Codon influence on protein expression in E. coli correlates with mRNA levels

• Published in: Nature (London) Vol. 529; no. 7586; pp. 358 - 363
• Main Authors: Boël, Grégory, Letso, Reka, Neely, Helen, Price, W. Nicholson, Wong, Kam-Ho, Su, Min, Luff, Jon D., Valecha, Mayank, Everett, John K., Acton, Thomas B., Xiao, Rong, Montelione, Gaetano T., Aalberts, Daniel P., Hunt, John F.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 21-01-2016; Nature Publishing Group
• Subjects: 631/326; 631/337/574; 631/553/2695; 631/61; Biochemistry, Molecular Biology; Biotechnology; Codon; Codon - genetics; DNA-Directed RNA Polymerases - metabolism; E coli; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - biosynthesis; Escherichia coli Proteins - genetics; Gene Expression Regulation, Bacterial - genetics; Generalized linear models; Genes; Genes, Synthetic - genetics; Genetic aspects; Half-Life; Humanities and Social Sciences; Kinetics; Life Sciences; Logistic Models; Messenger RNA; Microbiological research; Models, Genetic; Molecular Sequence Data; multidisciplinary; Odds Ratio; Peptide Chain Elongation, Translational; Physiology; Protein Biosynthesis - genetics; Protein expression; Proteins; Quantitative Methods; RNA Folding; RNA polymerase; RNA Stability; RNA, Bacterial - genetics; RNA, Bacterial - metabolism; RNA, Messenger - genetics; RNA, Messenger - metabolism; Science; Transcription, Genetic - genetics; Transfer RNA; Viral Proteins - metabolism
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/nature16509

Degeneracy in the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, has an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyse the sequence features influencing protein expression levels in 6,348 experiments using bacteriophage T7 polymerase to synthesize messenger RNA in
Escherichia coli
. Logistic regression yields a new codon-influence metric that correlates only weakly with genomic codon-usage frequency, but strongly with global physiological protein concentrations and also mRNA concentrations and lifetimes
in vivo
. Overall, the codon content influences protein expression more strongly than mRNA-folding parameters, although the latter dominate in the initial ~16 codons. Genes redesigned based on our analyses are transcribed with unaltered efficiency but translated with higher efficiency
in vitro
. The less efficiently translated native sequences show greatly reduced mRNA levels
in vivo
. Our results suggest that codon content modulates a kinetic competition between protein elongation and mRNA degradation that is a central feature of the physiology and also possibly the regulation of translation in
E. coli
.
In-depth analyses of protein expression studies are used to derive a new codon-influence metric that correlates with global protein levels, mRNA levels and mRNA lifetimes
in vivo
, indicating tight coupling between translation efficiency and mRNA stability; genes redesigned based on these analyses consistently yield high protein expression levels both
in vivo
and
in vitro
.
Improving protein translation
All living organisms use the same set of 64 possible triplet codons to encode 20 amino acids and stop signals during translation of mRNA into protein, but the frequency with which each codon is used varies between organisms. This variation has been thought to be the basis for often poor protein expression when a gene is translated in a heterologous system. John Hunt and colleagues have performed in-depth analysis of protein expression studies to derive a codon-influence metric. When genes are redesigned using this metric, the authors find no effect on their transcription, but much greater translation efficiency. They conclude that
in vivo
mRNA degradation and translation processes are in competition, and using the metric, protein production can be favoured. This approach may facilitate heterologous gene expression in many biotechnical or experimental settings.