In vivo misreading by tRNA overdose

Rpb5-H147R is an AT-GC transition replacing CAC(His) by CGC(Arg) at a conse rved and critical position of ABC27 (Rpb5p), one of the five common and ess ential subunits shared by all three eukaryotic RNA polymerases. This mutati on is viable at 25 degrees C, but has a lethal phenotype at 34 degrees C. A search for dosage-dependent suppressors identified five distinct clones th at all bear a copy of the tRNA(GUG)(His) gene. Suppression was also observe d with a small genomic insert bearing this tRNA gene and no other coding se quences, under conditions where there is a sevenfold increase in the cellul ar concentration of tRNA(GUG)(His). Overexpressing tRNA(ICG)(Arg), which no rmally decodes the suppressed CGC codon, counteracted suppression, Suppress ion is codon specific because it was abolished when replacing CGC by its sy nonymous codons CGA, CGU, or AGA, but was not detectably affected by severa l nucleotide substitutions modifying the surrounding sequence and is thus l argely insensitive to the nucleotide context. It is proposed that overexpre ssing tRNA(GUG)(His) extends its decoding properties from CAC(His) to the n oncognate CGC(Arg) codon through an illegitimate U.G pairing at the middle base of the anticodon. Accordingly, tRNA(GUG)(His) would compete with tRNA( ICG)(Arg) for chain elongation and generate a significant level of misreadi ng errors under normal growth conditions.