Ribosomal selection of mRNAs with degenerate initiation triplets

Abstract

To assess the influence of degenerate initiation triplets on mRNA recruitment by ribosomes, five mRNAs identical but for their start codon (AUG, GUG, UUG, AUU and AUA) were offered to a limiting amount of ribosomes, alone or in competition with an identical AUGmRNA bearing a mutation conferring different electrophoretic mobility to the product. Translational efficiency and competitiveness of test mRNAs toward this AUGmRNA were determined quantifying the relative amounts of the electrophoretically separated wt and mutated products synthesized in vitro and found to be influenced to different extents by the nature of their initiation triplet and by parameters such as temperature and nutrient availability in the medium. The behaviors of AUAmRNA, UUGmRNA and AUGmRNA were the same between 20 and 40°C whereas the GUG and AUUmRNAs were less active and competed poorly with the AUGmRNA, especially at low temperature. Nutrient limitation and preferential inhibition by ppGpp severely affected activity and competitiveness of all mRNAs bearing non-AUG starts, the UUGmRNA being the least affected. Overall, our data indicate that beyond these effects exclusively due to the degenerate start codons within an optimized translational initiation region, an important role is played by the context in which the rare start codons are present.

Retapamulin-Assisted Ribosome Profiling Reveals the Alternative Bacterial Proteome

“…The in-frame iTISs exploit various initiator codons that have been shown previously to be capable of directing translation initiation in E. coli (Chengguang et al, 2017;Hecht et al, 2017), although similar to the pTISs, the AUG codon is the most prevalent (Figure 3A). A SD-like sequence could be recognized upstream of many of the in-frame internal start codons (Table S1).…”

A frame-shifted gene, which rescued its function by non-natural start codons and its application in constructing synthetic gene circuits

“…However, no studies were performed by replacing or removing AUG (ATG) start codon. The start codons used in our study do not belong to the other lesser used and rare but known start codons in bacteria like GUG, UUG, CUG, AUU, AUC and AUA [14, 25]. Thus we defined them as non-natural start codons.…”

A Practical Guide for the Quality Evaluation of Fluobodies/Chromobodies

“…In eukaryotic systems, it is known that the binding of regulative proteins on upstream open reading frame sequences constitutes a modality for shifting the start codon and obtaining alternative protein synthesis [10]. In prokaryotes, the relevance of alternative start codons has been underestimated and their annotation has often been misreported or completely omitted [11,12], but it has recently been demonstrated that sub-optimal start codons, such as GUG and UUG, can be activated with mechanisms that can require compensatory mutations in the Shine-Dalgarno sequence [13,14]. The third hypothesis does not consider biological reasons but focuses on trivial technical aspects that could induce protein degradation, as in the case of mCherry, during either expression/purification or sample preparation and separation [7,15].…”