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. 1996 Feb 15;314(Pt 1):49–53. doi: 10.1042/bj3140049

Effects of nucleotide substitutions within the T-loop of precursor tRNAs on interaction with ATP/CTP:tRNA nucleotidyltransferases from Escherichia coli and yeast.

Z Li 1, K A Gillis 1, L A Hegg 1, J Zhang 1, D L Thurlow 1
PMCID: PMC1217051  PMID: 8660309

Abstract

Recognition of tRNA and tRNA-like substrates by the enzyme ATP/CTP:tRNA nucleotidyltransferase requires chemically intact nucleotides within the T-loop, especially at positions 57 and 58, which are invariant purines among naturally occurring tRNAs. To test the effects of base substitutions at these positions, which are distant from the site of catalysis, we synthesized mutant tRNA(Glu) molecules. These in vitro-synthesized RNAs also contained an extra 33 bases at the 5' end and lacked post-transcriptionally modified bases. The precursor tRNAs were used as substrates for nucleotidyltransferases from Escherichia coli and yeast. Substitution of cytidines at either position 57 or 58 had dramatic inhibitory effects on recognition by both enzymes, including raising the apparent Km and lowering the apparent Vmax.; substitution of an adenosine at position 57 or a uridine at position 58 inhibited the reaction only slightly by comparison. Our results demonstrate that the identities of nucleotides at positions 57 and 58 are relevant to recognition by nucleotidyltransferase, and that a purine is required at position 57. The extra bases at the 5' end and the lack of post-transcriptionally modified bases did not substantially inhibit interaction with the enzyme, as judged by the wild-type precursor tRNA(Glu) acting as an effective substrate for both enzymes in the presence of equal concentrations of appropriate tRNA substrates isolated from E. coli.

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Selected References

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

  1. Deutscher M. P., Evans J. A. Transfer RNA nucleotidyltransferase repairs all transfer RNAs randomly. J Mol Biol. 1977 Feb 5;109(4):593–597. doi: 10.1016/s0022-2836(77)80093-4. [DOI] [PubMed] [Google Scholar]
  2. Deutscher M. P. Ribonucleases, tRNA nucleotidyltransferase, and the 3' processing of tRNA. Prog Nucleic Acid Res Mol Biol. 1990;39:209–240. doi: 10.1016/s0079-6603(08)60628-5. [DOI] [PubMed] [Google Scholar]
  3. Deutscher M. P. Transfer RNA nucleotidyltransferase. Methods Enzymol. 1990;181:434–439. doi: 10.1016/0076-6879(90)81141-g. [DOI] [PubMed] [Google Scholar]
  4. Giegé R., Puglisi J. D., Florentz C. tRNA structure and aminoacylation efficiency. Prog Nucleic Acid Res Mol Biol. 1993;45:129–206. doi: 10.1016/s0079-6603(08)60869-7. [DOI] [PubMed] [Google Scholar]
  5. Hegg L. A., Thurlow D. L. Cytidines in tRNAs that are required intact by ATP/CTP:tRNA nucleotidyltransferases from Escherichia coli and Saccharomyces cerevisiae. Nucleic Acids Res. 1990 Oct 25;18(20):5975–5979. doi: 10.1093/nar/18.20.5975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Jinks-Robertson S., Gourse R. L., Nomura M. Expression of rRNA and tRNA genes in Escherichia coli: evidence for feedback regulation by products of rRNA operons. Cell. 1983 Jul;33(3):865–876. doi: 10.1016/0092-8674(83)90029-6. [DOI] [PubMed] [Google Scholar]
  7. Joshi R. L., Joshi S., Chapeville F., Haenni A. L. tRNA-like structures of plant viral RNAs: conformational requirements for adenylation and aminoacylation. EMBO J. 1983;2(7):1123–1127. doi: 10.1002/j.1460-2075.1983.tb01556.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Masiakowski P., Deutscher M. P. Dissection of the active site of rabbit liver tRNA nucleotidyltransferase. Specificity and properties of the tRNA and acceptor subsites determined with model acceptor substrates. J Biol Chem. 1980 Dec 10;255(23):11233–11239. [PubMed] [Google Scholar]
  9. Masiakowski P., Deutscher M. P. Separation of functionally distinct regions of a macromolecular substrate. Stimulation of tRNA nucleotidyltransferase by a nonreacting fragment of tRNA. J Biol Chem. 1979 Apr 25;254(8):2585–2587. [PubMed] [Google Scholar]
  10. McClain W. H., Guerrier-Takada C., Altman S. Model substrates for an RNA enzyme. Science. 1987 Oct 23;238(4826):527–530. doi: 10.1126/science.2443980. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Spacciapoli P., Doviken L., Mulero J. J., Thurlow D. L. Recognition of tRNA by the enzyme ATP/CTP:tRNA nucleotidyltransferase. Interference by nucleotides modified with diethyl pyrocarbonate or hydrazine. J Biol Chem. 1989 Mar 5;264(7):3799–3805. [PubMed] [Google Scholar]
  13. Spacciapoli P., Thurlow D. L. Purines in tRNAs required for recognition by ATP/CTP:tRNA nucleotidyltransferase from rabbit liver. J Mol Recognit. 1990 Aug;3(4):149–155. doi: 10.1002/jmr.300030403. [DOI] [PubMed] [Google Scholar]

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