Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1979 Nov;140(2):408–414. doi: 10.1128/jb.140.2.408-414.1979

Function of Modified Nucleosides 7-Methylguanosine, Ribothymidine, and 2-Thiomethyl-N6-(Isopentenyl)adenosine in Procaryotic Transfer Ribonucleic Acid

A Hoburg 1, H J Aschhoff 1, H Kersten 1, U Manderschied 2, H G Gassen 2
PMCID: PMC216664  PMID: 115845

Abstract

To elucidate subtle functions of transfer ribonucleic acid (tRNA) modifications in protein synthesis, pairs of tRNA's that differ in modifications at specific positions were prepared from Bacillus subtilis. The tRNA's differ in modifications in the anticodon loop, the extra arm, and the TU̷C loop. The functional properties of these species were compared in aminoacylation, as well as in initiation and peptide bond formation, at programmed ribosomes. These experiments demonstrated the following. (i) In tRNAfMet the methylation of guanosine 46 in the extra arm to 7-methylguanosine by the 7-methylguanosine–forming enzyme from Escherichia coli changes the aminoacylation kinetics for the B. subtilis methionyl-tRNA synthetase. In repeated experiments the Vmax value is decreased by one-half. (ii) tRNAfMet species with ribothymidine at position 54 (rT54) or uridine at position 54 (U54) were obtained from untreated or trimethoprim-treated B. subtilis. The formylated fMet-tRNAfMet species with U54 and rT54, respectively, function equally well in an in vitro initiation system containing AUG, initiation factors, and 70s ribosomes. The unformylated Met-tRNAtMet species, however, differ from each other: “Met-tRNAfMet rT” is inactive, whereas the U54 counter-upart effectively forms the initiation complex. (iii) Two isoacceptors, tRNA1Phe and tRNA2Phe, were obtained from B. subtilis. tRNA1Phe accumulates only under special growth conditions and is an incompletely modified precursor oftRNA2Phe: in the first position of the anticodon, guanosine replaces Gm, and next to the 3′ end of the anticodon (isopentenyl)adenosine replaces 2-thiomethyl-N6-(isopentenyl)adenosine. Both tRNA's behave identically in aminoacylation kinetics. In the factor-dependent AUGU3-directed formation of fMet-Phe, the undermodified tRNA1Phe is always less efficient at Mg2+ concentrations between 5 and 15 mM than its mature counterpart.

