![]() h Schematic representation of the role of the RTS in distal operon gene translation (ribosomes are not drawn to scale). Secondary mRNA structures of all clones are available in Supplementary data file 3. The red dot represents the RFP stop codon. ![]() g ΔG fold landscape around the stop codon and the mRNA secondary structure presented in the first window outside the stop codon-occupying ribosome footprint of two selected clones (111, 207). Correlation between GFP expression and ΔG fold of ( e) all ( n = 33) isolated variants, and ( f) a subset ( n = 8) presenting an AUG start codon at position +3 or +4. Spearman correlation was performed on the weighted averages of the six bins ( n = 6, ρ = 1, p value = 0.0028). The x and y axes error bars represent the 99% confidence interval and relative standard deviation, respectively. d Correlation between the population mean GFP expression levels and the weighted mean of ΔG fold of 3 × 10 3 unique sequences in each bin. c Sorting of 10 6 cells into color-coded bins with constant RFP and variable GFP levels (top) GFP distribution in 3000 cells from each bin after sorting (bottom). b GFP and RFP fluorescence of 10 5 cells. Finally, we show that RTSs are positively selected to insulate translation when re-initiation-avoidance is beneficial, yet are depleted where re-initiation could prove useful, principally in operon-clustered genes.Ī Synthetic operon design and the FACS scheme employed. We further report, on the basis of large-scale computational analysis, that such structures are abundant throughout bacteria. Using Escherichia coli transformed with a synthetic operon as a model system, we discover a stable mRNA secondary structure found near the stop codon, termed the ribosome termination structure (RTS), that controls the efficiency of translation re-initiation. We thus considered whether mRNA secondary structure could serve this role, given how mRNA structure can affect translation at the de novo initiation 6, 7 and elongation 8, 9 steps, and can also affect translational coupling between two neighboring genes on the same operon 5, 10, 11. Specifically, regulators that determine whether a ribosome dissociates from the mRNA or remains bound to re-initiate translation have yet to be discovered. Presently, the mechanisms regulating translation re-initiation are not well understood 3, 4, 5. Translation re-initiation, a scenario whereby the terminating proximal-ribosome does not dissociate from the mRNA after termination and instead re-initiates translation on the neighboring distal cistron, alleviates this problem. This distance is too small to simultaneously accommodate one ribosome terminating on the stop codon of the proximal gene and a second ribosome initiating de novo translation on the start codon of the distal gene 3. In bacterial operons, the intergenic distance between most of the neighboring cistrons is shorter than 25–30 nucleotides 2, 3. Indeed, the mechanisms which control translation initiation in operons remain a matter of debate. Specifically, how does translation initiation of a downstream operon gene occur without interference from the translating ribosome of the upstream gene? Despite our considerable understanding of protein translation in bacteria, this largely remains an unanswered question. However, in bacteria, where a single mRNA transcript can contain several genes clustered into an operon, translation initiation must account for the space between genes. ![]() When monocistronic mRNA encoding a single gene is translated, spatial considerations that could interfere with ribosome binding are largely irrelevant. ![]() To initiate protein translation, a ribosome binds and assembles an initiation complex in the area of the gene start codon 1. ![]()
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