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. 1998 Jun 1;17(11):3188–3196. doi: 10.1093/emboj/17.11.3188

Presence and location of modified nucleotides in Escherichia coli tmRNA: structural mimicry with tRNA acceptor branches.

B Felden 1, K Hanawa 1, J F Atkins 1, H Himeno 1, A Muto 1, R F Gesteland 1, J A McCloskey 1, P F Crain 1
PMCID: PMC1170657  PMID: 9606200

Abstract

Escherichia coli tmRNA functions uniquely as both tRNA and mRNA and possesses structural elements similar to canonical tRNAs. To test whether this mimicry extends to post-transcriptional modification, the technique of combined liquid chromatography/ electrospray ionization mass spectrometry (LC/ESIMS) and sequence data were used to determine the molecular masses of all oligonucleotides produced by RNase T1 hydrolysis with a mean error of 0.1 Da. Thus, this allowed for the detection, chemical characterization and sequence placement of modified nucleotides which produced a change in mass. Also, chemical modifications were used to locate mass-silent modifications. The native E.coli tmRNA contains two modified nucleosides, 5-methyluridine and pseudouridine. Both modifications are located within the proposed tRNA-like domain, in a seven-nucleotide loop mimicking the conserved sequence of T loops in canonical tRNAs. Although tmRNA acceptor branches (acceptor stem and T stem-loop) utilize different architectural rules than those of canonical tRNAs, their conformations in solution may be very similar. A comparative structural and functional analysis of unmodified tmRNA made by in vitro transcription and native E.coli tmRNA suggests that one or both of these post-transcriptional modifications may be required for optimal stability of the acceptor branch which is needed for efficient aminoacylation.

