title: Presence and location of modified nucleotides in Escherichia coli tmRNA: structural mimicry with tRNA acceptor branches.
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.
authors.affiliation: Howard Hughes Medical Institute, University of Utah, Salt Lake City, UT 84112, USA.
authors.forename: B
authors.initials: B
authors.lastname: Felden
authors.forename: K
authors.initials: K
authors.lastname: Hanawa
authors.forename: J F
authors.initials: JF
authors.lastname: Atkins
authors.forename: H
authors.initials: H
authors.lastname: Himeno
authors.forename: A
authors.initials: A
authors.lastname: Muto
authors.forename: R F
authors.initials: RF
authors.lastname: Gesteland
authors.forename: J A
authors.initials: JA
authors.lastname: McCloskey
authors.forename: P F
authors.initials: PF
authors.lastname: Crain
mesh_terms: D001482:Base Composition; D001483:Base Sequence; D002853:Chromatography, Liquid; D004926:Escherichia coli / Q000235:genetics*; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry; D011560:Pseudouridine / Q000737:chemistry; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics; D012329:RNA, Bacterial / Q000737:chemistry* / Q000378:metabolism*; D012343:RNA, Transfer / Q000737:chemistry* / Q000235:genetics*; D006163:Ribonuclease T1 / Q000378:metabolism; D014529:Uridine / Q000031:analogs & derivatives / Q000737:chemistry
chemical_list: D009841:Oligonucleotides; D012329:RNA, Bacterial; C096222:tmRNA; D011560:Pseudouridine; C009182:ribothymidine; D012343:RNA, Transfer; D006163:Ribonuclease T1; D014529:Uridine