Of Terms in Biology: "Synonymous Substitution", part 1|2

by Christoph
 

We are occasionally warned not to compare apples with pears (sorry, English speakers, not with oran­ges this time). Sure, both are pome fruits, and both plants be­long to the Rosa­ceae fa­mily, but they differ no­tab­ly in shape, feel, smell and taste. Geneticists may well need a similar warning when they sometimes use the terms silent mutation and synonymous substitution (or synonymous mutation) interchangeably.
 

Figure 1. (A) Paleo-experimental evolution consists of resurrecting an ancient gene, removing the modern form of the gene from an extant organism, and then inserting the ancestral form into the extant organism. For instance, the ancient gene from the gray node on the phylogeny can be resurrected and then inserted into the E. coli genome (red node) at the precise chro­mosomal location that the extant gene was knocked out. This synthetic/engineered organism is then evolved in the laboratory. Our ap­proach contrasts to other ap­proaches that are only able to use modern genes from an organism to replace its ortholog in a different extant or­ganism (say, inserting the gene from the extant or­ganism at the green node into the E. coli, red, or­gan­ism). Such an approach can be limiting if adaptive or neutral mutations that prevent interoperability occur­red along particular branches that connect the green and red nodes (B) Precise re­­pla­ce­­ment of a modern bacterial EF-Tu gene with its ~500 million year old an­cestor extends the bacterial doubling time by two-fold. Two genes, tufA and tufB, (varying by just one amino acid) code for EF-Tu proteins in E. coli. Precise repla­cement of endogenous EF-Tu requires both chromo­somal tufA and tufB to be disrupted. Deletions of tufA or tufB in the E. coli B strain have similar effects (~34 min) when deleted individually. The ancient EF (AnEF) has 21 (out of 392) amino acid differences with the modern EF-Tu protein. Measurements are performed in LB media at 37°C in triplicate. Modern E. coli B strain REL606 was obtained courtesy of R. E. Lenski (Michigan State University). Source. Frontispiece: Overall sche­matic of interaction between E. coli EF-Tu and ribo­so­me. Source

I could easily provide watertight definitions for both terms "silent mutation" and "synony­mous substitution." Perhaps illustrated with a nice Venn diagram that makes it clear from which perspective which of the two terms make sense, where their meanings overlap and where they do not. I prefer to go with two examples, though.
 

A synonymous substitution that does not remain silent   but needs an introduction. The work of Betül Kaçar from the Dept. of Bacteriology at the University of Wisconsin–Madison (UW–Madison) and her collaborators has long focused on the intriguing topic of merging ancestral sequence reconstruction ("paleogenomics") with experimental evolution. One of their pet molecules is elongation factor EF-Tu, a core component of the translational appara­tus in all bacteria, archaea and eukaryotes (eEF‑1 or EF‑1A in the latter two). EF-Tu shuttles amino acid‑charged tRNAs (aa‑tRNAs) to ribosomes and facilitates their binding to the ribo­somal A‑site (see frontispiece), and, given its crucial function, an ancestral EF‑Tu (AnEF) was certainly already part of LUCA's inventory.
 

To date, there is no known backup mechanism that would allow cells to dispense with the function of EF-Tu. And, despite their highly conserved sequences, it is usually impossible to functionally exchange evolved EF‑Tu alleles between distantly related species as, for example, mesophilic E. coli and thermophilic Thermus thermophilus (Figure 1A). However, De Tarafder et al. (2021) succeeded in getting two sequence-reconstructed 1.3- to 3.3-Gy-old ancestral EF-Tus to "work" with reasonable kinetic parameters in both contemporary E. coli and Thermus.
 

To round off the introduction and to come closer to the example, Betul Kaçar and Eric Gaucher had found that a sequence-reconstructed 0.5-Gy-old ancestral EF-Tu (AnEF) allows E. coli cells to grow with a doubling time of ~53 min. This corresponds to a two-fold increase of the doub­ling time compared to the tufA⁺ tufB⁺ parent strain (Figure 1B). E. coli has, like most bacteria, two genes encoding EF-Tu, tufA and tufB, with the two proteins being virtually identical. Therefore, one of the alleles had to be deleted to allow a fair comparison between the fitness of the ancestral AnEF and the contemporary EF-Tu (both share ~95% protein sequence identity). Strain constructs tufA⁺ ΔtufB and ΔtufA tufB⁺ with a single tuf gene both had doubling times of ~34 min, indicating that the ~53 min doubling time of the ΔtufA ΔtufB::anEF strain is not so bad after all. But could it be improved (=shortened) by experimental evolution in the lab?
 

