protein Prp8 in eukaryotes. Here, we determined a crystal structure of a full-length thermostable group II intron RT in complex with an RNA template-DNA primer duplex and incoming dNTP at 3.0-Å resolution. We find that the binding of template-primer and key aspects of the RT active site are surprisingly different from retroviral RTs, but remarkably similar to viral RNA-dependent RNA polymerases. The structure reveals a host of features not seen previously in RTs that may contribute to distinctive biochemical properties of group II intron RTs, and it provides a prototype for many related bacterial and eukaryotic non-LTR-retroelement RTs. It also reveals how protein structural features used for reverse transcription evolved to promote the splicing of both group II and spliceosomal introns.

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

“…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). A recent crystal structure of a full-length TGIRT enzyme (GsI-IIC RT, a form of which is sold commercially as TGIRT-III; InGex) in active conformation with bound substrates revealed that group II intron RTs are closely related to RNA-dependent RNA polymerases, as expected for an ancestral RT, and identified a series of novel structural features that may contribute to their high fidelity and processivity (Stamos et al 2017). These features include more constrained binding pockets than retroviral RTs for the templating RNA base, 3' end of the DNA primer, and the incoming dNTP, as well as a larger fingers region that enables more extensive contact with the template-primer substrate than is possible for retroviral RTs (Stamos et al 2017).…”

Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq

“…This difference may reflect that template-switching by retroviral RTs involves partial or complete dissociation of the enzyme from the initial template, enabling the completed cDNA to base pair to the new template for reinitiation of cDNA synthesis by the same or another RT. By contrast, non-LTR-retroelement RTs bind the donor and acceptor simultaneously without dissociating, leaving a relatively small binding pocket that may sterically hinder access to multiple overhang bases (41).…”

“…Comparison of GsI-IIC RT to other non-LTR-retroelement RTs. Non-LTR-retroelement RTs typically have an NTE with a conserved RT0 motif that likely contributes to a structurally similar binding pocket for the 3' end of the acceptor nucleic acid during template switching (5,19,41,42). Thus, it is unsurprising that many features of the template switching and NTA mechanisms of Gs-IIC RT are similar to those found previously for the MRP and R2 element RTs, including a prominent role for NTA in adding nucleotides to the 3' end of the cDNA that can base pair to the 3' end of acceptor RNAs (25,33,34).…”

“…Template switching may provide a means of reverse transcribing through such a nicked RNA template in vivo, enabling the synthesis of long cDNAs from discontinuous RNA templates, and would disfavor multiple NTAs in functionally important sequences, accounting for the kinetic preference for a single NTA. We note that template switching may play a similar role for RNA viruses, whose RNA-dependent RNA polymerases (RdRPs) are closely related to group II intron RTs, including a structurally homologous acceptor RNA binding pocket, termed motif G in viral RdRPs (41,60). This central function may be combined with additional functions specific to the life cycle of different non-LTR-retroelements and viruses, such as preferential template-switching to a 3'-tRNAlike structure in the cases of MRP RTs (25) and higher rates of internal template switches to generate recombinant viruses to evade host defenses or repair defective genomes (61).…”

“…Recently, we determined a crystal structure of a full-length thermostable group II intron RT (GsI-IIC RT) in complex with template-primer substrate and incoming dNTP that provided insight into the structural basis for template switching by non-LTR-retroelement RTs (41). This crystal structure revealed that the NTE and RT0 loop present in non-LTR-retroelement RTs likely form a binding pocket for the 3' end of an acceptor nucleic acid, a structure that does not exist in retroviral RTs.…”