Selection, Identification, and Characterization of Codon Pairs that Inhibit Translation and the Development of the RNA-ID Method to Measure the Effects of Cis-Regulatory Elements

Abstract

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biochemistry and Biophysics, 2013.It has been known for over 30 years that synonymous codon choice regulates translation, but the characteristics of codons that inhibit translation, and the factors that restrict their function, are unknown. In a systematic screen of 59 of 61 sense codons, our lab identified the arginine CGA codon as strongly inhibitory, and then determined that CGA inhibition was primarily due to A·I wobble decoding. Furthermore, I found that adjacent CGA codons are synergistically inhibitory relative to separated CGA codons, which presents the possibility that combinations of non-identical codons may regulate translation in ways that are not predictable from their individual codon behavior. To search for inhibitory codon combinations among the 3,721 codon pairs and 226,981 codon triplets, I developed the RNA-ID method to screen for cis-regulatory elements. RNA-ID utilizes a fluorescence-based, integrated reporter and coupled to flow cytometry to evaluate effects of sequences on expression in Saccharomyces cerevisiae. This method is useful because insertion of test sequences, using ligation independent cloning, is simple, expression is detectable over a 250-fold range, measurements are quantitative and dose-dependent, separation of specific populations is nearly complete, and results from a single sequence are reproducible. To find inhibitory codon combinations, a library of sequences, consisting of random 9 nucleotide inserts, was evaluated using RNA-ID. Only a small fraction of yeast (<0.2%) exhibited GFP/RFP at 6-10% the maximal level, indicating that very few sequences inhibit expression to this degree. Ninety-two strains from this group were studied. Five novel sequences that inhibit translation where identified based on suppression of the expression defect by appropriate tRNAs. The inhibitory sequences all contain at least one codon decoded by wobble, and in each case, the suppression of the inhibitory effect requires expression of a mutated, exact base-pairing tRNA. Thus, I conclude that wobble decoding is likely to be a critical factor in their inhibition of translation. In two cases, inhibition depends upon a pair of adjacent codons and upon the order of these codons, thus indicating that the intact codon pair is the inhibitory unit, consistent with the idea that they act within a single round of translation