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Biochem J. Jun 1, 2000; 348(Pt 2): 337–342.
PMCID: PMC1221071

Expression of an active form of recombinant Ty1 reverse transcriptase in Escherichia coli: a fusion protein containing the C-terminal region of the Ty1 integrase linked to the reverse transcriptase-RNase H domain exhibits polymerase and RNase H activities.

Abstract

Replication of the Saccharomyces cerevisiae Ty1 retrotransposon requires a reverse transcriptase capable of synthesizing Ty1 DNA. The first description of an active form of a recombinant Ty1 enzyme with polymerase and RNase H activities is reported here. The Ty1 enzyme was expressed as a hexahistidine-tagged fusion protein in Escherichia coli to facilitate purification of the recombinant protein by metal-chelate chromatography. Catalytic activity of the recombinant protein was detected only when amino acid residues encoded by the integrase gene were added to the N-terminus of the reverse transcriptase-RNase H domain. This suggests that the integrase domain could play a role in proper folding of reverse transcriptase. Several biochemical properties of the Ty1 enzyme were analysed, including the effect of MgCl(2), NaCl, temperature and of the chain terminator dideoxy GTP on its polymerase activity. RNase H activity was examined by monitoring the cleavage of a RNA-DNA template-primer. Our results suggest that the distance between the RNase H and polymerase active sites corresponds to the length of a 14-nucleotide RNA-DNA heteroduplex. The recombinant protein produced in E. coli should be useful for further biochemical and structural analyses and for a better understanding of the role of integrase in the activation of reverse transcriptase.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Varmus H. Retroviruses. Science. 1988 Jun 10;240(4858):1427–1435. [PubMed]
  • Boeke JD, Garfinkel DJ, Styles CA, Fink GR. Ty elements transpose through an RNA intermediate. Cell. 1985 Mar;40(3):491–500. [PubMed]
  • Eichinger DJ, Boeke JD. The DNA intermediate in yeast Ty1 element transposition copurifies with virus-like particles: cell-free Ty1 transposition. Cell. 1988 Sep 23;54(7):955–966. [PubMed]
  • Mellor J, Fulton SM, Dobson MJ, Wilson W, Kingsman SM, Kingsman AJ. A retrovirus-like strategy for expression of a fusion protein encoded by yeast transposon Ty1. Nature. 1985 Jan 17;313(5999):243–246. [PubMed]
  • Le Grice SF, Cameron CE, Benkovic SJ. Purification and characterization of human immunodeficiency virus type 1 reverse transcriptase. Methods Enzymol. 1995;262:130–144. [PubMed]
  • Boeke JD, Eichinger D, Castrillon D, Fink GR. The Saccharomyces cerevisiae genome contains functional and nonfunctional copies of transposon Ty1. Mol Cell Biol. 1988 Apr;8(4):1432–1442. [PMC free article] [PubMed]
  • Taube R, Loya S, Avidan O, Perach M, Hizi A. Reverse transcriptase of mouse mammary tumour virus: expression in bacteria, purification and biochemical characterization. Biochem J. 1998 Feb 1;329(Pt 3):579–587. [PMC free article] [PubMed]
  • Trentin B, Rebeyrotte N, Mamoun RZ. Human T-cell leukemia virus type 1 reverse transcriptase (RT) originates from the pro and pol open reading frames and requires the presence of RT-RNase H (RH) and RT-RH-integrase proteins for its activity. J Virol. 1998 Aug;72(8):6504–6510. [PMC free article] [PubMed]
  • Mathias SL, Scott AF, Kazazian HH, Jr, Boeke JD, Gabriel A. Reverse transcriptase encoded by a human transposable element. Science. 1991 Dec 20;254(5039):1808–1810. [PubMed]
  • Baudin F, Marquet R, Isel C, Darlix JL, Ehresmann B, Ehresmann C. Functional sites in the 5' region of human immunodeficiency virus type 1 RNA form defined structural domains. J Mol Biol. 1993 Jan 20;229(2):382–397. [PubMed]
  • Garfinkel DJ, Hedge AM, Youngren SD, Copeland TD. Proteolytic processing of pol-TYB proteins from the yeast retrotransposon Ty1. J Virol. 1991 Sep;65(9):4573–4581. [PMC free article] [PubMed]
