Synonymous but not the same: the causes and consequences of codon bias

Abstract

Despite their name, synonymous mutations have significant consequences for cellular processes in all taxa. As a result, an understanding of codon bias is central to fields as diverse as molecular evolution and biotechnology. Although recent advances in sequencing and synthetic biology have helped to resolve longstanding questions about codon bias, they have also uncovered striking patterns that suggest new hypotheses about protein synthesis. Ongoing work to quantify the dynamics of initiation and elongation is as important for understanding natural synonymous variation as it is for designing transgenes in applied contexts.

Figure 1. Codon bias within and between genomes

The Relative Synonymous Codon Usage (RSCU) is plotted for fifty randomly selected genes from each of nine species. RSCU ranges from 0 (when the codon is absent), through 1 (when there is no bias), to 6 (when a single codon is used in a six-codon family). Methionine, tryptophan and stop codons are omitted. Genes are in rows and codons are in columns, with C- and G-ending codons on the left side of each panel. Note the extensive heterogeneity of codon usage among human genes. Other measures of a gene’s codon bias include CAI (similarity of codon usage to a reference set of highly expressed genes); FOP (the frequency of “optimal” codons), and tAI (similarity of codon usage to the relative copy numbers of tRNA genes).

Figure 2. Relationships between initiation rate, elongation rate, ribosome density, and rate of protein synthesis for endogenous genes

The steady-state rate of protein synthesis and density of ribosomes bound on an mRNA both depend on the rates of initiation and elongation. When elongation is the rate-limiting step in a gene’s translation (case A), the message will be covered as densely as possible by ribosomes, and faster elongation will tend to increase the rate of protein synthesis. However, most endogenous genes are believed to be initiation-limited (cases B, C, D), so that their transcripts are not completely covered by ribosomes; this is evidenced by extensive variability in ribosome densities across endogenous mRNAs. For two initiation-limited genes with the same initiation rate, the mRNA with faster elongation (afforded by higher codon adaptation to tRNA pools, say) will exhibit a lower density of translating ribosomes (C versus B) but no greater rate of termination. Thus, when initiation is limiting, high codon adaptation should not be expected to increase the amount of protein produced per mRNA molecule. A lower density of ribosomes can also occur when two initiation-limited genes have the same elongation rate, but one has a slower initiation rate (D versus C). The extent to which variation in ribosome densities arises from variation in initiation versus elongation rates remains to be determined. In all cases shown here, like most endogenous genes, the gene’s mRNA does not account for a substantial proportion of total cellular mRNA, so that the rates of initiation and elongation do not substantially alter the pool of free ribosomes (cf Figure 4).

Figure 3. Effects of mRNA secondary structure on translation initiation in Bacteria

A) Structure in the ribosome binding site (RBS) usually inhibits initiation. However, initiation can occur when the structured element is positioned between the Shine-Dalgarno sequence (SD) and start codon (AUG), or 15 nucleotides downstream of the start codon, . B) Synonymous mutations in the region from nt −4 to +37 of a GFP gene alter predicting folding energies by up to 12 kcal/mol. 5′ mRNA folding energies below −10 kcal/mol strongly inhibits GFP expression in E. coli55. C) More than 40% of human genes have predicted 5′ folding energies below the −10 kcal/mol threshold, and are therefore expected to express poorly in E. coli without modification.

Figure 4. The elongation rate may influence the rate of protein synthesis for an over-expressed gene

Unlike for most endogenous genes, mRNA from an over-expressed transgene may account for a substantial proportion of total cellular mRNA. In this case, slow elongation (caused by poor codon adaptation to charged tRNA pools, say) can increase the density of bound ribosomes and thereby reduce the pool of available ribosomes in the cell. Such a depletion of available ribosomes will feed back to reduce the initiation rate of subsequent translating ribosomes on the message, thereby reducing the rate of protein synthesis. This is illustrated schematically by comparing over-expressed mRNA’s with slow elongation (top) and rapid elongation (bottom), but identical initiation sequences. Thus, the relationship between codon adaptation and the rate of protein synthesis per mRNA molecule may differ for an over-expressed transgene as compared to an endogenous gene (cf Figure 2).