Studies on Translation Speed and Protein Folding Efficiency in Bacteria and Eukaryotes

The mechanisms for de novo protein folding differ significantly between bacteria and eukaryotes, as evidenced by the often observed poor yields of native eukaryotic proteins upon recombinant production in bacterial systems. Polypeptide synthesis rates are faster in bacteria than in eukaryotes, but the effects of general variations in translation rates on protein folding efficiency have remained largely unexplored. By employing E. coli cells with mutant ribosomes whose translation speed can be modulated. In this work, it is showed that reducing polypeptide elongation rates leads to enhanced folding of diverse proteins of eukaryotic origin. These results suggest that in eukaryotes, protein folding necessitates slow translation rates. The degeneracy of the genetic code allows most amino acids to be encoded by multiple codons. The distribution of these so-called synonymous codons among protein coding iii sequences is not entirely random and multiple theories have arisen to explain the biological significance of such non-uniform codon selection. Most ideas revolve the notion that certain codons allow for faster or more efficient translation, whereas the presence of others result in slower translation rates. The presence of these different types of codons along a message is postulated in turn to confer variable rates of emergence of the nascent polypeptide from the ribosome, which may influence its capacity to fold towards the native state, among other properties. A metric to predict organism-specific polypeptide elongation rates of any mRNA based on whether each codon is decoded by tRNAs capable of Watson-Crick, non-Watson-Crick or both types of interactions was developed. By pulse-chase analyses in living E. coli cells it was demonstrated that sequence engineering based on these concepts predictably modulate translation rates due to changes in polypeptide elongation and show that such alterations significantly impact the folding of proteins of eukaryotic origin. Finally, this work shows 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.