Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1992 Oct 15;89(20):9652–9656. doi: 10.1073/pnas.89.20.9652

Reverse transcriptase of human immunodeficiency virus can use either human tRNA(3Lys) or Escherichia coli tRNA(2Gln) as a primer in an in vitro primer-utilization assay.

L A Kohlstaedt 1, T A Steitz 1
PMCID: PMC50190  PMID: 1384059

Abstract

Although the reverse transcriptase (RT) of human immunodeficiency virus (HIV) uses human tRNA(3Lys) as a primer of viral genome DNA synthesis in vivo, HIV RT binds Escherichia coli glutamine tRNA and in vitro-made human lysine tRNA with nearly equivalent affinities. We show that HIV RT can use either tRNA(3Lys) or tRNA(2Gln) as a primer for DNA synthesis in vitro without the addition of any other host or viral proteins. E. coli tRNA(2Gln) can serve as a primer for HIV RT if a primer-binding site sequence complementary to the 3' end of tRNA(2Gln) is at the 3' end of the template. With this reduced template, the specificity of binding the proper tRNA is due to base-pairing between a bound tRNA to the primer-binding site of the viral RNA template rather than sequence-specific recognition of tRNA(3Lys) by RT. If an 8-nucleotide viral sequence 3' to the primer-binding site is included in the template, then addition of Zn2+ or Co2+ is required for tRNA(3Lys)-primed synthesis, and tRNA(2Gln) now fails to prime synthesis. The latter result implies that a template sequence adjacent to the primer-binding site and containing 6 nucleotides complementary to the anticodon loop of human tRNA(3Lys) plays an active role in tRNA discrimination.

Full text

PDF
9652

Images in this article

9653Image
on p.9653
9654Image
on p.9654
9655Image
on p.9655
9655Image
on p.9655

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Araya A., Sarih L., Litvak S. Reverse transcriptase mediated binding of primer tRNA to the viral genome. Nucleic Acids Res. 1979 Aug 24;6(12):3831–3843. doi: 10.1093/nar/6.12.3831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barat C., Le Grice S. F., Darlix J. L. Interaction of HIV-1 reverse transcriptase with a synthetic form of its replication primer, tRNA(Lys,3). Nucleic Acids Res. 1991 Feb 25;19(4):751–757. doi: 10.1093/nar/19.4.751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barat C., Lullien V., Schatz O., Keith G., Nugeyre M. T., Grüninger-Leitch F., Barré-Sinoussi F., LeGrice S. F., Darlix J. L. HIV-1 reverse transcriptase specifically interacts with the anticodon domain of its cognate primer tRNA. EMBO J. 1989 Nov;8(11):3279–3285. doi: 10.1002/j.1460-2075.1989.tb08488.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. D'Aquila R. T., Summers W. C. HIV-1 reverse transcriptase/ribonuclease H: high level expression in Escherichia coli from a plasmid constructed using the polymerase chain reaction. J Acquir Immune Defic Syndr. 1989;2(6):579–587. [PubMed] [Google Scholar]
  5. Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Furfine E. S., Reardon J. E. Human immunodeficiency virus reverse transcriptase ribonuclease H: specificity of tRNA(Lys3)-primer excision. Biochemistry. 1991 Jul 23;30(29):7041–7046. doi: 10.1021/bi00243a001. [DOI] [PubMed] [Google Scholar]
  7. Goff S. P. Retroviral reverse transcriptase: synthesis, structure, and function. J Acquir Immune Defic Syndr. 1990;3(8):817–831. [PubMed] [Google Scholar]
  8. Kohlstaedt L. A., Wang J., Friedman J. M., Rice P. A., Steitz T. A. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science. 1992 Jun 26;256(5065):1783–1790. doi: 10.1126/science.1377403. [DOI] [PubMed] [Google Scholar]
  9. Omer C. A., Faras A. J. Mechanism of release of the avian rotavirus tRNATrp primer molecule from viral DNA by ribonuclease H during reverse transcription. Cell. 1982 Oct;30(3):797–805. doi: 10.1016/0092-8674(82)90284-7. [DOI] [PubMed] [Google Scholar]
  10. Perona J. J., Swanson R., Steitz T. A., Söll D. Overproduction and purification of Escherichia coli tRNA(2Gln) and its use in crystallization of the glutaminyl-tRNA synthetase-tRNA(Gln) complex. J Mol Biol. 1988 Jul 5;202(1):121–126. doi: 10.1016/0022-2836(88)90524-4. [DOI] [PubMed] [Google Scholar]
  11. Prats A. C., Sarih L., Gabus C., Litvak S., Keith G., Darlix J. L. Small finger protein of avian and murine retroviruses has nucleic acid annealing activity and positions the replication primer tRNA onto genomic RNA. EMBO J. 1988 Jun;7(6):1777–1783. doi: 10.1002/j.1460-2075.1988.tb03008.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Sampson J. R., Uhlenbeck O. C. Biochemical and physical characterization of an unmodified yeast phenylalanine transfer RNA transcribed in vitro. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1033–1037. doi: 10.1073/pnas.85.4.1033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Sarih L., Araya A., Litvak S. Characterization of the cDNA synthesized by avian retrovirus reverse transcriptase using 35 S avian myeloblastosis virus RNA and an exogenous bovine primer tRNA. FEBS Lett. 1988 Mar 28;230(1-2):61–66. doi: 10.1016/0014-5793(88)80642-2. [DOI] [PubMed] [Google Scholar]
  14. Schwartz D. E., Tizard R., Gilbert W. Nucleotide sequence of Rous sarcoma virus. Cell. 1983 Mar;32(3):853–869. doi: 10.1016/0092-8674(83)90071-5. [DOI] [PubMed] [Google Scholar]
  15. Vallee B. L., Auld D. S. Active-site zinc ligands and activated H2O of zinc enzymes. Proc Natl Acad Sci U S A. 1990 Jan;87(1):220–224. doi: 10.1073/pnas.87.1.220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Vioque A., Arnez J., Altman S. Protein-RNA interactions in the RNase P holoenzyme from Escherichia coli. J Mol Biol. 1988 Aug 20;202(4):835–848. doi: 10.1016/0022-2836(88)90562-1. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES