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. 2000 Oct;6(10):1403–1412. doi: 10.1017/s1355838200000947

NMR and biochemical characterization of recombinant human tRNA(Lys)3 expressed in Escherichia coli: identification of posttranscriptional nucleotide modifications required for efficient initiation of HIV-1 reverse transcription.

C Tisné 1, M Rigourd 1, R Marquet 1, C Ehresmann 1, F Dardel 1
PMCID: PMC1370011  PMID: 11073216

Abstract

Reverse transcription of HIV-1 viral RNA uses human tRNA(Lys)3 as a primer. Some of the modified nucleotides carried by this tRNA must play a key role in the initiation of this process, because unmodified tRNA produced in vitro is only marginally active as primer. To provide a better understanding of the contribution of base modifications in the initiation complex, we have designed a recombinant system that allows tRNA(Lys)3 expression in Escherichia coli. Because of their high level of overexpression, some modifications are incorporated at substoichiometric levels. We have purified the two major recombinant tRNA(Lys)3 subspecies, and their modified nucleotide contents have been characterized by a combination of NMR and biochemical techniques. Both species carry psis, Ds, T, t6A, and m7G. Differences are observed at position 34, within the anticodon. One fraction lacks the 5-methylaminomethyl group, whereas the other lacks the 2-thio group. Although the s2U34-containing recombinant tRNA is a less efficient primer, it presents most of the characteristics of the mammalian tRNA. On the other hand, the mnm5U34-containing tRNA has a strongly reduced activity. Our results demonstrate that the modifications that are absent in E. coli (m2G10, psi27, m5C48, m5C49, and m1A58) as well as the mnm5 group at position 34 are dispensable for initiation of reverse transcription. In contrast, the 2-thio group at position 34 seems to play an important part in this process.

