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Ty3 reverse transcriptase complexed with an RNA-DNA hybrid shows structural and functional asymmetry

Nature Structural & Molecular Biology volume 21, pages 389–396 (2014)Cite this article

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Abstract

Retrotransposons are a class of mobile genetic elements that replicate by converting their single-stranded RNA intermediate to double-stranded DNA through the combined DNA polymerase and ribonuclease H (RNase H) activities of the element-encoded reverse transcriptase (RT). Although a wealth of structural information is available for lentiviral and gammaretroviral RTs, equivalent studies on counterpart enzymes of long terminal repeat (LTR)–containing retrotransposons, from which they are evolutionarily derived, is lacking. In this study, we report the first crystal structure of a complex of RT from the Saccharomyces cerevisiae LTR retrotransposon Ty3 in the presence of its polypurine tract–containing RNA-DNA hybrid. In contrast to its retroviral counterparts, Ty3 RT adopts an asymmetric homodimeric architecture whose assembly is substrate dependent. Moreover, our structure and biochemical data suggest that the RNase H and DNA polymerase activities are contributed by individual subunits of the homodimer.

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Figure 1: Overall structure of Ty3 RT and the dimer interface.
Figure 2: Comparison of RNase H and connection subdomains.
Figure 3: Substrate binding.
Figure 4: Biochemical experiments.
Figure 5: Structural comparison of Ty3 and HIV-1 RTs.

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Acknowledgements

We thank W. Yang for critical reading of the manuscript, I. Ptasiewicz for excellent technical assistance, the staff of beamline 14-1 at Berliner Elektronenspeicherring-Gesellschaft für Synchrotronstrahlung for assistance with data collection, and J.M. Bujnicki for help in the preparation of the sequence alignments. This work was supported by grants from the Polish National Science Center (contract no. N N301 439738 to M.N.) and the FP7 HEALTHPROT project (contract no. 229676 to M.N.). S.F.J.L.G. is supported by the Intramural Research Program of the National Cancer Institute, US National Institutes of Health and by federal funds from National Institutes of Health contract no. HHSN261200800001E (M.K.B.). The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government. M.N. is a recipient of the Foundation for Polish Science 'Ideas for Poland' award. The research of M.N. was supported in part by an International Early Career Scientist grant from the Howard Hughes Medical Institute. The research was performed using Centre for Preclinical Research and Technology infrastructure (European Union project POIG.02.02.00-14-024/08-00).

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Authors and Affiliations

  1. Laboratory of Protein Structure, International Institute of Molecular and Cell Biology, Warsaw, Poland

    Elżbieta Nowak, Justyna Studnicka, Jakub Jurkowski & Marcin Nowotny

  2. Reverse Transcriptase Biochemistry Section, HIV Drug Resistance Program, Frederick National Laboratory, Frederick, Maryland, USA

    Jennifer T Miller, Marion K Bona & Stuart F J Le Grice

  3. Biophysics Core Facility, International Institute of Molecular and Cell Biology, Warsaw, Poland

    Roman H Szczepanowski

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  1. Elżbieta Nowak
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Contributions

E.N. obtained crystals of Ty3 RT–substrate complex; E.N. and M.N. solved and analyzed the structure; J.T.M., M.K.B. and J.S. performed biochemical experiments; E.N. and R.H.S. performed biophysical protein characterization; J.J. prepared the expression construct and conducted initial crystallization experiments; S.F.J.L.G. and M.N. designed the research and wrote the manuscript.

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Correspondence to Stuart F J Le Grice or Marcin Nowotny.

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Integrated supplementary information

Supplementary Figure 1 Experimental electron density maps and the content of the asymmetric unit.

(a) and (b) Samples of experimental electron density maps after selenium single wavelength anomalous diffraction (SAD) phasing (stereoviews). (a) The region around the DNA polymerase active site. (b) The central β-sheet of RNase H domain from subunit B. Maps are contoured at 1.5 and 1.2 σ. (c) Composition of the asymmetric unit. The two complexes present in the asymmetric unit are shown, colored as in Figure 1.

Supplementary Figure 2 Multiple sequence alignment of retrolement and retrovirus RT sequences.

Ty3/Gypsy retroelements: Boudicca (S. mansoni), Gypsy (D. melanogaster), Maggy (M. grisea), Ulysses (D. virilis), Real (A. alternata), TF2 (S. pombe), Ty3 and retroviruses: human immunodeficiency virus 1 (HIV-1), xenotropic murine leukemia-related virus (XMRV), human T-cell leukemia virus-1 (HTLV-I), prototype foamy virus (PFV), Rous sarcoma virus (RSV), mouse mammary tumor virus (MMTV). Retroelements were selected to represent the most diverse sequences based on phylogenetic analysis of the RT sequence. For all sequences the active site residues are highlighted in yellow. For Ty3 RT sequence residues involved in RNA stabilization are highlighted in red, and DNA interaction in blue. Dimer interface residues are highlighted in gray. Numbers on the top of the alignment correspond to Ty3 RT residues.

