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RNA control of reverse transcription in a diversity-generating retroelement

Nature volume 638, pages 1122–1129 (2025)Cite this article

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Abstract

Diversity-generating retroelements (DGRs) create massive protein sequence variation (up to 1030)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 types2. DGRs are especially enriched in the human gut microbiome2,3 and nano-sized microorganisms that seem to comprise most microbial life and maintain DGRs despite reduced genomes4,5. DGRs are also implicated in the emergence of multicellularity6,7. 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.

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Fig. 1: DGR and bRT–Avd:RNAΔ98 RNP.
Fig. 2: bRT–Avd and template:primer duplex.
Fig. 3: cPRT stop.
Fig. 4: Thumb ring and protein-binding RNA.
Fig. 5: RNA conformational variability and conservation.

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Data availability

The atomic coordinates and sharpened cryo-EM and half maps of bRT–Avd/RNAΔ98 in Active G:dCTP conformation (Extended Data Table 1; Complex 1) have been deposited in the PDB (https://www.rcsb.org/structure/) with code 8UB7 and in the EMDB (https://www.ebi.ac.uk/emdb/) with code EMD-42077, respectively; Active A:Empty conformation (Complex 2) with codes PDB 8UBB and EMD-42081, respectively; Active G:Empty conformation (Complex 3) with codes PDB 8UB9 and EMD-42079, respectively; Resting conformation (Complex 4) with codes PDB 8UBC and EMD-42082, respectively; Resting conformation (Complex 5) with codes PDB 8UBE and EMD-42084, respectively; Resting conformation (Complex 6) with codes PDB 8UBF and EMD-42085, respectively; Pre-Active 1 conformation (Complex 7) with codes PDB 8UBA and EMD-42080; Pre-Active 1 conformation (Complex 8) with codes PDB 8UB8 and EMD-42078; and Pre-Active 2 conformation (Complex 9) with codes PDB 8UBD and EMD-42083. Coordinates for Methylobacterium extorquens formaldehyde activating enzyme (PDB 1Y60), RNA-dependent RNA polymerase (PDB 3OL9), group II intron RT GsI-IIC (PDB 6AR1) and Avd (PDB 4DWL) are available in the PDB with the codes indicated.

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Acknowledgements

We thank R. Baker and A. Leschziner for initial cryo-EM characterization of complexes, T. Baker for computational resources and J. Meyers for data collection at Pacific Northwest Center for Cryo-EM (PNCC). Use of PNCC was supported by NIH grant no. U24GM129547. This work was supported by the National Institutes of Health, grant nos. R01 GM132720 (P.G.), R01 AI163327 (G.G.) and R01 GM033050-35 (T.B.). B.G.P. was supported by the Gordon and Betty Moore Foundation, the G. Unger Vetlesen Foundation and the Owens Family Foundation.

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  1. Sumit Handa

    Present address: 10X Genomics, Pleasanton, CA, USA

  2. These authors contributed equally: Sumit Handa, Tapan Biswas

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, USA

    Sumit Handa, Tapan Biswas, Jeet Chakraborty, Gourisankar Ghosh & Partho Ghosh

  2. Marine Biological Laboratory, Josephine Bay Paul Center, Woods Hole, MA, USA

    Blair G. Paul

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Contributions

S.H. and P.G. conceptualized the project. S.H., T.B., J.C., B.G.P. and P.G. carried out the investigation. S.H., T.B., B.G.P. and P.G. carried out visualization. P.G. and G.G. acquired funding. P.G. administered and supervised the project. S.H., T.B., B.G.P. and P.G. wrote the original draft and S.H., T.B., G.G., B.G.P. and P.G. reviewed and edited the draft.

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Correspondence to Partho Ghosh.

