Availability of a second upstream AUG can completely overcome inhibition of protein synthesis initiation engendered by mRNA secondary structure encompassing the start codon.

Secondary structure analysis of the mRNA from a nonproductive construct carrying the nonstructural gene 3 (NS3) of Japanese Encephalitis Virus revealed the presence of a potential 28 nucleotide long stem and loop beginning with the guanine of the initiation codon AUG that had a calculated stabilization energy of -13 kcal/mol (delta Gfzero). Provision of an additional AUG along with three codons upstream resulted in complete relief of inhibition. N-terminal amino acid sequence of the recombinant protein was consistent with initiation of protein synthesis having occurred from the upstream AUG. Similar levels of NS3 specific RNA in E. coli cells carrying the expressing and nonexpressing constructs and restoration of recombinant protein expression following deletion of segments beginning with the stem and loop confirmed the role of this structure in blocking expression at the level of translation initiation. Our approach exploits the ability of a ribosome in motion to open up downstream secondary structural elements of considerable stability and represents a novel and widely applicable strategy to overcome a block in translation initiation caused by mRNA secondary structure around the translation start site.