Full text

PDF
408

Selected References

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

  1. Arnold H. H. Initiation of protein synthesis in bacillus subtilis in the presence of trimethoprim or aminopterin. Biochim Biophys Acta. 1977 May 3;476(1):76–87. doi: 10.1016/0005-2787(77)90287-8. [DOI] [PubMed] [Google Scholar]
  2. Aschhoff H. J., Elten H., Arnold H. H., Mahal G., Kersten W., Kersten H. 7-Methylguanine specific tRNA-methyltransferase from Escherichia coli. Nucleic Acids Res. 1976 Nov;3(11):3109–3122. doi: 10.1093/nar/3.11.3109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Davanloo P., Sprinzl M., Watanabe K., Albani M., Kersten H. Role of ribothymidine in the thermal stability of transfer RNA as monitored by proton magnetic resonance. Nucleic Acids Res. 1979 Apr;6(4):1571–1581. doi: 10.1093/nar/6.4.1571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Egan B. Z. Separation of oligonucleotides by reversed-phase chromatography. Biochim Biophys Acta. 1973 Mar 19;299(2):245–252. doi: 10.1016/0005-2787(73)90347-x. [DOI] [PubMed] [Google Scholar]
  5. Erbe R. W., Nau M. M., Leder P. Translation and translocation of defined RNA messengers. J Mol Biol. 1969 Feb 14;39(3):441–460. doi: 10.1016/0022-2836(69)90137-5. [DOI] [PubMed] [Google Scholar]
  6. Gauss D. H., Grüter F., Sprinzl M. Compilation of tRNA sequences. Nucleic Acids Res. 1979 Jan;6(1):r1–r19. [PMC free article] [PubMed] [Google Scholar]
  7. Grosjean H., Söll D. G., Crothers D. M. Studies of the complex between transfer RNAs with complementary anticodons. I. Origins of enhanced affinity between complementary triplets. J Mol Biol. 1976 May 25;103(3):499–519. doi: 10.1016/0022-2836(76)90214-x. [DOI] [PubMed] [Google Scholar]
  8. Grunberg-Manago M., Gros F. Initiation mechanisms of protein syntehesis. Prog Nucleic Acid Res Mol Biol. 1977;20:209–284. doi: 10.1016/s0079-6603(08)60474-2. [DOI] [PubMed] [Google Scholar]
  9. Keith G., Rogg H., Dirheimer G., Menichi B., Heyham T. Post-transcriptional modification of tyrosine tRNA as a function of growth in Bacillus subtilis. FEBS Lett. 1976 Jan 15;61(2):120–123. doi: 10.1016/0014-5793(76)81017-4. [DOI] [PubMed] [Google Scholar]
  10. Leder P., Bursztyn H. Initiation of protein synthesis,I. Effect of formylation of methionyl-tRNA on codon recognition. Proc Natl Acad Sci U S A. 1966 Nov;56(5):1579–1585. doi: 10.1073/pnas.56.5.1579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Leifer W., Kreuzer T. Experiments and theoretical calculations for forming gradients for zonal rotor centrifugation. Anal Biochem. 1971 Nov;44(1):89–96. doi: 10.1016/0003-2697(71)90349-6. [DOI] [PubMed] [Google Scholar]
  12. Marinus M. G., Morris N. R., Söll D., Kwong T. C. Isolation and partial characterization of three Escherichia coli mutants with altered transfer ribonucleic acid methylases. J Bacteriol. 1975 Apr;122(1):257–265. doi: 10.1128/jb.122.1.257-265.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Menichi B., Heyman T. Study of tyrosine transfer ribonucleic acid modification in relation to sporulation in Bacillus subtilis. J Bacteriol. 1976 Jul;127(1):268–280. doi: 10.1128/jb.127.1.268-280.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Mohr S. C., Thach R. E. Application of ribonuclease T1 to the synthesis of oligoribonucleotides of defined base sequence. J Biol Chem. 1969 Dec 25;244(24):6566–6576. [PubMed] [Google Scholar]
  15. Noll M., Hapke B., Schreier M. H., Noll H. Structural dynamics of bacterial ribosomes. I. Characterization of vacant couples and their relation to complexed ribosomes. J Mol Biol. 1973 Apr 5;75(2):281–294. doi: 10.1016/0022-2836(73)90021-1. [DOI] [PubMed] [Google Scholar]
  16. Quigley G. J., Rich A. Structural domains of transfer RNA molecules. Science. 1976 Nov 19;194(4267):796–806. doi: 10.1126/science.790568. [DOI] [PubMed] [Google Scholar]
  17. Raettig R., Schmidt W., Mahal G., Kersten H., Arnold H. H. Purification and characterization of tRNAMet-f, tRNAPhe and tRNATyr2 from Baccillus subtilis. Biochim Biophys Acta. 1976 Jun 18;435(2):109–118. doi: 10.1016/0005-2787(76)90241-0. [DOI] [PubMed] [Google Scholar]
  18. Rich A., Schimmel P. R. Structural organization of complexes of transfer RNAs with aminoacyl transfer RNA synthetases. Nucleic Acids Res. 1977;4(5):1649–1665. doi: 10.1093/nar/4.5.1649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Roe B., Michael M., Dudock B. Function of N2 methylguanine in phenylalanine transfer RNA. Nat New Biol. 1973 Dec 5;246(153):135–138. doi: 10.1038/newbio246135a0. [DOI] [PubMed] [Google Scholar]
  20. Samuel C. E., Rabinowitz J. C. Initiation of protein synthesis by folate-sufficient and folate-deficient Streptococcus faecalis R. Biochemical and biophysical properties of methionine transfer ribonucleic acid. J Biol Chem. 1974 Feb 25;249(4):1198–1206. [PubMed] [Google Scholar]
  21. Schmidt W., Arnold H. H., Kersten H. Tetrahydrofolate-dependent biosynthesis of ribothymidine in transfer ribonucleic acids of Gram-positive bacteria. J Bacteriol. 1977 Jan;129(1):15–21. doi: 10.1128/jb.129.1.15-21.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schwarz U., Menzel H. M., Gassen H. G. Codon-dependent rearrangement of the three-dimensional structure of phenylalanine tRNA, exposing the T-psi-C-G sequence for binding to the 50S ribosomal subunit. Biochemistry. 1976 Jun 1;15(11):2484–2490. doi: 10.1021/bi00656a035. [DOI] [PubMed] [Google Scholar]
  23. Singhal R. P., Vold B. Changes in transfer ribonucleic acids of Bacillus subtilis during different growth phases. Nucleic Acids Res. 1976 May;3(5):1249–1262. doi: 10.1093/nar/3.5.1249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sprinzl M., Cramer F. The -C-C-A end of tRNA and its role in protein biosynthesis. Prog Nucleic Acid Res Mol Biol. 1979;22:1–69. doi: 10.1016/s0079-6603(08)60798-9. [DOI] [PubMed] [Google Scholar]
  25. Sprinzl M., Wagner T., Lorenz S., Erdmann V. A. Regions of tRNA important for binding to the ribosomal A and P sites. Biochemistry. 1976 Jul 13;15(14):3031–3039. doi: 10.1021/bi00659a015. [DOI] [PubMed] [Google Scholar]
  26. Stern L., Schulman L. H. Role of anticodon bases in aminoacylation of Escherichia coli methionine transfer RNAs. J Biol Chem. 1977 Sep 25;252(18):6403–6408. [PubMed] [Google Scholar]
  27. Suttle D. P., Haralson M. A., Ravel J. M. Initiation factor 3 requirement for the formation of initiation complexes with synthetic oligonucleotides. Biochem Biophys Res Commun. 1973 Mar 17;51(2):376–382. doi: 10.1016/0006-291x(73)91268-0. [DOI] [PubMed] [Google Scholar]
  28. Watanabe K., Oshima T., Nishimura S. CD spectra of 5-methyl-2-thiouridine in tRNA-Met-f from an extreme thermophile. Nucleic Acids Res. 1976 Jul;3(7):1703–1713. doi: 10.1093/nar/3.7.1703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Watanabe K., Oshima T., Saneyoshi M., Nishimura S. Replacement of ribothymidine by 5-methyl-2-thiouridine in sequence GT psi C in tRNA of an extreme thermophile. FEBS Lett. 1974 Jul 1;43(1):59–63. doi: 10.1016/0014-5793(74)81105-1. [DOI] [PubMed] [Google Scholar]
  30. Wintermeyer W., Zachau H. G. Tertiary structure interactions of 7-methylguanosine in yeast tRNA Phe as studied by borohydride reduction. FEBS Lett. 1975 Oct 15;58(1):306–309. doi: 10.1016/0014-5793(75)80285-7. [DOI] [PubMed] [Google Scholar]
  31. Yamada Y., Ishikura H. Nucleotide sequence of initiator tRNA from Bacillus subtilis. FEBS Lett. 1975 Jun 15;54(2):155–158. doi: 10.1016/0014-5793(75)80064-0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES