Investigating tRNA abundance as a determinant of decoding speed in Saccharomyces cerevisiae.

There are two main points that control the rate of translation, translation initiation which controls the rate of recruitment of ribosomes to the mRNA, and translation elongation which controls the speed with which ribosomes decode the mRNA. When initiation is not limiting, ribosomal queues can build up, reducing ribosomal clearance from the start codon. The dynamics of elongation control are largely determined by the decoding speed of individual codons. One way in which the system controls the rate and efficiency of translation is through codon bias, taking advantage of the differences in decoding speed. Our aim in this study is to investigate the role of tRNA abundance in determining decoding speed for the synonymous codons of threonine.

Using Saccharomyces cerevisiae, we build codon reporter constructs where a codon optimised Renilla luciferase gene was preceded by ten codon repeats of one of the four threonine codons, in such a way that the decoding speed of the codon repeats determined the expression levels of the luciferase. Our analyses showed that the observed protein levels of RLuc correlated with the predicted order of decoding speeds, based on our knowledge of threonyl-tRNA populations, in descending order ACT, ACA, ACC and ACG. However, RNA analyses suggest that only one out of the four threonine reporters, ACG10_Rluc was limited at the level of translation thus able to report on decoding speed. Our current data suggest that decoding speed reporters based on the principle tested here are of limited usefulness, because of unanticipated interactions between codon usage and mRNA levels.