Genes for highly abundant proteins in Escherichia coli avoid 5' codons that promote ribosomal initiation.

In many species highly expressed genes (HEGs) over-employ the synonymous codons that match the more abundant iso-acceptor tRNAs. Bacterial transgene codon randomization experiments report, however, that enrichment with such "translationally optimal" codons has little to no effect on the resultant protein level. By contrast, consistent with the view that ribosomal initiation is rate limiting, synonymous codon usage following the 5' ATG greatly influences protein levels, at least in part by modifying RNA stability. For the design of bacterial transgenes, for simple codon based in silico inference of protein levels and for understanding selection on synonymous mutations, it would be valuable to computationally determine initiation optimality (IO) scores for codons for any given species. One attractive approach is to characterize the 5' codon enrichment of HEGs compared with the most lowly expressed genes, just as translational optimality scores of codons have been similarly defined employing the full gene body. Here we determine the viability of this approach employing a unique opportunity: for Escherichia coli there is both the most extensive protein abundance data for native genes and a unique large-scale transgene codon randomization experiment enabling objective definition of the 5' codons that cause, rather than just correlate with, high protein abundance (that we equate with initiation optimality, broadly defined). Surprisingly, the 5' ends of native genes that specify highly abundant proteins avoid such initiation optimal codons. We find that this is probably owing to conflicting selection pressures particular to native HEGs, including selection favouring low initiation rates, this potentially enabling high efficiency of ribosomal usage and low noise. While the classical HEG enrichment approach does not work, rendering simple prediction of native protein abundance from 5' codon content futile, we report evidence that initiation optimality scores derived from the transgene experiment may hold relevance for in silico transgene design for a broad spectrum of bacteria. Author summary: Transgene experiments in Escherichia coli report that codon usage in the first few amino acids after the initiating ATG has a profound influence on the resulting protein level by promoting ribosomal initiation, rather than enabling speedy elongation. For the design of bacterial transgenes and for simple codon-based in silico inference of protein levels, it would be valuable to computationally determine initiation optimality scores for codons for any given species. An attractive approach is to characterize the 5' codon enrichment of highly expressed genes compared with the most lowly expressed genes, just as translational optimality scores of codons (affecting elongation) have been similarly defined employing the full gene body. Using unique resources provided for E. coli we show that, unexpectedly, this doesn't work: the 5' ends of highly expressed genes are enriched, compared to lowly expressed ones, in codons that objectively are associated with low initiation rates. This likely reflects conflicting selection pressures in highly expressed genes which can favour low initiation to promote low noise or high efficiency. While simple prediction of native protein abundance from 5' codon content is then somewhat futile, the objective initiation optimality scores may hold relevance for in silico transgene design for a broad spectrum of bacteria. [ABSTRACT FROM AUTHOR]