Effect of short 5' UTR on protein synthesis in two biological kingdoms

Efficient ribosomal protein synthesis is dependent on cis-acting elements in the 5′ untranslated region (UTR) of mRNAs. Between prokaryotes and eukaryotes, the sequence and location of these elements differ to the extent of not being functionally interchangeable. We explored the possibility of constructing bifunctional UTRs that could direct translation in both prokaryotes and eukaryotes. A variant of a UTR from ner of phage Mu (ner-ACC) enhanced protein synthesis in a rabbit reticulocyte lysate, and it was compared to a lacZ-CTA, containing the λ cro RBS and the Escherichia coli lacZ spacer. Several mutants in the −3 to −1 regions of both lacZ-CTA and ner-ACC were tested in rabbit reticulocyte lysate and E. coli to select UTRs that were optimized simultaneously for both biological kingdoms. The lacZ-ATC proved 217-fold more effective than ner-ACC in this cross-species ability to enhance translation. The lacZ-ACC and ner-ATC were 83- and 78-fold, respectively, better than ner-ACC. We conclude that short UTRs (12–15 nt in length) can be fine-tuned in the −9 to −1 regions to enhance protein synthesis concurrently in prokaryotes and eukaryotes. In related studies, we show that nt at the −3 to −1 region of mRNAs exert an enormous impact on synthesis of proteins in E. coli.