Thesis (Ph.D.)--University of Washington, 2016-06Synonymous codon choice modulates translation, but the properties of codons or codon combinations that result in impaired translation are not understood. We scored expression of 35,811 three-codon insertions in GFP in Saccharomyces cerevisiae and evaluated these variants for codon usage and RNA structure effects on GFP fluorescence levels. We have established that codon pairs affect translation elongation and efficiency in yeast in a manner distinct from the effects of individual codon tRNA abundance. Also, similar to previous studies in bacteria, we have found that the base-pairing status of nucleotides near the translation start site is likely to impair translation initiation. Both inhibitory codon pairs and 5’ mRNA structure can impose substantial limitations on translation efficiency through synonymous variation. For 17 inhibitory codon pairs, we show that it is the pair, rather than the dipeptide, the 6-base sequence, or the two individual codons, that is responsible for inhibition. Variants from the GFP insertion library that had an inhibitory pair had significantly lower expression than variants in which: the 6 base sequences were out of frame; the two codons were present but separated; or one of the codons of the pair was instead an optimal codon. We find that the inhibitory pairs act in translation, based on both suppression of inhibition by over expressed tRNA (11/12 tested) and the reduction in translation speeds relative to synonymous dipeptide sequences, as observed from ribosome occupancies along yeast transcripts. Furthermore, for 12 of the 17 pairs, preserving the order of codons in the pair was required for strong inhibitory effects. Thus the position of inhibitory pairs within the ribosome is likely a key factor in translation efficiency. Moreover, the identity of codons in inhibitory pairs is inconsistent with an inhibition mechanism governed primarily by limited tRNA supply. Rather, our data implicates wobble decoding and interactions between adjacent sites in the ribosome. The high-throughput experimental analysis described here has resulted in the direct and extensive identification of multiple inhibitory codon pairs, a quantitative analysis of their relative effects on translation in vivo, and tests of their activity as modulators of translation
