Multiple upstream start codons (AUG) in 5' untranslated region enhance translation efficiency of cry2Ac11 without...

Cry2Ac11, a 65 kDa insecticidal protein produced by Bacillus thuringiensis, shows toxicity against dipteran and lepidopteran larvae. It is encoded by cry2Ac11 gene ( orf3), which is part of an operon comprising orf1, orf2, and orf3. Orf2, a helper protein, helps in proper folding and prevents aberrant aggregation of newly produced molecules. In this study, we have elucidated the effect of different mutations in translation initiation region (TIR), particularly the ribosomal binding site and the start codon (RBS-ATG) on cry2Ac11 gene expression without helper protein. All recombinant constructs were expressed in acrystalliferous B. thuringiensis subsp israelensis 4Q7 under the control of strong chimeric promoter cyt1AP/STAB. Of all the mutants, mut/RBS2, with two consecutive AUGs after the spacer region in TIR, exhibited 89- and 2246-fold higher transcript levels compared with 4Q7-operSalI/RBS ( cry2Ac11 operon) and 4Q7-w-RBS ( cry2Ac11 gene), respectively. The analysis of mut/RBS2 messenger RNA (mRNA) structure in the RBS-AUG region showed the presence of RBS in the single-stranded part of the moderately stable hairpin loop. The high expression efficiency of Cry2Ac11 mutant without helper protein is a cumulative and cooperative result of chimeric promoter cyt1AP/STAB-SD with the optimal context of RBS-AUG region provided by multiple AUGs and stabilizer sequence at 3' ends.