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Extended data figures and tables

Extended Data Fig. 1 bRT-(Avd-Fae):RNAΔ98.

a. Schematic of bRT, Avd, Avd-Fae and DGR RNA and RNAΔ98. b. Synthesis of cDNA by bRT-Avd or bRT-(Avd-Fae) from DGR RNA or RNAΔ98. Reactions were incubated with dNTPs, including [α-32P]dCTP, for 2 h at 37 °C. Products were treated with RNase and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane M corresponds to radiolabeled, single-stranded DNA molecular mass markers, denoted by nucleotide (nt) length. The panels are from the same gel in which irrelevant data were deleted (marked by line). For gel source data, see Supplementary Fig. 1a. The gel is representative of two independent replicates. c. Frequency distribution of deoxynucleotides incorporated (thymidine, purple) or misincorporated (adenosine, orange; cytosine, red; guanosine, green) by bRT-Avd (T 88.9%, A 1.8%, C 8.7%, G 0.6%) or bRT-Avd-Fae (T 90.0%, A 0.9%, C 8.7%, G 0.4%) at TRG117A in RNAΔ98. TRG117A has a lower misincorporation frequency than other adenosines in TR14. d. Two views of local resolution map of bRT-Avd without RNAΔ98 shown (left, in same orientation as Fig. 2a). Resolution scale in Å below.

Extended Data Fig. 2 RNP Interactions and Active Conformations.

a bRT (red) superposed with the group II intron RT GsI-IIC (blue) in ‘right-hand’ view of polymerases. Subdomains of the RTs are indicated. GsI-IIC has a D domain that is not present in bRT or other DGR RTs. b. Interaction between bRT E40 and Avd R79 and R83. Avd R79A and R83A do not bind bRT and cause loss of function13. Coloring as in Fig. 2a. c. Interaction of bRT NTE with TRG117 and Avd. Interacting amino acids in bonds representation (carbon yellow for bRT, salmon for Avd, and cyan for TRG117; oxygen red; nitrogen blue; and phosphorus orange). d. Superposition of Active G:dCTP (blue, Complex 1 in Extended Data Table 1), Active A:Empty (red, Complex 2), and Active G:Empty RNPs (green, Complex 3). Root mean square deviation of 1.6 Å for 10,218 atoms between Active G:dCTP and Active A:Empty RNPs, and 0.7 Å for 10,265 atoms between Active G:dCTP and Active A:Empty RNPs. e. Model of bRT-Avd-Fae:RNAΔ98 complex. Fae (red, PDB 1Y60) was placed at the wide end of the Avd barrel for visual purposes. No interpretable density exists for Fae. Coloring as in Fig. 1d. Dotted line indicates potential location of intact TR.

Extended Data Fig. 3 DGR RNA Binding and cPRT Base Pairing.

a-d. Electrophoretic mobility shift assay (EMSA) of (a) DGR RNA, (b) DGR RNA Δavd368-379, (c) DGR RNA ΔSp96-140, and (d) non-DGR RNA with varying concentrations of bRT-Avd (as indicated at top of each panel). RNA was resolved by 4% native PAGE. Arrowheads indicate position of shifted bands. To the side of each gel is shown LC/MS-MS analysis of shifted EMSA bands. Lower quantities of RNA at higher bRT-Avd concentrations in the non-DGR RNA sample likely indicate non-specific aggregation of bRT-Avd with RNA. For gel source data, see Supplementary Fig. 1b–e. Representative gel from two independent replicates is shown. e. Schematic of (1, WT) 12 potential bps in the avd-Sp duplex; (2) substitutions in Sp that disrupt the 12 bps; (3) complementary substitutions in avd that restore the 12 bps; (4) substitutions in Sp that disrupt six Watson-Crick bps; and (5) complementary substitutions in avd that restore the six Watson-Crick bps. Solid bars are Watson-Crick base pairs, and open circles wobble base pairs. f. Top, cDNA synthesis by bRT-Avd with (1) wild-type or (2-5) mutant DGR RNA. The lane numbers correspond to the numbering in panel e. Products were treated with RNase and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane M corresponds to radiolabeled, single-stranded DNA molecular mass markers, denoted by nucleotide (nt) length. Arrowheads indicate the positions of ~90- and ~120-nt cDNAs. A representative gel from three independent replicates is shown. Bottom, input RNA for cDNA synthesis resolved by 6% denaturing PAGE. Lane M corresponds to DNA molecular mass markers, denoted by nt length. DGR RNA runs at a position higher than the 300 nt marker, likely due to retention of secondary structure in the RNA. The numbers correspond to the numbering of sequences depicted in panel e. A representative gel from three independent replicates is shown. For gel source data, see Supplementary Fig. 1f.