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Selected References

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  1. Burnett B. P., McHenry C. S. Posttranscriptional modification of retroviral primers is required for late stages of DNA replication. Proc Natl Acad Sci U S A. 1997 Jul 8;94(14):7210–7215. doi: 10.1073/pnas.94.14.7210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Carbon J. A., Hung L., Jones D. S. A reversible oxidative in activation of specific transfer RNA species. Proc Natl Acad Sci U S A. 1965 May;53(5):979–986. doi: 10.1073/pnas.53.5.979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Commans S., Lazard M., Delort F., Blanquet S., Plateau P. tRNA anticodon recognition and specification within subclass IIb aminoacyl-tRNA synthetases. J Mol Biol. 1998 May 15;278(4):801–813. doi: 10.1006/jmbi.1998.1711. [DOI] [PubMed] [Google Scholar]
  4. Heerschap A., Haasnoot C. A., Hilbers C. W. Nuclear magnetic resonance studies on yeast tRNAPhe. III. Assignments of the iminoproton resonances of the tertiary structure by means of nuclear Overhauser effect experiments at 500 MHz. Nucleic Acids Res. 1983 Jul 11;11(13):4501–4520. doi: 10.1093/nar/11.13.4501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Isel C., Ehresmann C., Keith G., Ehresmann B., Marquet R. Initiation of reverse transcription of HIV-1: secondary structure of the HIV-1 RNA/tRNA(3Lys) (template/primer). J Mol Biol. 1995 Mar 24;247(2):236–250. doi: 10.1006/jmbi.1994.0136. [DOI] [PubMed] [Google Scholar]
  6. Isel C., Keith G., Ehresmann B., Ehresmann C., Marquet R. Mutational analysis of the tRNA3Lys/HIV-1 RNA (primer/template) complex. Nucleic Acids Res. 1998 Mar 1;26(5):1198–1204. doi: 10.1093/nar/26.5.1198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Isel C., Marquet R., Keith G., Ehresmann C., Ehresmann B. Modified nucleotides of tRNA(3Lys) modulate primer/template loop-loop interaction in the initiation complex of HIV-1 reverse transcription. J Biol Chem. 1993 Dec 5;268(34):25269–25272. [PubMed] [Google Scholar]
  8. Isel C., Westhof E., Massire C., Le Grice S. F., Ehresmann B., Ehresmann C., Marquet R. Structural basis for the specificity of the initiation of HIV-1 reverse transcription. EMBO J. 1999 Feb 15;18(4):1038–1048. doi: 10.1093/emboj/18.4.1038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lanchy J. M., Ehresmann C., Le Grice S. F., Ehresmann B., Marquet R. Binding and kinetic properties of HIV-1 reverse transcriptase markedly differ during initiation and elongation of reverse transcription. EMBO J. 1996 Dec 16;15(24):7178–7187. [PMC free article] [PubMed] [Google Scholar]
  10. Lanchy J. M., Isel C., Keith G., Le Grice S. F., Ehresmann C., Ehresmann B., Marquet R. Dynamics of the HIV-1 reverse transcription complex during initiation of DNA synthesis. J Biol Chem. 2000 Apr 21;275(16):12306–12312. doi: 10.1074/jbc.275.16.12306. [DOI] [PubMed] [Google Scholar]
  11. Lanchy J. M., Keith G., Le Grice S. F., Ehresmann B., Ehresmann C., Marquet R. Contacts between reverse transcriptase and the primer strand govern the transition from initiation to elongation of HIV-1 reverse transcription. J Biol Chem. 1998 Sep 18;273(38):24425–24432. doi: 10.1074/jbc.273.38.24425. [DOI] [PubMed] [Google Scholar]
  12. Le Grice S. F., Grüninger-Leitch F. Rapid purification of homodimer and heterodimer HIV-1 reverse transcriptase by metal chelate affinity chromatography. Eur J Biochem. 1990 Jan 26;187(2):307–314. doi: 10.1111/j.1432-1033.1990.tb15306.x. [DOI] [PubMed] [Google Scholar]
  13. Leroy J. L., Bolo N., Figueroa N., Plateau P., Guérón M. Internal motions of transfer RNA: a study of exchanging protons by magnetic resonance. J Biomol Struct Dyn. 1985 Feb;2(5):915–939. doi: 10.1080/07391102.1985.10507609. [DOI] [PubMed] [Google Scholar]
  14. Mak J., Kleiman L. Primer tRNAs for reverse transcription. J Virol. 1997 Nov;71(11):8087–8095. doi: 10.1128/jvi.71.11.8087-8095.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Marquet R., Isel C., Ehresmann C., Ehresmann B. tRNAs as primer of reverse transcriptases. Biochimie. 1995;77(1-2):113–124. doi: 10.1016/0300-9084(96)88114-4. [DOI] [PubMed] [Google Scholar]
  16. Meinnel T., Mechulam Y., Fayat G. Fast purification of a functional elongator tRNAmet expressed from a synthetic gene in vivo. Nucleic Acids Res. 1988 Aug 25;16(16):8095–8096. doi: 10.1093/nar/16.16.8095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Motorin Y., Grosjean H. Multisite-specific tRNA:m5C-methyltransferase (Trm4) in yeast Saccharomyces cerevisiae: identification of the gene and substrate specificity of the enzyme. RNA. 1999 Aug;5(8):1105–1118. doi: 10.1017/s1355838299982201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Paillart J. C., Marquet R., Skripkin E., Ehresmann B., Ehresmann C. Mutational analysis of the bipartite dimer linkage structure of human immunodeficiency virus type 1 genomic RNA. J Biol Chem. 1994 Nov 4;269(44):27486–27493. [PubMed] [Google Scholar]
  19. Roy S., Papastavros M. Z., Sanchez V., Redfield A. G. Nitrogen-15-labeled yeast tRNAPhe: double and two-dimensional heteronuclear NMR of guanosine and uracil ring NH groups. Biochemistry. 1984 Sep 11;23(19):4395–4400. doi: 10.1021/bi00314a024. [DOI] [PubMed] [Google Scholar]
  20. Skripkin E., Isel C., Marquet R., Ehresmann B., Ehresmann C. Psoralen crosslinking between human immunodeficiency virus type 1 RNA and primer tRNA3(Lys). Nucleic Acids Res. 1996 Feb 1;24(3):509–514. doi: 10.1093/nar/24.3.509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sullivan M. A., Cannon J. F., Webb F. H., Bock R. M. Antisuppressor mutation in Escherichia coli defective in biosynthesis of 5-methylaminomethyl-2-thiouridine. J Bacteriol. 1985 Jan;161(1):368–376. doi: 10.1128/jb.161.1.368-376.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Szewczak A. A., Kellogg G. W., Moore P. B. Assignment of NH resonances in nucleic acids using natural abundance 15N-1H correlation spectroscopy with spin-echo and gradient pulses. FEBS Lett. 1993 Aug 2;327(3):261–264. doi: 10.1016/0014-5793(93)81000-p. [DOI] [PubMed] [Google Scholar]
  23. Wallis N. G., Dardel F., Blanquet S. Heteronuclear NMR studies of the interactions of 15N-labeled methionine-specific transfer RNAs with methionyl-tRNA transformylase. Biochemistry. 1995 Jun 13;34(23):7668–7677. doi: 10.1021/bi00023a013. [DOI] [PubMed] [Google Scholar]
  24. Yan X., Xue H., Liu H., Hang J., Wong J. T., Zhu G. NMR studies of Bacillus subtilis tRNA(Trp) hyperexpressed in Escherichia coli. Assignment of imino proton signals and determination of thermal stability. J Biol Chem. 2000 Mar 10;275(10):6712–6716. doi: 10.1074/jbc.275.10.6712. [DOI] [PubMed] [Google Scholar]

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