The fitness landscape of a tRNA gene

Abstract

Fitness landscapes describe the genotype-fitness relationship and represent major determinants of evolutionary trajectories. However, the vast genotype space, coupled with the difficulty of measuring fitness, has hindered the empirical determination of fitness landscapes. Combining precise gene replacement and next-generation sequencing, we quantified Darwinian fitness under a high-temperature challenge for more than 65,000 yeast strains, each carrying a unique variant of the single-copy tRNA(CCU)(Arg) gene at its native genomic location. Approximately 1% of single point mutations in the gene were beneficial and 42% were deleterious. Almost half of all mutation pairs exhibited statistically significant epistasis, which had a strong negative bias, except when the mutations occurred at Watson-Crick paired sites. Fitness was broadly correlated with the predicted fraction of correctly folded transfer RNA (tRNA) molecules, thereby revealing a biophysical basis of the fitness landscape.

Fig. 1

Determining the fitness landscape of the yeast ${\text{tRNA}}_{\text{CCU}}^{\text{Arg}}$ gene. Chemically synthesized ${\text{tRNA}}_{\text{CCU}}^{\text{Arg}}$ gene variants are fused with the marker gene URA3 before placed at the native ${\text{tRNA}}_{\text{CCU}}^{\text{Arg}}$ locus. The tRNA variant-carrying cells are competed. Fitness of each ${\text{tRNA}}_{\text{CCU}}^{\text{Arg}}$ genotype relative to wild-type is calculated from the relative frequency change of paired-end sequencing reads covering the tRNA gene variant during competition. See also Fig. S1 and (15).

Fig. 2

Yeast ${\text{tRNA}}_{\text{CCU}}^{\text{Arg}}$ gene fitness landscape. (A) Average fitness upon a mutation at each site. White circles indicate invariant sites. (B–D) Fitness distributions of (B) N1, (C) N2, and (D) N3 mutants, respectively. (E) Mean observed fitness (black circles) decreases with mutation number. Red circles show mean expected fitness without epistasis (right shifted for viewing). Error bars show one standard deviation. (F) Fraction of the 200 eukaryotic ${\text{tRNA}}_{\text{CCU}}^{\text{Arg}}$ genes with the same nucleotide as yeast at a given site decreases with the average fitness upon mutation at the site in yeast. Each dot represents one of the 69 examined tRNA sites. (G) Fraction of times that a mutant nucleotide appears in the 200 sequences increases with the fitness of the mutant in yeast. Each dot represents a N1 mutant. In (F) and (G), ρ, rank correlation coefficient; P, P-value from t-tests.

Fig. 3

Epistasis (ρ) in fitness between point mutations in the ${\text{tRNA}}_{\text{CCU}}^{\text{Arg}}$ gene is negatively biased. (A) Epistasis between point mutations. Lower-right triangle shows all pairwise epistasis (white = not estimated), while upper-left triangle shows statistically significant epistasis (white = no estimation or insignificant). ${\text{tRNA}}_{\text{CCU}}^{\text{Arg}}$ secondary structure is plotted linearly. Parentheses and crosses show stem and loop sites, respectively. Same color indicates sites in the same loop/stem. Each site has three mutations. (B) Distributions of pairwise epistasis (gray) and statistically significant pairwise epistasis (blue) among 12,985 mutation pairs. (C) Mean epistasis between first and second mutations increases with the fitness cost of the first mutation. (D) Mean fitness cost of the second mutation decreases with the fitness cost of the first mutation. In (C) and (D), Pearson’s correlation (r), associated P value, and the linear regression (red) are shown. (E–F) Distributions of epistasis (gray) and statistically significant epistasis (blue) between pairs of mutations that (E) convert a Watson-Crick (WC) base pair to another WC pair or (F) break a WC pair in stems. In (B), (E), and (F), the vertical red line shows zero epistasis.

Fig. 4

${\text{tRNA}}_{\text{CCU}}^{\text{Arg}}$ folding offers a mechanistic explanation of the fitness landscape. (A) Relationship between the predicted proportion of tRNA molecules that are functional (P_func) for a genotype and its fitness. Genotypes (with P_func ≥ 10⁻⁴) are ranked by P_func and grouped into 20 equal-size bins; mean P_func and mean fitness ± SE of each bin are presented. The red dot represents all variants with P_func < 10⁻⁴. (B) LOESS regression curves between P_func and fitness for N1, N2, and N3 mutants, respectively, with dashed lines indicating 95% confidence intervals. (C) Quantile-quantile plot between epistasis predicted from P_func values using N1 and N2 LOESS curves and observed epistasis. The ith dot from the left shows the ith smallest predicted epistasis value (y-axis) and ith smallest observed epistasis value (x-axis). Red diagonal line shows the ideal situation of y = x. Above and left of the plot are frequency distributions of observed and predicted epistasis, respectively. Red horizontal and vertical lines indicate zero epistasis.