Defining bottlenecks and physiological impact of an orthogonal translation initiation system

Abstract

Reengineering translation initiation provides a powerful route to develop new translation systems that enable precise control of protein synthesis. While many engineered translation systems show promise, their orthogonality and impact on host physiology is largely uncharacterized, limiting broader application. Here, we develop an initiator-tRNA with an AAC anticodon mutation (i-tRNA-AAC), enabling translation initiation at a GUU start codon. Using fluorescence assays, proteomics, and tRNA sequencing, we assess the i-tRNA-AAC initiation orthogonality and effects on the host, guiding the optimization of its translational efficiency. We find the i-tRNA-AAC mutant initiates translation exclusively from its GUU start codon and is improved by overexpression of valyl-tRNA synthetase and methionyl-tRNA formyltransferase. However, this intervention perturbs aminoacylation and base modifications of endogenous tRNAs along with proteome-wide changes likely due to increased valine demand. Our findings demonstrate how an orthogonal translation initiation system reshapes host physiology and reveals the adaptive responses that accompany translational reprogramming.