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

Abstract

Dimers of tRNAs can form through quasi self-complementary anticodon-anticodon interactions, for example at neutral pH in yeast tRNA Asp(GUC) and at pH4.5 in Escherichia coli tRNA Gly(GCC) through a partially protonated interaction. The requirements for tRNA oligomerization, and the factors that prevented higher orders of structures forming were ex amined with unmodified wild-type and variant E. coli tRNAs Gly(GCC) . Non-denaturing agarose gel electro phoresis was used as a rapid screening method. A number of tRNA Gly(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, di valent and multivalent cations (magnesium and so dium 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.