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

Abstract

Protein synthesis efficiency is highly dependent on 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 inE. coli. Increasing A and/or decreasing guanosine (G) content in this region results in substantial increases in protein expression level bothin vivoandin vitrothat are correlated with steady-state mRNA concentrationin vivo, and this effect 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 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 inE. coli.

Global regulation via modulation of ribosome pausing by the ABC-F protein EttA

“…Most of the constructions with a yfp gene express the native YFP venus except for the mgtL and mgtA 1-5aa constructions which express an optimized YFP 28 and for the yfp mutant presented in Supplementary Fig. 7a where the first 6 codons systematically use the synonymous codon with the highest number of adenosine 96 .…”