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. 2019 Nov 12;116(48):24206–24213. doi: 10.1073/pnas.1905990116

Fig. 3.

Fig. 3.

Evolutionary adaptation of kleenX174 to high-growth rate results in a reversion (2939C > T) and increased gene H expression. (A) The mutation 2939C > T observed in 2 independent adaptation experiments both restores the putative start codon of cryptic ORF 36 (GCG > GTG) and silently changes the third codon of gene H (GGC > GGT, Gly > Gly). (B) Evolved kleenX174(2939C > T) grows faster than ancestral kleenX174 in liquid culture (population doublings per hour). (C) The kleenX174(2939C > T) mutation also recovers plaque size. (D) RNA structure predictions of gene H sequence variants from wild-type øX174, kleenX174, and mutant kleenX174(2939C > T). NUPACK lowest-energy RNA structures generated from an 83-nt window surrounding the gene H start codon. (E) Protein H production from kleenX174(2939C > T) is increased compared to ancestral kleenX174 genome. Protein H produced from synthetic dsDNA templates (Datasets S2–S4) containing either wild-type øX174, kleenX174, or kleenX174(2939C > T) gene H sequence plus 20-bp upstream sequence identical to genome background, flanked by T7 promoter (PT7) and terminator sequences (T7 Term). PURExpress reactions run with 0.8 nM template were separated on SDS/PAGE followed by fluorescence detection of BODIPY-FL tagged lysine incorporated into proteins produced during the transcription/translation reaction. Error bars represent 1 SD (n = 3).