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The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a ‘copy and paste’ mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p) 1 . ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer 2 , 3 , autoimmunity 4 , 5 and ageing 6 , 7 , making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p ‘core’ (residues 238–1061, including the RT domain) by X-ray crystallography and cryo-electron microscopy in several conformational states. Our analyses identified two previously undescribed folded domains, extensive contacts to RNA templates and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed-ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production 6 – 8 . In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on the evolutionary history of L1 and enable rational drug development targeting L1. X-ray crystallography, cryo-electron microscopy, structural modelling, biochemistry, cell biology, and evolutionary analysis enable characterization of ORF2p, the reverse transcriptase of the ancient ‘parasitic’ LINE-1 retrotransposon that has written around one-third of the human genome.

Bacteria defend themselves from viral predation using diverse immune systems, many of which target foreign DNA for degradation1. Defence-associated reverse transcriptase (DRT) systems provide an intriguing counterpoint to this strategy by using DNA synthesis instead2,3. We and others recently showed that DRT2 systems use an RNA template to assemble a de novo gene that encodes the antiviral effector protein Neo4,5. It remains unclear whether similar mechanisms of defence are used by other related DRT families. Here, we show that DRT9 systems defend against phage using DNA homopolymer synthesis. Viral infection triggers polydeoxyadenylate (poly-dA) accumulation in the cell, driving abortive infection and population-level immunity. Cryo-electron microscopy structures reveal how a non-coding RNA serves as both a structural scaffold and reverse transcription template to direct hexameric complex assembly and poly-dA synthesis. Notably, biochemical and functional experiments identify tyrosine residues within the reverse transcriptase itself that probably prime DNA synthesis, leading to the formation of protein–DNA covalent adducts. Synthesis of poly-dA by DRT9 in vivo is regulated by the competing activities of phage-encoded triggers and host-encoded silencers. Collectively, our study identifies a nucleic-acid-driven defence system that expands the paradigm of bacterial immunity and broadens the known functions of reverse transcriptases.A bacterial reverse transcriptase synthesizes polydeoxyadenylate to provide population-level immunity against viral infection.

The prime editor system composed of Streptococcus pyogenes Cas9 nickase (nSpCas9) and engineered Moloney murine leukaemia virus reverse transcriptase (M-MLV RT) collaborates with a prime editing guide RNA (pegRNA) to facilitate a wide variety of precise genome edits in living cells 1 . However, owing to a lack of structural information, the molecular mechanism of pegRNA-guided reverse transcription by the prime editor remains poorly understood. Here we present cryo-electron microscopy structures of the SpCas9–M-MLV RTΔRNaseH–pegRNA–target DNA complex in multiple states. The termination structure, along with our functional analysis, reveals that M-MLV RT extends reverse transcription beyond the expected site, resulting in scaffold-derived incorporations that cause undesired edits at the target loci. Furthermore, structural comparisons among the pre-initiation, initiation and elongation states show that M-MLV RT remains in a consistent position relative to SpCas9 during reverse transcription, whereas the pegRNA–synthesized DNA heteroduplex builds up along the surface of SpCas9. On the basis of our structural insights, we rationally engineered pegRNA variants and prime-editor variants in which M-MLV RT is fused within SpCas9. Collectively, our findings provide structural insights into the stepwise mechanism of prime editing, and will pave the way for the development of a versatile prime editing toolbox. Cryo-electron microscopy structures of the prime editor bound to a prime editing guide RNA and target DNA, in the pre-initiation, initiation and elongation and termination states, provide insights into the mechanism by which prime editing occurs.

Reverse transcriptases (RTs) have well-established roles in the replication and spread of retroviruses and retrotransposons. However, recent evidence suggests that RTs have been conscripted by cells for diverse roles in antiviral defence. Here we determine structures of a type I-A retron, which explain how RNA, DNA, RT, HNH-nuclease and four molecules of a structure maintenance of chromosome (SMC)-family ATPase assemble into a 364 kDa complex that provides phage defence. We show that phage-encoded nucleases trigger degradation of the retron-associated DNA, leading to activation of the HNH nuclease. The HNH nuclease cleaves tRNA Ser , stalling protein synthesis and arresting viral replication. Taken together, these data reveal diverse and paradoxical roles for RTs in the perpetuation and elimination of genetic parasites. In this study, the authors determine the structure of a Type I-A retron from E. coli FORC82 and reveal the functional interplay between Reverse Transcriptases (RTs) and Structural Maintenance of Chromosomes (SMC) ATPases.’

