A Systematic Analysis of Codon Effects on Translation Efficiency: Identification and Mechanistic Examination of the Inhibitory Effects of the Arginine Codon CGA

Abstract

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biochemistry and Biophysics, 2011.The particular choice of codons used to encode a polypeptide influences translational efficiency, accuracy, reading frame maintenance, and nascent polypeptide folding. Although the codons that are implicated in efficient translation are well established, there has been no comprehensive analysis of individual codon effects on expression in any organism. To systematically study the effects of codons on translation efficiency, 10 repeats of each codon were inserted upstream of the firefly luciferase reporter in two positions, either near the translation start or in the middle of a fusion construct. The arginine codon CGA was identified as a strong inhibitor of translation efficiency. CGA-mediated inhibition of expression occurs with as few as three arginine residues, acts on at least three downstream genes, and is dependent upon CGA codon dosage. Two CGA codons result in a 2-fold reduction in expression and 5 CGA codons result in a 25-fold reduction, which are surprising results in view of the prevailing idea that individual codons effects on expression are relatively small. Moreover, CGA-mediated inhibition results from inefficient translation, since an anticodon-matched isoaccepting tRNA species efficiently rescues expression. This result implies that inefficient I•A wobble decoding, not limiting tRNA amount, is responsible for CGA codon effects. Thus, decoding interactions within the ribosome modulate the efficiency of translation. An examination of the effects of CGA codon repeats on both mRNA and protein species supports a model in which CGA codons elicit ribosome stalling during translation. First, insertion of CGA codons results in a new RNA, whose properties are consistent with cleavage near the CGA codons. Similar RNA cleavage products are detected when ribosomes stall due to strong RNA secondary structures. Second, the presence of CGA codons results in a truncated polypeptide, the amount of which is dramatically increased in the absence of Ltn1p, an E3 ubiquitin ligase that recognizes ribosomes stalled due to the absence of a stop codon. Thus, the presence of the polypeptide and its susceptibility to Ltn1-mediated degradation are expected if ribosomes stall at CGA codons. These results provide the basis for determining the mechanisms by which CGA codons exert their effects on translation