  • Martin-Rendon E, Hurd DW, Marfany G, Kingsman SM, Kingsman AJ. Identification of proteolytic cleavage sites within the gag-analogue protein of Ty1 virus-like particles. Mol Microbiol. 1996 Dec;22(5):1035–1043. [PubMed]
  • Merkulov GV, Swiderek KM, Brachmann CB, Boeke JD. A critical proteolytic cleavage site near the C terminus of the yeast retrotransposon Ty1 Gag protein. J Virol. 1996 Aug;70(8):5548–5556. [PMC free article] [PubMed]
  • Moore SP, Garfinkel DJ. Expression and partial purification of enzymatically active recombinant Ty1 integrase in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1994 Mar 1;91(5):1843–1847. [PMC free article] [PubMed]
  • Kirchner J, Sandmeyer SB. Ty3 integrase mutants defective in reverse transcription or 3'-end processing of extrachromosomal Ty3 DNA. J Virol. 1996 Jul;70(7):4737–4747. [PMC free article] [PubMed]
  • Hippenmeyer PJ, Grandgenett DP. Requirement of the avian retrovirus pp32 DNA binding protein domain for replication. Virology. 1984 Sep;137(2):358–370. [PubMed]
  • Kenna MA, Brachmann CB, Devine SE, Boeke JD. Invading the yeast nucleus: a nuclear localization signal at the C terminus of Ty1 integrase is required for transposition in vivo. Mol Cell Biol. 1998 Feb;18(2):1115–1124. [PMC free article] [PubMed]
  • Moore SP, Rinckel LA, Garfinkel DJ. A Ty1 integrase nuclear localization signal required for retrotransposition. Mol Cell Biol. 1998 Feb;18(2):1105–1114. [PMC free article] [PubMed]
  • Jacobo-Molina A, Ding J, Nanni RG, Clark AD, Jr, Lu X, Tantillo C, Williams RL, Kamer G, Ferris AL, Clark P, et al. Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 A resolution shows bent DNA. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6320–6324. [PMC free article] [PubMed]
  • Kohlstaedt LA, Wang J, Friedman JM, Rice PA, Steitz TA. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science. 1992 Jun 26;256(5065):1783–1790. [PubMed]
  • Huang H, Chopra R, Verdine GL, Harrison SC. Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance. Science. 1998 Nov 27;282(5394):1669–1675. [PubMed]
  • Wöhrl BM, Moelling K. Interaction of HIV-1 ribonuclease H with polypurine tract containing RNA-DNA hybrids. Biochemistry. 1990 Nov 6;29(44):10141–10147. [PubMed]
  • Gopalakrishnan V, Peliska JA, Benkovic SJ. Human immunodeficiency virus type 1 reverse transcriptase: spatial and temporal relationship between the polymerase and RNase H activities. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10763–10767. [PMC free article] [PubMed]
  • Kati WM, Johnson KA, Jerva LF, Anderson KS. Mechanism and fidelity of HIV reverse transcriptase. J Biol Chem. 1992 Dec 25;267(36):25988–25997. [PubMed]
  • Götte M, Fackler S, Hermann T, Perola E, Cellai L, Gross HJ, Le Grice SF, Heumann H. HIV-1 reverse transcriptase-associated RNase H cleaves RNA/RNA in arrested complexes: implications for the mechanism by which RNase H discriminates between RNA/RNA and RNA/DNA. EMBO J. 1995 Feb 15;14(4):833–841. [PMC free article] [PubMed]
  • Götte M, Maier G, Onori AM, Cellai L, Wainberg MA, Heumann H. Temporal coordination between initiation of HIV (+)-strand DNA synthesis and primer removal. J Biol Chem. 1999 Apr 16;274(16):11159–11169. [PubMed]
  • Furfine ES, Reardon JE. Reverse transcriptase.RNase H from the human immunodeficiency virus. Relationship of the DNA polymerase and RNA hydrolysis activities. J Biol Chem. 1991 Jan 5;266(1):406–412. [PubMed]
  • Poch O, Sauvaget I, Delarue M, Tordo N. Identification of four conserved motifs among the RNA-dependent polymerase encoding elements. EMBO J. 1989 Dec 1;8(12):3867–3874. [PMC free article] [PubMed]
  • Xiong Y, Eickbush TH. Origin and evolution of retroelements based upon their reverse transcriptase sequences. EMBO J. 1990 Oct;9(10):3353–3362. [PMC free article] [PubMed]

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