Prokaryotic defense-associated reverse transcriptase (DRT) systems confer host resistance to viral infection through DNA synthesis; however, the molecular mechanisms underlying their function remain poorly understood. Here, we demonstrate that DRT4, a single-gene anti-phage defense system, synthesizes single-stranded DNA (ssDNA) products of random sequences in a template-independent manner. High-resolution cryo-EM structures of DRT4 in multiple functional states elucidate its oligomeric architecture, catalytic metal ion coordination, and substrate/DNA product binding, offering mechanistic insights into its promiscuous polymerization activity. Structural and biochemical analyses further identify a conserved tyrosine residue that acts as the priming site for the initiation of DNA synthesis. Upon phage infection, a phage-encoded DNA-binding protein, ORF55, protects the 3’ end of the DRT4-synthesized ssDNA from host exonuclease degradation, likely resulting in toxic ssDNA accumulation that leads to cell death. Remarkably, ORF55 also activates DRT6, a structural homolog of DRT4, suggesting a conserved activation mechanism among related DRT systems. These findings provide structural and mechanistic insights into DRT4-mediated antiviral defense, establishing a distinct paradigm for antiviral reverse transcriptase in bacterial immunity. Prokaryotic defense-associated reverse transcriptase (DRT) systems confer resistance to viral infection through DNA synthesis. Here, the authors dissect DRT4 mechanism of function and show that ORF55, a phage-encoded DNA-binding protein, protects the 3’ end of the DRT4- synthesized ssDNA, activating the anti-phage defense program.

Diversity-generating retroelements (DGRs) create massive protein sequence variation (up to 10 30 ) 1 in ecologically diverse microorganisms. A recent survey identified around 31,000 DGRs from more than 1,500 bacterial and archaeal genera, constituting more than 90 environment types 2 . DGRs are especially enriched in the human gut microbiome 2 , 3 and nano-sized microorganisms that seem to comprise most microbial life and maintain DGRs despite reduced genomes 4 , 5 . DGRs are also implicated in the emergence of multicellularity 6 , 7 . Variation occurs during reverse transcription of a protein-encoding RNA template coupled to misincorporation at adenosines. In the prototypical Bordetella bacteriophage DGR, the template must be surrounded by upstream and downstream RNA segments for complementary DNA synthesis to be carried out by a complex of the DGR reverse transcriptase bRT and associated protein Avd. The function of the surrounding RNA was unknown. Here we show through cryogenic electron microscopy that this RNA envelops bRT and lies over the barrel-shaped Avd, forming an intimate ribonucleoprotein. An abundance of essential interactions in the ribonucleoprotein precisely position an RNA homoduplex in the bRT active site for initiation of reverse transcription. Our results explain how the surrounding RNA primes complementary DNA synthesis, promotes processivity, terminates polymerization and strictly limits mutagenesis to specific proteins through mechanisms that are probably conserved in DGRs belonging to distant taxa. This study presents cryogenic electron microscopy structural analysis of a diversity-generating retroelement (DGR) reverse transcription system from Bordetella bacteriophage, with results indicating that RNA surrounding the template controls reverse transcription through mechanisms that are conserved across distant taxa.

