ИСТИНА

Regulation of gene expression at the level of translation accounts for up to three orders of magnitude in its efficiency. Previously, we systematically compared the impact of several known mRNA ribosome binding site (RBS) features, such as length and location of the Shine Dalgarno sequence (SD), secondary structure, start codon etc, on translation initiation; the experiments were done in a system with internal control based on dual cerulean and red (CER/RFP) fluorescent proteins. Based on these results, we proposed a model for estimating translation efficiency and predict translation efficiency for large set of natural 5’-UTRs. Among a set of 50 natural 5’-UTRs cloned into the CER/RFP reporter system we’ve found several outliers that possesses much worse or much better translation efficiencies than could be predicted. Consequently, we concluded that known elements composing bacterial RBS are non sufficient to adequately describe translation efficiency. We chose a set of outlier 5’-UTRs to investigate what makes them different from those whose ribosome binding efficency was successfully predicted. Selected 5’-UTRs were randomly mutated within the reporter plasmid and E. coli cells transformed with this library were sorted according to the ratio of the CER and RFP fluorescence, subjected to next generation sequencing (NGS) and analysis. Several unexpected features which strongly affect bacterial translation were found. To investigate mRNA RBS elements contributing to translation efficiency further we created an unbiased set of reporter construct libraries exceeding a million randomized RBS variants. After sorting and NGS we not only investigated a detailed anatomy of classic bacterial RBS, but also found several completely unusual sequences, which provide strong translation efficiency.