In Vivo Evolution of a Catalytic RNA Couples Trans-Splicing to Translation
Figure 3
Enrichment of specific mutations during the evolution.
A, The number of mutations detected over all 21 cycles of evolution is plotted for each of the 414 nucleotide positions in the ribozyme. The graph represents a total of 2438 mutations. The structural elements corresponding to the nucleotide positions according to [51] are given at the top. The short helices P3, P7, and P9.0 are not labeled due to space constraints. The position of the P6b loop is indicated by an empty triangle. B, The frequency of mutations that appeared at least 15 times in total is shown over the cycles of evolution. The nucleotide position of these mutations is given in the left-most column. Ten sequences were obtained for each cycle, with blue denoting at least 2 mutations and red denoting at least four mutations detected for that cycle and nucleotide position. For cycles 3–8, where the evolution was split into two branches, the color-coding is averaged over both branches. C, The sequencing chromatogram of the evolved plasmid library after cycle 21 shows the enrichment of mutations 238, 239, and 240 in the ribozyme population (top), compared to the parent sequence (bottom). Note that at position 236, “C” (blue) represents only a small proportion of the evolved pool.