DNA is inherently limited by its four natural nucleotides. Efforts to expand the genetic alphabet, by addition of an unnatural base pair, promise to expand the biotechnological applications available for DNA as well as being an essential first step towards expansion of the genetic code. We have conducted two independent screens of hydrophobic unnatural nucleotides to identify novel candidate base pairs that are well recognized by a natural DNA polymerase. From a pool of 3600 candidate base pairs, both screens identified the same base pair, dSICS:dMMO2, which we report here. Using a series of related analogs, we performed a detailed structure-activity relationship analysis, which allowed us to identify the essential functional groups on each nucleobase. From the results of these studies, we designed an optimized base pair, d5SICS:dMMO2, which is efficiently and selectively synthesized by Kf within the context of natural DNA.
A general mass spectrometry-based screen for unusually hydrophobic cellular small-molecule RNA conjugates revealed geranylated RNA in E. coli, Enterobacter aerogenes, Pseudomonas aeruginosa, and Salmonella thyphimurium. The geranyl group is conjugated to the sulfur atom in two 5-methylaminomethyl-2-thiouridine nucleotides. These geranylated nucleotides occur in the first anticodon position of tRNAGluUUC, tRNALysUUU, tRNAGlnUUG at a frequency of up to 6.7% (~400 geranylated nucleotides per cell). RNA geranylation levels can be increased or abolished by mutation or deletion of the selU (ybbB) gene in E. coli, and purified SelU protein in the presence of geranyl pyrophosphate and tRNA can produce geranylated tRNA. The presence or absence of the geranyl group in tRNAGluUUC, tRNALysUUU, and tRNAGlnUUG affects codon bias and frameshifting during translation. These RNAs represent the first reported examples of oligoisoprenylated cellular nucleic acids.
To what extent are evolutionary outcomes determined by a population's recent environment, and to what extent do they depend on historical contingency and random chance? Here we apply a unique experimental system to investigate evolutionary reproducibility and path dependence at the protein level. We combined phage-assisted continuous evolution with high-throughput sequencing to analyze evolving protein populations as they adapted to divergent and then convergent selection pressures over hundreds of generations. Independent populations of T7 RNA polymerase genes were subjected to one of two selection histories ("pathways") demanding recognition of distinct intermediate promoters followed by a common final promoter. We observed distinct classes of solutions with unequal phenotypic activity and evolutionary potential evolve from the two pathways, as well as from replicate populations exposed to identical selection conditions. Mutational analysis revealed specific epistatic interactions that explained the observed path dependence and irreproducibility. Our results reveal in molecular detail how protein adaptation to different environments, as well as stochasticity among populations evolved in the same environment, can both generate evolutionary outcomes that preclude subsequent convergence. directed evolution | evolutionary biology | tape of life
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