Genetic code redundancy allows most amino acids to be encoded by multiple codons which are non-randomly distributed along coding sequences. An accepted theory explaining the biological significance of such non-uniform codon selection is that codons are translated at different speeds. Thus, varying codon placement along a message may confer variable rates of polypeptide emergence from the ribosome, which may influence the capacity to fold towards the native state. Previous studies report conflicting results regarding whether certain codons correlate with particular structural or folding properties of the encoded protein. This is partly due to different criteria traditionally utilized for predicting translation speeds of codons, including their usage frequencies and the concentration of tRNA species capable of decoding them, which do not always correlate. Here, we developed a metric to predict organism-specific relative translation rates of codons based on the availability of tRNA decoding mechanisms: Watson-Crick, non-Watson-Crick or both types of interactions. We determine translation rates of messages by pulse-chase analyses in living E. coli cells and show that sequence engineering based on these concepts predictably modulates translation rates in a manner that is superior to codon usage frequency, occur during the elongation phase, and significantly impacts folding of the encoded polypeptide. Finally, we demonstrate that sequence harmonization based on expression host tRNA pools, designed to mimic ribosome movement of the original organism, can significantly increase the folding of the encoded polypeptide. These results illuminate how genetic code degeneracy may function to specify properties beyond amino acid encoding, including folding.

The genetic code is said to be redundant in that the same amino acid residue can be encoded by multiple, so-called synonymous, codons. If all properties of synonymous codons were entirely equivalent, one would expect that they would be equally distributed along protein coding sequences. However, many studies over the last three decades have demonstrated that their distribution is not entirely random. It has been postulated that certain codons may be translated by the ribosome faster than others and thus their non-random distribution dictates how fast the ribosome moves along particular segments of the mRNA. The reasons behind such segmental variability in the rates of protein synthesis, and thus polypeptide emergence from the ribosome, have been explored by theoretical and experimental approaches. Predictions of the relative rates at which particular codons are translated and their impact on the nascent chain have not arrived at unequivocal conclusions. This is probably due, at least in part, to variation in the basis for classification of codons as “fast” or “slow”, as well as variability in the number and types of genes and proteins analyzed. Recent methodological advances have allowed nucleotide-resolution studies of ribosome residency times in entire transcriptomes, which confirm the non-uniform movement of ribosomes along mRNAs and shed light on the actual determinants of rate control. Moreover, experiments have begun to emerge that systematically examine the influence of variations in ribosomal movement and the fate of the emerging polypeptide chain.

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Treatment of clinical isolates of human pathogenic bacteria, which were known to be resistant to multiple commonly-used antibiotics, with refined leukocyte extracts from the American alligator (Alligator mississippiensis) resulted in a time-and concentration-dependent inhibition of bacterial proliferation. The alligator leukocyte extract exhibited the strongest antibacterial effect on Pseudomonas aeruginosa followed by Enterococcus faecium and then Klebsiella pneumonia. The antibacterial activities were acid-soluble, heat-stable at 70 o C for one h, sensitive to protease treatment, and did not require divalent metal ions for antibacterial activity. Collectively, these data strongly suggest that the molecule(s) responsible for the observed antibacterial activities are small, cationic antimicrobial peptides.

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