Nonsense suppression by near-cognate tRNAs employs alternative base pairing at codon positions 1 and 3

Premature termination codons (PTCs) in an mRNA ORF inactivate gene function by causing production of a truncated protein and destabilization of the mRNA. Readthrough of a PTC allows ribosomal A-site insertion of a near-cognate tRNA, leading to synthesis of a full-length protein from otherwise defective mRNA. To understand the mechanism of such nonsense suppression, we developed a yeast system that allows purification and sequence analysis of full-length readthrough products arising as a consequence of endogenous readthrough or the compromised termination fidelity attributable to the loss of Upf (up-frameshift) factors, defective release factors, or the presence of the aminoglycoside gentamicin. Unlike classical wobble models, our analyses showed that three of four possible near-cognate tRNAs could mispair at position 1 or 3 of nonsense codons and that, irrespective of whether readthrough is endogenous or induced, the same sets of amino acids are inserted. We identified the insertion of Gln, Tyr, and Lys at UAA and UAG, whereas Trp, Arg, and Cys were inserted at UGA, and the frequency of insertion of individual amino acids was distinct for specific nonsense codons and readthrough-inducing agents. Our analysis suggests that the use of genetic or chemical means to increase readthrough does not promote novel or alternative mispairing events; rather, readthrough effectors cause quantitative enhancement of endogenous mistranslation events. Knowledge of the amino acids incorporated during readthrough not only elucidates the decoding process but also may allow predictions of the functionality of readthrough protein products.