PRESENCE AND LOCATION OF MODIFIED NUCLEOTIDES IN ESCHERICHIA-COLI TMRNA - STRUCTURAL MIMICRY WITH TRANSFER-RNA ACCEPTOR BRANCHES

Escherichia coli tmRNA functions uniquely as both tRNA and mRNA and po ssesses structural elements similar to canonical tRNAs, To test whethe r this mimicry extends to post-transcriptional modification, the techn ique of combined liquid chromatography/ electrospray ionization mass s pectrometry (LC/ESIMS) and sequence data were used to determine the mo lecular masses of all oligonucleotides produced by RNase T-1 hydrolysi s with a mean error of 0.1 Da, Thus, this allowed for the detection, c hemical characterization and sequence placement of modified nucleotide s which produced a change in mass. Also, chemical modifications were u sed to locate mass-silent modifications. The native E.coli tmRNA conta ins two modified nucleosides, 5-methyluridine and pseudouridine, Both modifications are located within the proposed tRNA-like domain, in a s even-nucleotide loop mimicking the conserved sequence of T loops in ca nonical tRNAs, Although tmRNA acceptor branches (acceptor stem and T s tem-loop) utilize different architectural rules than those of canonica l tRNAs, their conformations in solution may be very similar. A compar ative structural and functional analysis of unmodified tmRNA made by i ll vitro transcription and native E,coli tmRNA suggests that one or bo th of these post-transcriptional modifications may be required for opt imal stability of the acceptor branch which is needed for efficient am inoacylation.