Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 May;177(10):2858–2862. doi: 10.1128/jb.177.10.2858-2862.1995

An anticodon sequence mutant of Escherichia coli initiator tRNA: possible importance of a newly acquired base modification next to the anticodon on its activity in initiation.

D Mangroo 1, P A Limbach 1, J A McCloskey 1, U L RajBhandary 1
PMCID: PMC176959  PMID: 7751297

Abstract

Initiator tRNAs from eubacteria and chloroplasts lack a base modification next to the anticodon. This is in contrast to virtually all other tRNAs from these sources. We show that a mutant Escherichia coli initiator tRNA which has an anticodon sequence change from CAU to CUA now has a 2-methylthio-N6-(delta 2-isopentenyl)adenosine (ms2i6A) modification, produced by posttranscriptional modification of A, next to the anticodon. This newly acquired base modification may be important for the function of the mutant tRNA in initiation. In a miaA mutant strain of E. coli defective in biosynthesis of ms2i6A, the mutant initiator tRNA is 10- to 12-fold less active in initiation. The mutant tRNA is aminoacylated and formylated normally in the miaA strain. Thus, the absence of the base modification affects the activity of the mutant tRNA at a step subsequent to its formylation.

Full Text

The Full Text of this article is available as a PDF (289.3 KB).

Selected References

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

  1. Bouadloun F., Srichaiyo T., Isaksson L. A., Björk G. R. Influence of modification next to the anticodon in tRNA on codon context sensitivity of translational suppression and accuracy. J Bacteriol. 1986 Jun;166(3):1022–1027. doi: 10.1128/jb.166.3.1022-1027.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brombach M., Pon C. L. The unusual translational initiation codon AUU limits the expression of the infC (initiation factor IF3) gene of Escherichia coli. Mol Gen Genet. 1987 Jun;208(1-2):94–100. doi: 10.1007/BF00330428. [DOI] [PubMed] [Google Scholar]
  3. Crain P. F. Preparation and enzymatic hydrolysis of DNA and RNA for mass spectrometry. Methods Enzymol. 1990;193:782–790. doi: 10.1016/0076-6879(90)93450-y. [DOI] [PubMed] [Google Scholar]
  4. Donahue T. F., Cigan A. M., Pabich E. K., Valavicius B. C. Mutations at a Zn(II) finger motif in the yeast eIF-2 beta gene alter ribosomal start-site selection during the scanning process. Cell. 1988 Aug 26;54(5):621–632. doi: 10.1016/s0092-8674(88)80006-0. [DOI] [PubMed] [Google Scholar]
  5. Dube S. K., Marcker K. A., Clark B. F., Cory S. Nucleotide sequence of N-formyl-methionyl-transfer RNA. Nature. 1968 Apr 20;218(5138):232–233. doi: 10.1038/218232a0. [DOI] [PubMed] [Google Scholar]
  6. Eisenberg S. P., Yarus M., Soll L. The effect of an Escherichia coli regulatory mutation on transfer RNA structure. J Mol Biol. 1979 Nov 25;135(1):111–126. doi: 10.1016/0022-2836(79)90343-7. [DOI] [PubMed] [Google Scholar]
  7. Gefter M. L., Russell R. L. Role modifications in tyrosine transfer RNA: a modified base affecting ribosome binding. J Mol Biol. 1969 Jan 14;39(1):145–157. doi: 10.1016/0022-2836(69)90339-8. [DOI] [PubMed] [Google Scholar]
  8. Gualerzi C. O., Pon C. L. Initiation of mRNA translation in prokaryotes. Biochemistry. 1990 Jun 26;29(25):5881–5889. doi: 10.1021/bi00477a001. [DOI] [PubMed] [Google Scholar]
  9. Harada F., Gross H. J., Kimura F., Chang S. H., Nishimura S., RajBhandary U. L. 2-Methylthio N6-(delta 2-isopentenyl) adenosine: a component of E. coli tyrosine transfer RNA. Biochem Biophys Res Commun. 1968 Oct 24;33(2):299–306. doi: 10.1016/0006-291x(68)90784-5. [DOI] [PubMed] [Google Scholar]
  10. Hartz D., Binkley J., Hollingsworth T., Gold L. Domains of initiator tRNA and initiation codon crucial for initiator tRNA selection by Escherichia coli IF3. Genes Dev. 1990 Oct;4(10):1790–1800. doi: 10.1101/gad.4.10.1790. [DOI] [PubMed] [Google Scholar]
  11. Laten H., Gorman J., Bock R. M. Isopentenyladenosine deficient tRNA from an antisuppressor mutant of Saccharomyces cerevisiae. Nucleic Acids Res. 1978 Nov;5(11):4329–4342. doi: 10.1093/nar/5.11.4329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. McClain W. H. Rules that govern tRNA identity in protein synthesis. J Mol Biol. 1993 Nov 20;234(2):257–280. doi: 10.1006/jmbi.1993.1582. [DOI] [PubMed] [Google Scholar]
