Yeast Suppressor Mutations and Transfer RNA Processing

Transfer RNA molecules are essential for protein synthesis in all organisms. Once mature-sized tRNAs are produced, further interactions with enzymes or structural proteins are necessary for their use in protein biosynthesis: transport from the nucleus to the cytoplasm, aminoacylation by the cognate aminoacyl-tRNA synthetase, binding of initiation or elongation factors, and ribosomal interactions during translation and proofreading. If the suppressor tRNA locus has not been cloned, the loss of suppressor function in red or pink colonies must be mapped to the locus of the tRNA gene by further analysis. This can be achieved by mating the mutant strain with appropriate tester strains, such as a strain containing the suppressor-inactive, wild-type anticodon for the tRNA in question. Primer-directed mutagenesis allows specific changes to be made within the tRNA gene coding sequence which can be tested for their effect on tRNA expression. To determine transcription efficiency, lower temperature, shorter reaction time, and lower template concentration are necessary to slow RNA processing reactions so that only the dimeric transcript is formed, leading to more accurate quantification.