Application of sorting and next generation sequencing to study 5΄-UTR influence on translation efficiency in Escherichia coli

Abstract

Yield of protein per translated mRNA may vary by four orders of magnitude. Many studies analyzed the influence of mRNA features on the translation yield. However, a detailed understanding of how mRNA sequence determines its propensity to be translated is still missing. Here, we constructed a set of reporter plasmid libraries encoding CER fluorescent protein preceded by randomized 5΄ untranslated regions (5΄-UTR) and Red fluorescent protein (RFP) used as an internal control. Each library was transformed into Escherchia coli cells, separated by efficiency of CER mRNA translation by a cell sorter and subjected to next generation sequencing. We tested efficiency of translation of the CER gene preceded by each of 48 natural 5΄-UTR sequences and introduced random and designed mutations into natural and artificially selected 5΄-UTRs. Several distinct properties could be ascribed to a group of 5΄-UTRs most efficient in translation. In addition to known ones, several previously unrecognized features that contribute to the translation enhancement were found, such as low proportion of cytidine residues, multiple SD sequences and AG repeats. The latter could be identified as translation enhancer, albeit less efficient than SD sequence in several natural 5΄-UTRs.

Influence of the spacer region between the Shine–Dalgarno box and the start codon for fine‐tuning of the translation efficiency in Escherichia coli

“…The spacer region might be a part of a secondary structure that may mask other components of the translation initiation site, such as the SD box or the start codon. Indeed, formation of a secondary structure inhibits the initiation of translation (Smit and van Duin, 1990; Osterman et al , 2012; Evfratov et al , 2017). To analyse the influence of the spacer sequence on the folding energy of the translation initiation site, and hence on the translation efficiency, we modelled secondary structures for all mRNA sequences in our data set, using a window encompassing the entire 22 nt 5’‐UTR and the first 50 nt of the coding region.…”

“…Plasmid pRFPCER (Osterman et al , 2012) was used as the host vector for randomized library construction (Fig. S1), similar to our previously published protocol (Evfratov et al , 2017) presented in the supporting information (Fig. S1B).…”

Pervasive Translation in Mycobacterium tuberculosis

“…This raises the question of whether pervasive translation has a function in bacteria. We propose that it is an inevitable consequence of the relatively low specificity associated with the selection of start codons by initiating ribosomes: at least five possible start codon choices (AUG, GUG, UUG, AUU, AUC) ( 9, 26 ), limited sequence specificity in the Shine-Dalgarno sequence ( 27 ), and considerable flexibility in the Shine-Dalgarno to start codon distance ( 28 ). The cost of active spurious start codons must be balanced with the beneficial functions that these features are providing in their larger context.…”

Optimization of carbon and energy utilization through differential translational efficiency

“…The mRNA features carried at the 5′UTR and at the coding region (Fig. 5 ) can both influence translation 43 – 45 . We currently lack insight into how static mRNA features in bacteria influence TE in a condition-specific manner.…”