Base composition at the start of the coding sequence controls the balance between translation initiation and mRNA degradation in E. coli .

Protein synthesis efficiency is highly dependent on the mRNA coding sequence. Furthermore, there is extensive evidence of a correlation between mRNA stability and protein expression level, though the mechanistic determinants remain unclear. Using yellow fluorescent protein (YFP) as a reporter gene, we herein demonstrate that adenosine (A) abundance in the first six codons is a critical determinant for achieving high protein synthesis in E. coli. Increasing A and/or decreasing guanosine (G) content in this region with synonymous codons results in substantial increases in protein expression level both in vivo and in vitro that are correlated with steady-state mRNA concentration in vivo. The change in mRNA concentration is attributable to changes in the stability of the mRNA that are directly coupled to its translation efficiency. Increasing A content promotes mRNA incorporation into the functional 70S ribosomal initiation complex without altering its affinity for the 30S ribosomal subunit. These results support a model in which base composition in the first six codons modulates local mRNA folding energy and single-strandedness to control the balance between productive translation initiation versus degradation of mRNAs bound to the 30S ribosomal subunit. Based on these findings, we developed a short N-terminal coding sequence that optimizes translation initiation efficiency for protein production in E. coli.