From reporters to endogenous genes: the impact of the first five codons on translation efficiency in Escherichia coli

Abstract

Translation initiation is a critical step in the regulation of protein synthesis, and it is subjected to different control mechanisms, such as 5' UTR secondary structure and initiation codon context, that can influence the rates at which initiation and consequentially translation occurs. For some genes, translation elongation also affects the protein synthesis rate. Recently, it was proposed that the identity of codons three to five, called short translational ramp, have a strong influence on translation elongation and protein expression. By the use of a GFP library where nearly all combinations of nucleotides at these positions were created, it was demonstrated that some of nucleotides combinations increased GFP expression up to four orders of magnitude by enhancing their translation efficiency (TE). While it is clear that the short ramp can influence protein expression levels of artificial constructs, its impact on physiological proteins is still unknown. In this work, we aimed to investigate the relevance of the short translational ramp on a physiological context. Through bioinformatics analysis, we identified the nucleotide combinations from the GFP library on Escherichia coli genes and examined their correlation with TE. We observed that E. coli genes were enriched with nucleotide compositions that enhanced protein expression on the GFP library, but, surprisingly, it seems to affect the TE only marginally.Nevertheless, our data indicate that different enterobacteria present similar nucleotide composition enrichment as E. coli, suggesting an evolutionary pressure towards the conservation of the short translational ramp.

Importance of the 5′ regulatory region to bacterial synthetic biology applications

“…However, a recent study by Moreira et al . (2019) supports that these findings are universally applicable. Moreira et al .…”

“…It is unclear whether the findings are universally applicable, since Verma et al used only GFP in their study. However, a recent study by Moreira et al (2019) supports that these findings are universally applicable. Moreira et al found that naturally highly abundant native E. coli proteins are enriched with the AA compositions that improved protein production according to Verma et al (2019).…”

Model‐driven design of synthetic N‐terminal coding sequences for regulating gene expression in yeast and bacteria

“…Recent investigations on NCS toolbox development have mainly been conducted in prokaryotes, such as E. coli and B. subtilis , where high‐throughput datasets and statistical analysis were applied. [ 3–8,37–40 ] Abundant evidence shows that both N‐terminal fragments from endogenous genes and randomly synthesized NCSs regulate gene translation levels efficiently and effectively. [ 3,4,6,8 ] However, an NCS toolbox for S. cerevisiae has not yet been developed.…”

Ramping Recombinant Protein Expression in Bacteria

“…4 Interestingly, native E. coli (and Saccharomyces cerevisiae) ORFs are enriched in the high-scoring sequences at codons 3− 5 from the eGFP library. 1 Based on a computational analysis, Moreira et al 5 found that two other enterobacteria (Klebsiella oxytoca and Enterobacter asburiae) are also enriched for highscoring sequences at codons 3−5, indicating a strong positive selection for particular sequences in this region. The idea that codons 3−5 function as key translation success factors is bolstered by another observation: native E. coli ORFs with high eGFP-scoring sequences 1 are associated with slightly higher translation efficiencies and protein abundances as determined from ribosome profiling and proteomic inventory data, respectively.…”

“…The idea that codons 3−5 function as key translation success factors is bolstered by another observation: native E. coli ORFs with high eGFP-scoring sequences 1 are associated with slightly higher translation efficiencies and protein abundances as determined from ribosome profiling and proteomic inventory data, respectively. 5 Why was a stronger correlation not observed between the codon 3−5 sequences and native protein synthesis rates? Bacterial translation is choreographed by a complex suite of determinants, making it difficult to unmask the exclusive contribution of the N-terminal sequence to systemwide expression of endogenous proteins without fully parsing potential counterbalancing determinants (e.g., mRNA-specific differences in coupling of transcription and translation, variable length of 5′-UTRs 5 ).…”