Figure 2. Fitness and growth characteristics of AnEF_C45T. (A) Schematic of AnEF_C45T. (B) Fitness phenotype be­tween endogenous and isogenic constructs (n = 5, AN­OVA, Tukey’s HSD). Modified from Source

This question was addressed in the new study by McGrath et al. (2023), who let the ΔtufA ΔtufB::anEF strain adapt through continuous cultivation over 3,000 generations (with sampling every 500 generations for whole‑genome sequencing, and, in parallel, evolving the ΔtufA tufB⁺ strain as control).
 

At generation 2,000, a mutation appeared in the anEF gene coding region that reached fixation by generation 2,500. They found by sequencing that a C→T mutation (transition) at position 45 of anEF changes a valine codon from GUC to GUU (V15V) (Figure 2A). Both the GUC and GUU codons are recognized by the same tRNA species in E. coli, tRNA^Val (valV, valW), whose 5'‑G in the GUC anticodon can basepair with both the 3'-C and 3'-U in the codon (a textbook case of a wobble base pair). This is thus a typical "synomymous substitu­tion" as it does not change the encoded amino acid, and AnEF_C45T would be expected to be phenotypically "silent." But why is it not "silent," apparently, but beneficial rather as it had swept through the entire population?
 

Because the ΔtufA ΔtufB::anEF strain had accumulated other mutations in addition to the C45T mutation in anEF during continuous cultivation (see below), the authors had to dissect the ef­fects of the C45T mutation from those of the "evolved genetic background" on fitness. To stu­dy this, they engineered four strains, two non‑evolved ('ancestor') and two 'evolved', each pair expressing either the original AnEF_C45 or the 'evolved' AnEF_C45T (Figure 2B). And this time they did not measure differences in doubling times (see Figure 1B) but differences in fitness via coculture competition assays (using the 'ancestral' and 'evolved' ΔtufA tufB⁺ strains, respectively, as controls).
 

Replacement of native E. coli EF-Tu (tufB⁺) with the 0.5-Gy-old EF-Tu (anEF_C45) (orange) causes a 13% decrease in fitness (Figure 2B, orange), which upon 3,000 generations of lab evolution is recovered by AnEF_C45T (red) and increased by 60% relative to the ancestor (note that in Figure 2B the fitness of "Ancestor AnEF_C45" is set to 1 for an easier visual comparison). Substitution of the synonymous mutation with the native codon in AnEF_T45C significantly decreases the fitness of the evolved strain by 30% (grey) whereas whereas introducing the synonymous mutation AnEF_C45T in the ancestral background has no relative fitness benefit (cyan). This demonstrates that the fitness effect of the synonymous mutation C45T in anEF depends on the evolved genetic background.
 

Figure 3. AnEF protein and mRNA levels are increased in strains carrying AnEF_C45T. (A) qPCR quantification of AnEF mRNA between constructs (n = 3, t-test). (B) West­ern blot quantification of AnEF protein between endo­genous and isogenic constructs (n = 3, t-test). Modified from Source

However, as usual, it seems to be a bit more complicated than just the "evolved genetic back­ground" being responsible for the improved fitness of strains carrying the AnEF_C45T allele. McGrath et al. (2023) found a moderate but statistically significant increase in mRNA levels (Figure 3A) and in protein levels (Figure 3B) for AnEF_C45T compared to AnEF_C45 in both the "Ancestor" and "Evolved" strains. They suspect that, similar to what Brandis et al. (2016) found for tufB in Salmonella, a synonymous mutation near the start codon, here anEF_C45T, promotes an open conformation of the mRNA that leads to increased AnEF expression. My tentative projection of the anEF_C45T mRNA sequence onto the secondary structure proposed by Brandis et al. (2016) supports this.
 

During its evolution in the lab for 3,000 generations, the 'Ancestor' AnEF_C45-strain (ΔtufA ΔtufB ::anEF) strain had accumulated seven non-synonymous mutations − the above mentioned "evolved genetic background" − in addition to the synonymous mutation anEF_C45T. None of these mutations did immediately point to a connection to the AnEF_C45T mutation, but some had also occurred in the long-term evolution exper­i­ment (LTEE) of E. coli B strain REL606, the parent of the "Ancestor" AnEF_C45-strain. It is thus reasonable to assume that these mutations reflect the adaptation of the population to the specific growth regime of the experiment, which selects for better growth (=shorter doubling times) over time.
 

Figure 4. (D) Muller plot demonstrating the genotype dy­namics across laboratory evolution with 3,000 gene­rations. Plotted are the mutations in genes that reached a minimum frequency of 25%, and highlighted in red is the selection frequency of the anEF gene. Source

From the Muller plot in Figure 4 that shows the genotype dynamics over time one can almost "read" that by generation 2,000 enough growth-enhancing mutations had accumulated for the improved expression of AnEF_C45T to become effective. To put it this way: the synonymous mu­tation C45T in the anEF gene would remain a silent mutation if it could not whisper in chorus along with other mutations in the host genome.
 

In the forthcoming part 2, I will present an example of Twin synonymous substitutions that remain silent.
 

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