Supplementary Figure 3 Structure-based alignment of RTs from Ty3 and HIV-1 (PDB ID: 1RTD16).

Identical residues are marked with '*' and similar ones with ':'. Secondary structure elements are shown as tubes (helices) and arrows (strands) and labeled and colored as in Figure 1. Part of the C-terminal region of Ty3 RNase H domain adopts different conformation in subunits A and B and therefore, two secondary structure assignments are given and fragments not observed in the structure are marked with '~'. Active site residues of the DNA polymerase and RNase H (for Ty3) domains are highlighted in yellow. Note the similarity in structural organization between the HIV-1 RT p66 connection subdomain and the Ty3 RNase H domain.

Supplementary Figure 4 Structure-based alignment of RNase H domains from HIV-1 and Ty3 RTs.

The alignment is marked as in Supplementary Figure 3 and secondary structure elements labeled as in Fig 1 and 2.

Supplementary Figure 5 RNase H active sites and model of the interaction with the substrate.

(a) Comparison of RNase H active sites (stereoview) shown in yellow (Ty3 RT), blue (B. halodurans RNase H1, PDB ID: 1ZBI27), and salmon (human RNase H1 PDB ID: 2QK9 (ref 28)). Two Mg2+ ions in the Bh-RNase H1 structure are shown as green spheres and the RNA strands from human and bacterial RNases H1 in salmon and red, respectively. The attacking nucleophilic water is shown as a red sphere. (b) Model of the interaction of the Ty3 subunit B RNase H domain with the scissile phosphate. RNase H domains and the nucleic acid are in cartoon representation and the rest of the structure in wire representation. Subunit A is shown in dark gray and yellow for RNase H and subunit B in light gray and light yellow for RNase H. Brown cartoon represents the RNase H domain modeled to interact with the scissile phosphate in position –13. Model with 12 nt distance from the 3′ end of the DNA strand was equally plausible and did not to include any steric clashes. RNA is in magenta and DNA in blue.

Supplementary Figure 6 Comparison of the DNA polymerase active sites of Ty3 and HIV-1 RT (PDB ID: 1RTD16) (stereoview).

Ty3 RT residues are shown in red (palm) and blue (fingers) and HIV-1 in grey. Metal ions and the incoming dNTP from HIV-1 structure are shown in gray. RNA-DNA from Ty3 structure is shown in magenta (RNA) and marine (DNA) ladder. dsDNA from the HIV-1 structure is shown as light blue ladder. The last nucleotide of the DNA located at the active site is shown with sticks.

Supplementary Figure 7 Substrate binding an oligomerization of Ty3 RT variants – analytical ultracentrifugation, sedimentation velocity analysis.

(a) Black trace - RNA-DNA hybrid alone, calculated MW of 18.8 kDa (assuming v-bar =0.54 ml g-1, value the most commonly used for nucleic acids), s=1.88 S, value normalized for 20 °C and water s20,w =1.74 S. Red - R140A R203A variant alone, MW of 49.1 (v-bar 0.7352), s =2.2 S and s20,w = 3.5 S. (b) Magenta - RNA-DNA with R140A R203A, estimated MW of 86 kDa, s= 3.5 S and s20,w =5.6 S. Green - DNA-RNA with R441A R442A, MW of 127 kDa, s= 4.2 S and s20,w =6.6 S. Blue – DNA-RNA with WT, estimated MW of 121 kDa, s =4.3 S and normalized s20,w =6.9 S. Excess of unbound proteins, WT and R441A R442A variant, with the MW of 56 kDa and 57 kDa, respectively, are marked with corresponding colors. The shift in the position of the peak for R441A R442A may be a result of a conformational change relative to the wild type protein.

Supplementary Figure 8 Uncropped images used in Fig. 4c and d.

(a) Ty3 RT DNA polymerase assay on HIV-1 RNA genome. The lanes used in Fig 4c are indicated with a blue line in the bottom of the gel. P-fluorescently labeled primer. (b) A sequencing reference gel with the reaction for HIV-1 RT used to identify the polymerase reaction products. dA – sequencing ladder, Mut – RNase H-deficient HIV-1 RT, WT – wild type HIV-1 RT. The position of the TAR hairpin (TAR), which causes stalling of the Ty3 RT is indicated on the left of the gel. (c) Uncropped gel presented in Fig 4d. (d) A reference Ty3 RT RNase H assay. Wild type protein was incubated with a fluorescently labeled hybrid and the reaction was stopped after 0.5, 10 and 20 minutes (indicated on top of the gel). Three different amounts of RNA size markers with the same fluorescent label were applied to the lanes 'markers'. Their sizes are indicated and the corresponding cut positions from the 3′-end of the DNA are given in parentheses.

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Nowak, E., Miller, J., Bona, M. et al. Ty3 reverse transcriptase complexed with an RNA-DNA hybrid shows structural and functional asymmetry. Nat Struct Mol Biol 21, 389–396 (2014). https://doi.org/10.1038/nsmb.2785

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