Co-translational Folding Mechanisms Research Articles

Synonymous codon substitutions perturb cotranslational protein folding in vivo and impair cell fitness

In the cell, proteins are synthesized from N to C terminus and begin to fold during translation. Cotranslational folding mechanisms are therefore linked to elongation rate, which varies as a function of synonymous codon usage. However, synonymous codon substitutions can affect many distinct cellular processes, which has complicated attempts to deconvolve the extent to which synonymous codon usage can promote or frustrate proper protein folding in vivo. Although previous studies have shown that some synonymous changes can lead to different final structures, other substitutions will likely be more subtle, perturbing predominantly the protein folding pathway without radically altering the final structure. Here we show that synonymous codon substitutions encoding a single essential enzyme lead to dramatically slower cell growth. These mutations do not prevent active enzyme formation; instead, they predominantly alter the protein folding mechanism, leading to enhanced degradation in vivo. These results support a model in which synonymous codon substitutions can impair cell fitness by significantly perturbing cotranslational protein folding mechanisms, despite the chaperoning provided by the cellular protein homeostasis network.

%MinMax: A versatile tool for calculating and comparing synonymous codon usage and its impact on protein folding.

Most amino acids can be encoded by more than one synonymous codon, but these are rarely used with equal frequency. In many coding sequences the usage patterns of rare versus common synonymous codons is nonrandom and under selection. Moreover, synonymous substitutions that alter these patterns can have a substantial impact on the folding efficiency of the encoded protein. This has ignited broad interest in exploring synonymous codon usage patterns. For many protein chemists, biophysicists and structural biologists, the primary motivation for codon analysis is identifying and preserving usage patterns most likely to impact high-yield production of functional proteins. Here we describe the core functions and new features of %MinMax, a codon usage calculator freely available as a web-based portal and downloadable script (http://www.codons.org). %MinMax evaluates the relative usage frequencies of the synonymous codons used to encode a protein sequence of interest and compares these results to a rigorous null model. Crucially, for analyzing codon usage in common host organisms %MinMax requires only the coding sequence as input; with a user-input codon frequency table, %MinMax can be used to evaluate synonymous codon usage patterns for any coding sequence from any fully sequenced genome. %MinMax makes no assumptions regarding the impact of transfer ribonucleic acid concentrations or other molecular-level interactions on translation rates, yet its output is sufficient to predict the effects of synonymous codon substitutions on cotranslational folding mechanisms. A simple calculation included within %MinMax can be used to harmonize codon usage frequencies for heterologous gene expression.

The Effect of Codon Translation Rates on Cotranslational Protein Folding Mechanisms of Arbitrary Complexity

Bacterial cells use the naturally occurring variability in the rate at which different codons are translated to guide the folding of nascent proteins into ordered, biologically-active structures during their synthesis by the ribosome. Predicting how codon translation rates effect cotranslational protein folding mechanisms is therefore of fundamental biological interest. Here, we demonstrate that cotranslational folding mechanisms sampling an arbitrarily large number of states can be accurately modeled by treating this problem using the Markov chain formalism. This allows a general equation to be derived that describes the probability that a nascent protein is in any one of these conformational or thermodynamic states as a function of translation rates of individual codons in an mRNA molecules' open reading frame, which we show is accurate in modeling molecular dynamics simulations of cotranslational folding. Using this framework we demonstrate that there exists scenarios in which, contrary to conventional wisdom, fast-translating codons can actually increase the amount of cotranslational folding that occurs. This approach can be applied to the cotranslational folding of cytosolic and membrane proteins, and possibly the processing of nascent chains by auxiliary factors such as chaperones and enzymes.