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.