Molecular bases for strong phenotypic effects of single-site synonymous substitutions in the E.coli ccdB toxin gene

Single-site synonymous mutations typically have only minor or no effects on gene function. Here, we estimate the effects on cell growth of ~200 single-site synonymous mutations in an operonic context by mutating each position of ccdB, the 101-residue long cytotoxin of the ccdAB Toxin-Antitoxin (TA) operon to all possible degenerate codons. Phenotypes were assayed by transforming the mutant library into CcdB sensitive and resistant E. coli strains, isolating plasmid pools, and subjecting them to deep sequencing. Since autoregulation is a hallmark of TA operons, phenotypes obtained for ccdB synonymous mutants after transformation in a RelE toxin reporter strain followed by deep sequencing provided information on the amount of CcdAB complex formed. Synonymous mutations in the N-terminal region involved in translation initiation showed the strongest non-neutral phenotypic effects. We observe an interplay of numerous factors, namely, location of the codon, codon usage, t-RNA abundance, formation of anti-Shine Dalgarno sequences, transcript secondary structure, and evolutionary conservation in determining phenotypic effects of ccdB synonymous mutations. Incorporation of an N-terminal, hyperactive synonymous mutation, in the background of the single-site synonymous mutant library sufficiently increased translation initiation, such that mutational effects on either folding or termination of translation became more apparent. Introduction of putative pause sites not only affects the translational rate but might also alter the folding kinetics of the protein in vivo. This information is useful in optimizing heterologous gene expression in E. coli and understanding the molecular bases for alteration in gene expression that arise due to synonymous mutations. SIGNIFICANCE STATEMENTSynonymous mutations which do not change amino-acid identity, typically have only minor or no effects on gene function. Using sensitive genetic screens in the context of the ccdAB bacterial toxin-antitoxin operon, we demonstrate that many single-site synonymous mutations of the ccdB toxin gene display significant phenotypic effects in an operonic context. The largest effects were seen for synonymous mutations in the N-terminal region involved in translation initiation. Synonymous mutations that affected either folding or translation termination were also identified. Lack of translational pausing due to synonymous mutations in hydrophobic residue stretches, was found to decrease the amount of properly folded CcdB protein. In summary the study provides novel insights into diverse mechanisms by which synonymous mutations modulate gene function.