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Selected References

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

  1. Bakin A., Ofengand J. Four newly located pseudouridylate residues in Escherichia coli 23S ribosomal RNA are all at the peptidyltransferase center: analysis by the application of a new sequencing technique. Biochemistry. 1993 Sep 21;32(37):9754–9762. doi: 10.1021/bi00088a030. [DOI] [PubMed] [Google Scholar]
  2. 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]
  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. Denman R., Colgan J., Nurse K., Ofengand J. Crosslinking of the anticodon of P site bound tRNA to C-1400 of E.coli 16S RNA does not require the participation of the 50S subunit. Nucleic Acids Res. 1988 Jan 11;16(1):165–178. doi: 10.1093/nar/16.1.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Felden B., Atkins J. F., Gesteland R. F. tRNA and mRNA both in the same molecule. Nat Struct Biol. 1996 Jun;3(6):494–494. doi: 10.1038/nsb0696-494. [DOI] [PubMed] [Google Scholar]
  6. Felden B., Himeno H., Muto A., Atkins J. F., Gesteland R. F. Structural organization of Escherichia coli tmRNA. Biochimie. 1996;78(11-12):979–983. doi: 10.1016/s0300-9084(97)86720-x. [DOI] [PubMed] [Google Scholar]
  7. Felden B., Himeno H., Muto A., McCutcheon J. P., Atkins J. F., Gesteland R. F. Probing the structure of the Escherichia coli 10Sa RNA (tmRNA). RNA. 1997 Jan;3(1):89–103. [PMC free article] [PubMed] [Google Scholar]
  8. Fenn J. B., Mann M., Meng C. K., Wong S. F., Whitehouse C. M. Electrospray ionization for mass spectrometry of large biomolecules. Science. 1989 Oct 6;246(4926):64–71. doi: 10.1126/science.2675315. [DOI] [PubMed] [Google Scholar]
  9. Gu X. R., Santi D. V. The T-arm of tRNA is a substrate for tRNA (m5U54)-methyltransferase. Biochemistry. 1991 Mar 26;30(12):2999–3002. doi: 10.1021/bi00226a003. [DOI] [PubMed] [Google Scholar]
  10. Hahner S., Lüdemann H. C., Kirpekar F., Nordhoff E., Roepstorff P., Galla H. J., Hillenkamp F. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI) of endonuclease digests of RNA. Nucleic Acids Res. 1997 May 15;25(10):1957–1964. doi: 10.1093/nar/25.10.1957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Himeno H., Sato M., Tadaki T., Fukushima M., Ushida C., Muto A. In vitro trans translation mediated by alanine-charged 10Sa RNA. J Mol Biol. 1997 May 23;268(5):803–808. doi: 10.1006/jmbi.1997.1011. [DOI] [PubMed] [Google Scholar]
  12. Ho N. W., Gilham P. T. Reaction of pseudouridine and inosine with N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethylcarbodiimide. Biochemistry. 1971 Sep 28;10(20):3651–3657. [PubMed] [Google Scholar]
  13. Hou Y. M., Schimmel P. A simple structural feature is a major determinant of the identity of a transfer RNA. Nature. 1988 May 12;333(6169):140–145. doi: 10.1038/333140a0. [DOI] [PubMed] [Google Scholar]
  14. Jain S. K., Gurevitz M., Apirion D. A small RNA that complements mutants in the RNA processing enzyme ribonuclease P. J Mol Biol. 1982 Dec 15;162(3):515–533. doi: 10.1016/0022-2836(82)90386-2. [DOI] [PubMed] [Google Scholar]
  15. Kammen H. O., Marvel C. C., Hardy L., Penhoet E. E. Purification, structure, and properties of Escherichia coli tRNA pseudouridine synthase I. J Biol Chem. 1988 Feb 15;263(5):2255–2263. [PubMed] [Google Scholar]
  16. Kealey J. T., Santi D. V. High-level expression and rapid purification of tRNA (m5U54)-methyltransferase. Protein Expr Purif. 1994 Apr;5(2):149–152. doi: 10.1006/prep.1994.1023. [DOI] [PubMed] [Google Scholar]
  17. Keiler K. C., Waller P. R., Sauer R. T. Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA. Science. 1996 Feb 16;271(5251):990–993. doi: 10.1126/science.271.5251.990. [DOI] [PubMed] [Google Scholar]
  18. Komine Y., Kitabatake M., Yokogawa T., Nishikawa K., Inokuchi H. A tRNA-like structure is present in 10Sa RNA, a small stable RNA from Escherichia coli. Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9223–9227. doi: 10.1073/pnas.91.20.9223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kowalak J. A., Pomerantz S. C., Crain P. F., McCloskey J. A. A novel method for the determination of post-transcriptional modification in RNA by mass spectrometry. Nucleic Acids Res. 1993 Sep 25;21(19):4577–4585. doi: 10.1093/nar/21.19.4577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lee S. Y., Bailey S. C., Apirion D. Small stable RNAs from Escherichia coli: evidence for the existence of new molecules and for a new ribonucleoprotein particle containing 6S RNA. J Bacteriol. 1978 Feb;133(2):1015–1023. doi: 10.1128/jb.133.2.1015-1023.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. McClain W. H., Foss K. Changing the identity of a tRNA by introducing a G-U wobble pair near the 3' acceptor end. Science. 1988 May 6;240(4853):793–796. doi: 10.1126/science.2452483. [DOI] [PubMed] [Google Scholar]
  22. McCloskey J. A., Crain P. F. The RNA modification database--1998. Nucleic Acids Res. 1998 Jan 1;26(1):196–197. doi: 10.1093/nar/26.1.196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nurse K., Wrzesinski J., Bakin A., Lane B. G., Ofengand J. Purification, cloning, and properties of the tRNA psi 55 synthase from Escherichia coli. RNA. 1995 Mar;1(1):102–112. [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Ramos A., Varani G. Structure of the acceptor stem of Escherichia coli tRNA Ala: role of the G3.U70 base pair in synthetase recognition. Nucleic Acids Res. 1997 Jun 1;25(11):2083–2090. doi: 10.1093/nar/25.11.2083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ray B. K., Apirion D. Characterization of 10S RNA: a new stable rna molecule from Escherichia coli. Mol Gen Genet. 1979 Jul 2;174(1):25–32. doi: 10.1007/BF00433301. [DOI] [PubMed] [Google Scholar]
  27. Sprinzl M., Horn C., Brown M., Ioudovitch A., Steinberg S. Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Res. 1998 Jan 1;26(1):148–153. doi: 10.1093/nar/26.1.148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Suddath F. L., Quigley G. J., McPherson A., Sneden D., Kim J. J., Kim S. H., Rich A. Three-dimensional structure of yeast phenylalanine transfer RNA at 3.0angstroms resolution. Nature. 1974 Mar 1;248(5443):20–24. doi: 10.1038/248020a0. [DOI] [PubMed] [Google Scholar]
  29. Tamura K., Asahara H., Himeno H., Hasegawa T., Shimizu M. Identity elements of Escherichia coli tRNA(Ala). J Mol Recognit. 1991 Jul-Dec;4(4):129–132. doi: 10.1002/jmr.300040404. [DOI] [PubMed] [Google Scholar]
  30. Tu G. F., Reid G. E., Zhang J. G., Moritz R. L., Simpson R. J. C-terminal extension of truncated recombinant proteins in Escherichia coli with a 10Sa RNA decapeptide. J Biol Chem. 1995 Apr 21;270(16):9322–9326. doi: 10.1074/jbc.270.16.9322. [DOI] [PubMed] [Google Scholar]
  31. Ushida C., Himeno H., Watanabe T., Muto A. tRNA-like structures in 10Sa RNAs of Mycoplasma capricolum and Bacillus subtilis. Nucleic Acids Res. 1994 Aug 25;22(16):3392–3396. doi: 10.1093/nar/22.16.3392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Williams K. P., Bartel D. P. Phylogenetic analysis of tmRNA secondary structure. RNA. 1996 Dec;2(12):1306–1310. [PMC free article] [PubMed] [Google Scholar]
  33. Williams K. P., Bartel D. P. The tmRNA Website. Nucleic Acids Res. 1998 Jan 1;26(1):163–165. doi: 10.1093/nar/26.1.163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Zwieb C., Larsen N., Wower J. The tmRNA database (tmRDB). Nucleic Acids Res. 1998 Jan 1;26(1):166–167. doi: 10.1093/nar/26.1.166. [DOI] [PMC free article] [PubMed] [Google Scholar]

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