Silent substitutions predictably alter translation elongation rates and protein folding efficiencies

Abstract

Genetic code redundancy allows most amino acids to be encoded by multiple codons that 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 toward 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 Escherichia 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, which 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.

Figure 1. Incorporation of tRNA gene information and nature of codon:anticodon base pairing allows the prediction of relative translation elongation rates

(a) Predicted gene content for tRNAs capable of decoding the standard genetic code according to gtrnadb.ucsc.edu is plotted for each codon in histogram form (as indicated) by each domain of life in different patterns (as indicated). The length of each box represents the extent to which genes for tRNAs capable of decoding the corresponding codon are present in a domain. For Met or Trp, 100% of genera examined in each domain are predicted to contain a single species of tRNA genes to decode these codons (and thus the length of these bars corresponds to “100% exclusivity”). (b) Predicted relative protein synthesis rates (see main text and Supplementary Methods) in E. coli for Luc sequences lacking codons decoded by wobble-based tRNA interactions (Luc_fast, orange), containing the most frequent E. coli codons (Luc_cbf, blue) or the unmodified firefly coding sequence (Luc_WT, gray).

Figure 2. Avoidance of wobble-based interactions during mRNA decoding results in acceleration of translation elongation rates in vivo

(a) Pulse-chase analyses (left panels) in live E. coli cells synthesizing recombinant Luc from the indicated constructs and plots (right panels) depicting the appearance of incorporated [³⁵S]methionine in full length Luc produced from the indicated constructs (colored dots), curves for the theoretical appearance of methionines with four calculated constant translation rates of the indicated constructs (colored lines) and calculated theoretical appearance of methionines according to our predicted variable rates (x symbols), which demonstrate that Luc_fast is translated faster than Luc_WT and Luc_cbf. (b) Pulse-chase analyses (left panels) and plots (right panels) as in a, for the Luc_WT-fast and Luc_WT-cbf constructs, as indicated, demonstrating that the observed effects on rates are not due to changes in translation initiation.

Figure 3. Synonymous sequence based acceleration influences the folding of the encoded polypeptide

Specific activities of protein products identical in primary sequence produced from Luc_WT, Luc_fast and Luc_cbf, as indicated (top panel). The value of the protein from Luc_WT was set to 100. Error bars represent S.E.M. SDS-PAGE of total (T), soluble (S) and insoluble (P) recombinant protein produced in E. coli from the indicated sequence-engineered constructs (bottom panel).

Figure 4. Mimicking eukaryotic tRNA population via synonymous sequence engineering of mRNA enhances folding efficiency of recombinant proteins in bacterial host

(a) Plots of predicted relative translation elongation rates for Luc_WT when expressed in E. coli (top panel) or D. melanogaster (middle panel) and the harmonized Luc_re sequence when expressed in E. coli (bottom panel). (b) Specific activities (top panel) and solubility analysis (bottom panel) of protein products identical in primary sequence produced from Luc_WT and Luc_re, as in Figure 3.