These ‘translational ramps’ were postulated to be causally responsible for elevated expression of genes rich in rare codons at the CDS’s 5′-end. It was postulated that rare codons (with low-abundant cognate tRNAs) may have been evolutionary selected for within the N-terminal CDS to slow down early translation elongation and reduce premature termination due to clashing ribosomes ( 39, 49–59). The first hypothesis is related to the fact that cellular tRNA concentrations correlate with the occurrence frequency of their cognate codons ( 46–48). These observations led to two different hypotheses that differ fundamentally in terms of the underlying causality. Genome-wide analyses of Escherichia coli and other organisms revealed an overrepresentation of rare codons in the first five to ten triplets of the CDS in native genes, and their occurrence in this region was found to coincide with high expression ( 30, 42–45). Moreover, codon usage was found to influence translation. For example, stable secondary structures around the start codon were found to hinder translation, while structures further up- or downstream had less pronounced effects ( 36). Further, the influence of mRNA secondary structures was studied under the hypothesis that the required unfolding of such structures during translation initiation might decrease expression ( 15, 21, 28–41). Remarkably, in some cases SD-like motifs are not required for translation, an observation hinting at the existence of other mechanisms besides ‘canonical’ translation initiation ( 23–27). For example, the distance between SD motif and start codon, the type of start codon, and interactions between distant 5′-UTR parts and the ribosome play important roles ( 14–22). However, since Shine and Dalgarno's discovery in 1973 ( 9), various additional influencing factors and sequence determinants affecting translation initiation were identified. The 3′-end of the 16S rRNA hybridises with the Shine-Dalgarno (SD) motif, a conserved five to eight nucleotide (nt) sequence located upstream of the start codon, which facilitates translation ( 9–13). At the onset of this step, the 30S ribosomal subunit attaches to the ribosome binding site (RBS) in the 5′-untranslated region (5′-UTR) upstream of the coding sequence (CDS). In prokaryotes, initiation of translation is the rate-limiting step in the translational process, during which ribosomes assemble on the mRNA to start the templated elongation of the nascent polypeptide ( 4–8). To this end, genetic parts that influence translation are modified to alter absolute and relative expression levels to engineer biosystems through control of individual genes, pathways, and even entire metabolic networks ( 1–3). Translation is a key step of gene expression and an important engineering target in synthetic biology. Our study highlights the indispensability of ultradeep sequence-function mapping to accurately determine the contribution of parts and phenomena involved in gene regulation. Moreover, the obtained large-scale data provide clear experimental evidence for a base-pairing interaction between initiator tRNA and mRNA beyond the anticodon-codon interaction, an effect that is often masked for individual sequences and therefore inaccessible to low-throughput approaches. We find that 5′-UTR and CDS individually account for 53% and 20% of variance in translation, respectively, and show conclusively that, contrary to a common hypothesis, tRNA abundance does not explain expression changes between CDSs with different synonymous codons. This allows us to disentangle and precisely quantify effects of various sequence determinants of translation. Herein, we systematically assess the dynamic translation from over 1.2 million 5′-UTR-CDS pairs in Escherichia coli to investigate their collective effect using a new method for ultradeep sequence-function mapping. However, the complex interaction of 5′-UTR and CDS has so far only been studied for few sequences leading to non-generalisable and partly contradictory conclusions. In bacteria, 5′-untranslated region (5′-UTR) and coding sequence (CDS) are well-known mRNA parts controlling translation and thus cellular protein levels. Translation is a key determinant of gene expression and an important biotechnological engineering target.
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