Sequence and structure of naturally-occurring tRNA transcripts and site-directed variants are significant barriers to forming oligomers beyond dimers

摘要

Dimers of tRNAs can form through quasi self-complementary anticodon-anticodon interactions, for example at neutral pH in yeast tRNAAsp(GUC) and at pH4.5 inEscherichia coli tRNAGly(GCC) through a partially protonated interaction. The requirements for tRNA oligomerization, and the factors that prevented higher orders of structures forming were examined with unmodified wild-type and variant E. coli tRNAsGly(GCC). Non-denaturing agarose gel electrophoresis was used as a rapid screening method. A number of tRNAGly(GCC) variants with nucleotide substitutions in the loop regions formed dimers, but surprisingly there was no evidence that distinct higher oligomers formed in any of the variants tested. The dimer interfaces of two of the variants were delineated by competitive inhibition with complementary DNA oligonucleotides. Components of an oligomerization facilitating buffer, containing monovalent, divalent and multivalent cations (magnesium and sodium ions and spermine), were tested separately and in combination, to optimize oligomerization and its detection using agarose gel electrophoresis. A rationale for the requirement for magnesium for dimerization is suggested from its role in RNA loop-loop interactions. Sequence specific variant tRNAs that can rapidly form heterodimers with damaging infectious RNA are potential therapeutic agents against viral mechanisms by acting as base pairing inhibitors.