Retrons are prokaryotic genetic elements involved in anti-phage defense and consist of a non-coding RNA, a reverse transcriptase (RT), and various effector proteins. Retron-Eco7 (previously known as Retron-Ec78) from Escherichia coli encodes two effector proteins (the PtuA ATPase and the PtuB nuclease) and degrades the host tRNA Tyr upon phage infection, thereby protecting host cells against invading phages. However, its defense mechanism remains elusive. Here, we report the cryo-electron microscopy (cryo-EM) structures of the Retron-Eco7 complex, comprising the RT, multicopy single-stranded DNA (msDNA), PtuA, and PtuB. The Retron-Eco7 structures reveal that the RT–msDNA complex associates with two PtuA–PtuB complexes, potentially inhibiting their nuclease activity and suppressing bacterial growth arrest prior to phage infection. Furthermore, the phage-encoded D15 nuclease acts as a trigger for the Retron-Eco7 system and cleaves the msDNA bound to the complex, facilitating the dissociation of PtuA–PtuB from RT–msDNA. Our data indicate that msDNA cleavage by D15 is the initial step required for the specific cleavage of the host tRNA Tyr by the PtuA–PtuB nuclease, which leads to abortive infection. Overall, this study provides mechanistic insights into the Retron-Eco7 system and highlights the diversity of prokaryotic anti-phage defense mechanisms. The Retron-Eco7 is a genetic element involved in anti-phage defense that encodes two effector proteins (PtuA and PtuB) and cleaves the host tRNA. Here, the authors solved the Retron-Eco7 complex structure using cryo-EM.

We report here crystal structures of a reverse transcriptase RTX, which was evolved in vitro from the B family polymerase KOD, in complex with either a DNA duplex or an RNA–DNA hybrid. Compared with the apo, binary, and ternary complex structures of the original KOD polymerase, the 16 substitutions that result in the function of copying RNA to DNA do not change the overall protein structure. Only six substitutions occur at the substrate-binding surface, and the others change domain–domain interfaces in the polymerase to enable RNA–DNA hybrid binding and reverse transcription. Most notably, F587L at the Palm and Thumb interface stabilizes the open and apo conformation of the Thumb. The intrinsically flexible Thumb domain seems to play a major role in accommodating the RNA–DNA hybrid product distal to the active site. This is reminiscent of naturally occurring RNA-dependent DNA polymerases, including telomerase, which have a dramatically augmented Thumb domain, and of reverse transcriptase, which extends its Thumb with the RNase H domain.

Engineering of a bifunctional enzyme that combines DNA-dependent DNA polymerase and reverse transcriptase (RT) activities is a highly promising biotechnological goal, as it would enable one-enzyme RT-PCR. For this purpose, we selected the high-fidelity Pyrococcus furiosus (Pfu) DNA polymerase as engineering scaffold. The selection of amino acid residues for replacement was carried out based on a multi-sequence alignment of diverse DNA polymerases and literature data, which allowed us to target amino acids, which presumably are triggers of the RT activity appearance. Six mutant variants of the Pfu enzyme were created and their activity was analyzed. Through enzymatic screening, we identified the Pfu-M6 variant, which exhibits dual DNA-dependent and RNA-dependent DNA polymerase activity. This thermostable enzyme retains its inherent DNA polymerase function and has acquired the ability to catalyze reverse transcription under standard PCR conditions, which allows the created mutant form to be used for efficient amplification of DNA starting from an RNA template.

The average human genome contains a small cohort of active L1 retrotransposons that encode two proteins (ORF1p and ORF2p) required for their mobility (i.e., retrotransposition). Prior studies demonstrated that human ORF1p, L1 RNA, and an ORF2p-encoded reverse transcriptase activity are present in ribonucleoprotein (RNP) complexes. However, the inability to physically detect ORF2p from engineered human L1 constructs has remained a technical challenge in the field. Here, we have employed an epitope/RNA tagging strategy with engineered human L1 retrotransposons to identify ORF1p, ORF2p, and L1 RNA in a RNP complex. We next used this system to assess how mutations in ORF1p and/or ORF2p impact RNP formation. Importantly, we demonstrate that mutations in the coiled-coil domain and RNA recognition motif of ORF1p, as well as the cysteine-rich domain of ORF2p, reduce the levels of ORF1p and/or ORF2p in L1 RNPs. Finally, we used this tagging strategy to localize the L1-encoded proteins and L1 RNA to cytoplasmic foci that often were associated with stress granules. Thus, we conclude that a precise interplay among ORF1p, ORF2p, and L1 RNA is critical for L1 RNP assembly, function, and L1 retrotransposition.