Non-Canonical Start Codons Reinitiate Translation in N-Terminal Truncated Kv Channels

Abstract

Genetic incorporation of unnatural amino acids (UAA) is a powerful tool to investigate protein structure and function. Through nonsense suppression of stop codons, UAAs are incorporated into proteins using chemically charged tRNAs or orthogonal tRNA/tRNA synthetase (tRNA/RS) pairs. Previously, we successfully incorporated a fluorescent UAA into the voltage gated-potassium (Kv) channel. The nonsense stop codons were located either central or C-terminal, and no leak expression was detected in the absence of the UAA, reflecting orthogonality of the tRNA/RS pair used. Here, we show that when stop codons are introduced in the near N-terminal region of the same Kv channel, a considerable amount of leak expression results from translation reinitiation. Since reinitiation only can occur when the distance between the first methionine and the inserted stop codon is short, it explains the position-dependent leak expression. We demonstrate that reinitiation occurs at several non-canonical (non-AUG) start codons, and leak expression was decreased from 25% to less than 1% through silent mutations of start codons. When using UAAs, it is crucial to have control of the protein translation process in order to get reliable results, and we emphasize that caution should be taken when UAAs are incorporated in the N-terminal region of any protein. This is particularly important if the N-terminal has a functional role where truncation generates a modulated phenotype which could be wrongly interpreted to be caused by the inserted UAA. To avoid reinitiation-mediated leak expression, we therefore recommend removing both canonical and non-canonical start codons.