Evaluation of 244,000 synthetic sequences reveals design principles to optimize translation in Escherichia coli.
Guillaume CambrayJoao C GuimaraesAdam Paul ArkinPublished in: Nature biotechnology (2018)
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 using an epigenetic assay. Codon composition has a sizable impact on translatability, but only in comparatively rare elongation-limited transcripts. We propose a set of design principles to improve translation efficiency that would benefit from more accurate prediction of secondary structures in vivo.
Keyphrases
- gene expression
- escherichia coli
- amino acid
- dna methylation
- high resolution
- rna seq
- crispr cas
- metabolic syndrome
- staphylococcus aureus
- high throughput
- pulmonary embolism
- genetic diversity
- mass spectrometry
- pseudomonas aeruginosa
- skeletal muscle
- living cells
- small molecule
- single cell
- weight loss
- multidrug resistant
- circulating tumor cells
- wild type