  13. McClain W. H., Schneider J., Gabriel K. Distinctive acceptor-end structure and other determinants of Escherichia coli tRNAPro identity. Nucleic Acids Res. 1994 Feb 11;22(3):522–529. doi: 10.1093/nar/22.3.522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Petrullo L. A., Gallagher P. J., Elseviers D. The role of 2-methylthio-N6-isopentenyladenosine in readthrough and suppression of nonsense codons in Escherichia coli. Mol Gen Genet. 1983;190(2):289–294. doi: 10.1007/BF00330653. [DOI] [PubMed] [Google Scholar]
  15. Pomerantz S. C., McCloskey J. A. Analysis of RNA hydrolyzates by liquid chromatography-mass spectrometry. Methods Enzymol. 1990;193:796–824. doi: 10.1016/0076-6879(90)93452-q. [DOI] [PubMed] [Google Scholar]
  16. Prather N. E., Mims B. H., Murgola E. J. supG and supL in Escherichia coli code for mutant lysine tRNAs+. Nucleic Acids Res. 1983 Dec 10;11(23):8283–8286. doi: 10.1093/nar/11.23.8283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Raftery L. A., Yarus M. Systematic alterations in the anticodon arm make tRNA(Glu)-Suoc a more efficient suppressor. EMBO J. 1987 May;6(5):1499–1506. doi: 10.1002/j.1460-2075.1987.tb02392.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. RajBhandary U. L. Initiator transfer RNAs. J Bacteriol. 1994 Feb;176(3):547–552. doi: 10.1128/jb.176.3.547-552.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. RajBhandary U. L., Kumar A. A formylatable methionine transfer ribonucleic acid from yeast: comparison of coding properties and sequences around the anticodon with Escherichia coli formylatable methionine transfer RNA. J Mol Biol. 1970 Jun 28;50(3):707–711. doi: 10.1016/0022-2836(70)90095-1. [DOI] [PubMed] [Google Scholar]
  20. Saks M. E., Sampson J. R., Abelson J. N. The transfer RNA identity problem: a search for rules. Science. 1994 Jan 14;263(5144):191–197. doi: 10.1126/science.7506844. [DOI] [PubMed] [Google Scholar]
  21. Seong B. L., RajBhandary U. L. Escherichia coli formylmethionine tRNA: mutations in GGGCCC sequence conserved in anticodon stem of initiator tRNAs affect initiation of protein synthesis and conformation of anticodon loop. Proc Natl Acad Sci U S A. 1987 Jan;84(2):334–338. doi: 10.1073/pnas.84.2.334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Smith D., Yarus M. tRNA-tRNA interactions within cellular ribosomes. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4397–4401. doi: 10.1073/pnas.86.12.4397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sprinzl M., Dank N., Nock S., Schön A. Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Res. 1991 Apr 25;19 (Suppl):2127–2171. doi: 10.1093/nar/19.suppl.2127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Tsang T. H., Buck M., Ames B. N. Sequence specificity of tRNA-modifying enzymes. An analysis of 258 tRNA sequences. Biochim Biophys Acta. 1983 Nov 17;741(2):180–196. doi: 10.1016/0167-4781(83)90058-1. [DOI] [PubMed] [Google Scholar]
  25. Tsui H. C., Leung H. C., Winkler M. E. Characterization of broadly pleiotropic phenotypes caused by an hfq insertion mutation in Escherichia coli K-12. Mol Microbiol. 1994 Jul;13(1):35–49. doi: 10.1111/j.1365-2958.1994.tb00400.x. [DOI] [PubMed] [Google Scholar]
  26. Vacher J., Grosjean H., Houssier C., Buckingham R. H. The effect of point mutations affecting Escherichia coli tryptophan tRNA on anticodon-anticodon interactions and on UGA suppression. J Mol Biol. 1984 Aug 5;177(2):329–342. doi: 10.1016/0022-2836(84)90460-1. [DOI] [PubMed] [Google Scholar]
  27. Varshney U., Lee C. P., RajBhandary U. L. Direct analysis of aminoacylation levels of tRNAs in vivo. Application to studying recognition of Escherichia coli initiator tRNA mutants by glutaminyl-tRNA synthetase. J Biol Chem. 1991 Dec 25;266(36):24712–24718. [PubMed] [Google Scholar]
  28. Varshney U., Lee C. P., Seong B. L., RajBhandary U. L. Mutants of initiator tRNA that function both as initiators and elongators. J Biol Chem. 1991 Sep 25;266(27):18018–18024. [PubMed] [Google Scholar]
  29. Varshney U., RajBhandary U. L. Initiation of protein synthesis from a termination codon. Proc Natl Acad Sci U S A. 1990 Feb;87(4):1586–1590. doi: 10.1073/pnas.87.4.1586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Yanofsky C. Mutations affecting tRNATrp and its charging and their effect on regulation of transcription termination at the attenuator of the tryptophan operon. J Mol Biol. 1977 Jul 15;113(4):663–677. doi: 10.1016/0022-2836(77)90229-7. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES