Analyzing and enhancing mRNA translational efficiency in an Escherichia coli in vitro expression system

Abstract

The dependence of efficiency of translation initiation on mRNA sequence parameters was investigated in an Escherichia coli in vitro expression system. We designed a large-scale expression experiment focussing on the influence of sequence variations in the translated region (TR) of the mRNA without changing the 5'-untranslated region (5'-UTR). The level of translated protein from 756 expression constructs was measured and the influence of a large number of possible effector attributes was statistically analyzed. Base exchanges immediately adjacent to the start codon up to nucleotide (+)25 had a profound effect on translational efficiency. Correlation analysis revealed a significant dependence on base pair probability and G+C content on the expression level, indicating that mRNA secondary structure in this region hampers translation. Using our training data, we developed a methodology to predict and improve the translation efficiency of open reading frames (ORFs).