Genome

Protein biosynthesis

Epigenetics

Polysome

Codon usage bias

Gene

Protein secondary structure

Biology

Messenger RNA

Translation (biology)

Start codon

Translational efficiency

Genetics

Ribosome

Computational biology

Biochemistry

Nature Biotechnology

Large and systematic mRNA library design disentangles the complex sequence determinants of translation efficiency in bacteria. Comparative analyses of natural and mutated sequences have been used to probe mechanisms of gene expression, but small sample sizes may produce biased outcomes. We applied an unbiased design-of-experiments approach to disentangle factors suspected to affect translation efficiency in E. coli. We precisely designed 244,000 DNA sequences implementing 56 replicates of a full factorial design to evaluate nucleotide, secondary structure, codon and amino acid properties in combination. For each sequence, we measured reporter transcript abundance and decay, polysome profiles, protein production and growth rates. Associations between designed sequences properties and these consequent phenotypes were dominated by secondary structures and their interactions within transcripts. We confirmed that transcript structure generally limits translation initiation and demonstrated its physiological cost u...

Evaluation of 244,000 synthetic sequences reveals design principles to optimize translation in Escherichia coli