Extended Data Fig. 4 Mutations in DGR RNA.

a. Top, cDNAs synthesized by bRT-Avd from wild-type or mutated DGR RNAs. Products were treated with RNase and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane M corresponds to radiolabeled, single-stranded DNA molecular mass markers, denoted by nucleotide (nt) length. Arrowheads correspond to cDNAs. Bottom, input RNAs for cDNA synthesis resolved by 6% denaturing PAGE. Lane M corresponds to DNA molecular mass markers, denoted by nt length. A representative gel from three independent replicates shown. An irrelevant lane is blanked out from the gels. For gel source data, see Supplementary Fig. 1g. b. cPRT activity of mutant DGR RNASp(Δ71-78) relative to wild-type DGR RNA. Data are from experiments that were repeated three independent times, and means and standard deviations shown. c and d. cDNAs synthesized by bRT-Avd from wild-type or DGR RNAs mutated in the Thumb ring (c) or bRT-binding RNA (d). Products were treated with RNase and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane M corresponds to radiolabeled, single-stranded DNA molecular mass markers, denoted by nt length. Arrowheads correspond to cDNAs. Bottom of each panel shows input RNAs for cDNA synthesis resolved by 6% denaturing PAGE. Lane M corresponds to DNA molecular mass markers, denoted by nt length. A representative gel from three independent replicates shown. For gel source data for panels c and d, see Supplementary Fig. 1h,i, respectively.

Extended Data Fig. 5 Flipped-out bases in Avd-binding loop.

a. Binding of flipped-out base SpU3 to a crevice between Avd1 and Avd2. b. Binding of flipped-out base SpU7 to a crevice between Avd3 and Avd4.

Extended Data Fig. 6 RNP Alternative Conformations.

a. bRT-Avd:RNAΔ98 in Resting conformation from Complex 4 (Extended Data Table 1). Coloring here and in other panels as in Fig. 1d. b. Resting conformation from Complex 5. c. Resting conformation from Complex 6. d. Pre-Active 1 conformation from Complex 7. e. Pre-Active 1 conformation from Complex 8. f. Pre-Active 2 conformation from Complex 9.

Extended Data Fig. 7 Pre-Active Conformation.

a. Schematic of continuous Sp strand in the cPRT Stop and TR:Sp duplex in the Pre-Active conformation (Extended Data Table 1, Complex 7), depicted as in Fig. 2c. b. Interactions of SpG57 and C58 with TRG117 and avdG380, respectively. bRT amino acids that contact SpG57 are shown. Coloring as in Fig. 2a for carbon; phosphorus orange, oxygen red, and nitrogen blue.

Extended Data Fig. 8 Conservation of RNA elements.

a-c. Sequence alignment consensus profiles for putative (left) cPRT Stop duplex and (right) Avd-binding stem loop for (a) Bacillota, (b) Pseudomonadota, and (c) Cyanobacteriota DGRs. The number of genomes in each alignment is indicated in parentheses. d. RNA structural features of the BΦ DGR predicted by MxFold252 as compared to data from the cryo-EM structure. The sequence upstream (i.e., avd) and downstream (i.e., Sp) of TR are in green and orange, respectively.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
Full size table
Extended Data Table 2 Q-scores and map correlation coefficients
Full size table

Supplementary information

Supplementary Information

Supplementary Figs. 1–14.

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Supplementary Table 1

Conservation of RNA structural elements.

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Handa, S., Biswas, T., Chakraborty, J. et al. RNA control of reverse transcription in a diversity-generating retroelement. Nature 638, 1122–1129 (2025). https://doi.org/10.1038/s41586-024-08405-w

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