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). (C) 2004 Elsevier Inc. All rights reserved.