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Cited by 31Chromosomal and Genetic VariationsEnzyme Structure and FunctionHIV/AIDS drug development and treatment

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

Open access
Nowak, Elżbieta, Miller, Jennifer T, Bona, Marion K, Studnicka, Justyna, Szczepanowski, Roman H, Jurkowski, Jakub, Le Grice, Stuart F J, Nowotny, Marcin·2014·Nature Structural & Molecular Biology
DOI 10.1038/nsmb.2785PMC PMC6321743
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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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Crossref · 10.1038/nsmb.2785

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  • Catalytic activity of rnhA ribonuclease H proteins requires an active-site DEDD motif and a conserved active-site histidine.

    “For the catalytic activity of rnhA RNH proteins, site-directed mutagenesis, thermal stability and structural determination studies have indicated that an active-site DEDD motif and conserved active-site histidine are required [43-50]”

    From Retroviral RNase H: structure, mechanism, and inhibition (2021) · cites this paper as [47]

    The Results (Biochemical characterization; page unavailable) identify the Ty3 reverse transcriptase D426N variant as RNase H-deficient, supporting a role for an aspartate residue in this enzyme’s activity. The paper does not establish that rnhA proteins require a DEDD motif or a conserved active-site histidine. (LLM, conf=0.78)

    Experimental — accuracy not yet measured

  • Endonuclease reactions such as ribonuclease H require divalent metal ions, either Mg2+ or Mn2+, as cofactors.

    “Endonuclease reactions, such as that of RNH, require divalent metal ions, either Mg2+ or Mn2+, as co-factors [47].”

    From Retroviral RNase H: structure, mechanism, and inhibition (2021) · cites this paper as [47]

    The Online Methods (RNase H assays; page unavailable) include MgCl2 in the RNase H hydrolysis reactions, supporting the magnesium part of the claim. The paper does not establish that a divalent metal ion is required, or report Mn2+ as an alternative cofactor for RNase H. (LLM, conf=0.84)

    Experimental — accuracy not yet measured

  • Two models for metal coordination in the HIV-1 ribonuclease H endonuclease reaction have been proposed: one involving a single ion and one involving two metal ions.

    “In HIV-1 RNH, two different models for metal coordination in the endonuclease reaction have been proposed, one that invokes binding by a single ion [47,59] (Figure 3c and 3d) and one that invokes binding by two metal ions [50,60-62] (Figure 3b).”

    From Retroviral RNase H: structure, mechanism, and inhibition (2021) · cites this paper as [47]

    The Results section on the RNase H domain discusses substrate cleavage and compares Ty3 with HIV-1 RT, but does not describe competing single-ion and two-metal-ion coordination models. Searches for those models and for metal-dependent catalysis found no passage in our copy that addresses the claim. (LLM, conf=0.76)

    Experimental — accuracy not yet measured

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Cites

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Research works printed but not linked: 1

  1. [6]Sandmeyer SB, Aye M & Menees TM Ty3: a position-specific, gypsylike element in Saccharomyces cerevisiae. (eds. Craig NL, Craigie R, Gellert M & Lambowitz AM) 663– 682 (ASM Press, Washington DC, 2002).

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Authors
Nowak, E., Miller, J. T., Bona, M. K., Studnicka, J., Szczepanowski, R. H., Jurkowski, J., Le Grice, S. F. J., & Nowotny, M.
Affiliations
  • International Institute of Molecular and Cell Biology (PL)
  • National Institutes of Health (US)
Corresponding author
Nowotny, Marcin
ORCID
Venue
Nature Structural & Molecular Biology
Publisher
Springer Science and Business Media LLC
Published
2014-03-09
Volume
21
Issue
4
Pages
389-396
ISSN
  • 1545-9985 (ISSN-L)
  • 1545-9993
DOI
10.1038/nsmb.2785
PMC
PMC6321743
OpenAlex
W1979841652
Cited by
31
References
44
Citations by year
2024: 1 · 2023: 3 · 2022: 1 · 2021: 6 · 2019: 3 · 2018: 2 · 2017: 5 · 2016: 1 · 2015: 8 · 2014: 1
FWCI
5.83
Type
journal article
Language
en
Open access
Licence
www.springer.com/tdm (tdm)
Locations
  • Nature Structural & Molecular Biology (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
  • HZB Repository (Helmholtz-Zentrum Berlin für Materialien und Energie GmbH (HZB)) (submittedVersion)
Funders
  • Howard Hughes Medical Institute
  • National Institutes of Health
  • National Cancer Institute (HHSN261200800001E), Z01BC010493)
Topics
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Chromosomal and Genetic Variations
  • Physical Sciences › Materials Science › Materials Chemistry › Enzyme Structure and Function
  • Health Sciences › Medicine › Infectious Diseases › HIV/AIDS drug development and treatment
Keywords
retrotransposons, LTR retrotransposons, polypurine tract, RNA-DNA hybrids, crystal structure, RNase H activity, DNA polymerase activity, Saccharomyces cerevisiae, mobile genetic elements
MeSH terms
Binding Sites, DNA, Models, Molecular, RNA-Directed DNA Polymerase, Ribonuclease H, Protein Structure, Tertiary, Crystallography, X-Ray, Retroelements, Dimerization, Saccharomyces cerevisiae Proteins

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