RNA Structural Determinants of Optimal Codons Revealed by MAGE-Seq

Synonymous codon choices at the beginning of genes optimize 5′ RNA structures for enhanced translation initiation, but less is known about mechanisms that drive codon optimization downstream within the gene. To understand what determines codon choices across a gene, we generated 12,726 in situ codon mutants in the Escherichia coli essential gene infA and measured their fitness by combining multiplex automated genome engineering mutagenesis with amplicon deep sequencing (MAGE-seq). Correlating predicted 5′ RNA structure with fitness revealed that codons even far from the start of the gene are deleterious if they disrupt the native 5′ RNA conformation. These long-range structural interactions generate context-dependent rules that constrain codon choices beyond intrinsic codon preferences. Genome-wide RNA folding predictions confirm that natural codon choices far from the start codon are optimized in part to prevent disruption of native structures near the 5′ UTR. Our results shed light on natural codon distributions and should improve engineering of gene expression for synthetic biology applications. [Display omitted] •Systematic mutagenesis of an essential gene reveals context-dependent codon preferences•5′ RNA structure determines codon preferences far from the start codon•Synonymous codons that disrupt the native 5′ RNA structure are more deleterious•MAGE-seq enables rapid and quantitative phenotyping of mutant libraries in E. coli Kelsic et al. develop and apply MAGE-seq to identify RNA structures that determine optimal codon preferences in an essential E. coli gene.