The nonenzymatic replication of RNA is a potential transitional stage between the prebiotic chemistry of nucleotide synthesis and the canonical RNA world in which RNA enzymes (ribozymes) catalyze replication of the RNA genomes of primordial cells. However, the plausibility of nonenzymatic RNA replication is undercut by the lack of a protocell-compatible chemical system capable of copying RNA templates containing all four nucleotides. We show that short 5′-activated oligonucleotides act as catalysts that accelerate primer extension, and allow for the one-pot copying of mixed sequence RNA templates. The fidelity of the primer extension products resulting from the sequential addition of activated monomers, when catalyzed by activated oligomers, is sufficient to sustain a genome long enough to encode active ribozymes. Finally, by immobilizing the primer and template on a bead and adding individual monomers in sequence, we synthesize a significant part of an active hammerhead ribozyme, forging a link between nonenzymatic polymerization and the RNA world.DOI: http://dx.doi.org/10.7554/eLife.17756.001

The nonenzymatic replication of RNA oligonucleotides is thought to have played a key role in the origin of life prior to the evolution of ribozyme-catalyzed RNA replication. Although the copying of oligo-C templates by 2-methylimidazole-activated G monomers can be quite efficient, the copying of mixed sequence templates, especially those containing A and U, is particularly slow and error-prone. The greater thermodynamic stability of the 2-thio-U(s2U):A base pair, relative to the canonical U:A base pair, suggests that replacing U with s2U might enhance the rate and fidelity of the nonenzymatic copying of RNA templates. Here we report that this single atom substitution in the activated monomer improves both the kinetics and the fidelity of nonenzymatic primer extension on mixed-sequence RNA templates. In addition, the mean lengths of primer extension products obtained with s2U is greater than those obtained with U, augmenting the potential for nonenzymatic replication of heritable function-rich sequences. We suggest that noncanonical nucleotides such as s2U may have played a role during the infancy of the RNA world by facilitating the nonenzymatic replication of genomic RNA oligonucleotides.

Over evolutionary timescales, the logic and pattern of cell-type specific gene expression can remain constant, yet the molecular mechanisms underlying such regulation can drift between alternative forms. Here, we document a new example of this principle in the regulation of the haploid-specific genes in a small clade of fungal species. For most ascomycete fungal species, transcription of these genes is repressed in the a/α cell type by a heterodimer of two homeodomain proteins, Mata1 and Matα2. We show that in the species Lachancea kluyveri, most of the haploid-specific genes are regulated in this way, but repression of one haploid-specific gene (GPA1) requires, in addition to Mata1 and Matα2, a third regulatory protein, Mcm1. Model building, based on x-ray crystal structures of the three proteins, rationalizes the requirement for all three proteins: no single pair of the proteins is optimally arranged, and we show that no single pair can bring about repression. This case study exemplifies the idea that the energy of DNA binding can be “shared out” in different ways and can result in different DNA-binding solutions across different genes—while maintaining the same overall pattern of gene expression.