Cryo-EM structure and functional landscape of an RNA polymerase ribozyme.

The emergence of an RNA replicase capable of self-replication is considered an important stage in the origin of life. RNA polymerase ribozymes (PR) - including a variant that uses trinucleotide triphosphates (triplets) as substrates - have been created by in vitro evolution and are the closest functional analogues of the replicase, but the structural basis for their function is poorly understood. Here we use single-particle cryogenic electron microscopy (cryo-EM) and high-throughput mutation analysis to obtain the structure of a triplet polymerase ribozyme (TPR) apoenzyme and map its functional landscape. The cryo-EM structure at 5-Å resolution reveals the TPR as an RNA heterodimer comprising a catalytic subunit and a noncatalytic, auxiliary subunit, resembling the shape of a left hand with thumb and fingers at a 70° angle. The two subunits are connected by two distinct kissing-loop (KL) interactions that are essential for polymerase function. Our combined structural and functional data suggest a model for templated RNA synthesis by the TPR holoenzyme, whereby heterodimer formation and KL interactions preorganize the TPR for optimal primer-template duplex binding, triplet substrate discrimination, and templated RNA synthesis. These results provide a better understanding of TPR structure and function and should aid the engineering of more efficient PRs.

Ribozyme-catalysed RNA synthesis using triplet building blocks.

RNA-catalyzed RNA replication is widely believed to have supported a primordial biology. However, RNA catalysis is dependent upon RNA folding, and this yields structures that can block replication of such RNAs. To address this apparent paradox, we have re-examined the building blocks used for RNA replication. We report RNA-catalysed RNA synthesis on structured templates when using trinucleotide triphosphates (triplets) as substrates, catalysed by a general and accurate triplet polymerase ribozyme that emerged from in vitro evolution as a mutualistic RNA heterodimer. The triplets cooperatively invaded and unraveled even highly stable RNA secondary structures, and support non-canonical primer-free and bidirectional modes of RNA synthesis and replication. Triplet substrates thus resolve a central incongruity of RNA replication, and here allow the ribozyme to synthesise its own catalytic subunit '+' and '-' strands in segments and assemble them into a new active ribozyme.

Nucleic acids: function and potential for abiogenesis.

The emergence of functional cooperation between the three main classes of biomolecules - nucleic acids, peptides and lipids - defines life at the molecular level. However, how such mutually interdependent molecular systems emerged from prebiotic chemistry remains a mystery. A key hypothesis, formulated by Crick, Orgel and Woese over 40 year ago, posits that early life must have been simpler. Specifically, it proposed that an early primordial biology lacked proteins and DNA but instead relied on RNA as the key biopolymer responsible not just for genetic information storage and propagation, but also for catalysis, i.e. metabolism. Indeed, there is compelling evidence for such an 'RNA world', notably in the structure of the ribosome as a likely molecular fossil from that time. Nevertheless, one might justifiably ask whether RNA alone would be up to the task. From a purely chemical perspective, RNA is a molecule of rather uniform composition with all four bases comprising organic heterocycles of similar size and comparable polarity and pK a values. Thus, RNA molecules cover a much narrower range of steric, electronic and physicochemical properties than, e.g. the 20 amino acid side-chains of proteins. Herein we will examine the functional potential of RNA (and other nucleic acids) with respect to self-replication, catalysis and assembly into simple protocellular entities.

Simple peptides derived from the ribosomal core potentiate RNA polymerase ribozyme function.

The emergence of functional interactions between nucleic acids and polypeptides was a key transition in the origin of life and remains at the heart of all biology. However, how and why simple non-coded peptides could have become critical for RNA function is unclear. Here, we show that putative ancient peptide segments from the cores of both ribosomal subunits enhance RNA polymerase ribozyme (RPR) function, as do derived homopolymeric peptides comprising lysine or the non-proteinogenic lysine analogues ornithine or, to a lesser extent, diaminobutyric acid, irrespective of chirality or chiral purity. Lysine decapeptides enhance RPR function by promoting holoenzyme assembly through primer-template docking, accelerate RPR evolution, and allow RPR-catalysed RNA synthesis at near physiological (≥1 mM) Mg2+ concentrations, enabling templated RNA synthesis within membranous protocells. Our results outline how compositionally simple, mixed-chirality peptides may have augmented the functional potential of early RNAs and promoted the emergence of the first protocells.

A synthetic approach to abiogenesis.

Synthetic biology seeks to probe fundamental aspects of biological form and function by construction (resynthesis) rather than deconstruction (analysis). Here we discuss how such an approach could be applied to assemble synthetic quasibiological systems able to replicate and evolve, illuminating universal properties of life and the search for its origins.

In-ice evolution of RNA polymerase ribozyme activity.

Mechanisms of molecular self-replication have the potential to shed light on the origins of life. In particular, self-replication through RNA-catalysed templated RNA synthesis is thought to have supported a primordial 'RNA world'. However, existing polymerase ribozymes lack the capacity to synthesize RNAs approaching their own size. Here, we report the in vitro evolution of such catalysts directly in the RNA-stabilizing medium of water ice, which yielded RNA polymerase ribozymes specifically adapted to sub-zero temperatures and able to synthesize RNA in ices at temperatures as low as -19 °C. The combination of cold-adaptive mutations with a previously described 5' extension operating at ambient temperatures enabled the design of a first polymerase ribozyme capable of catalysing the accurate synthesis of an RNA sequence longer than itself (adding up to 206 nucleotides), an important stepping stone towards RNA self-replication.

Ribozyme-catalyzed transcription of an active ribozyme.

A critical event in the origin of life is thought to have been the emergence of an RNA molecule capable of replicating a primordial RNA "genome." Here we describe the evolution and engineering of an RNA polymerase ribozyme capable of synthesizing RNAs of up to 95 nucleotides in length. To overcome its sequence dependence, we recombined traits evolved separately in different ribozyme lineages. This yielded a more general polymerase ribozyme that was able to synthesize a wider spectrum of RNA sequences, as we demonstrate by the accurate synthesis of an enzymatically active RNA, a hammerhead endonuclease ribozyme. This recapitulates a central aspect of an RNA-based genetic system: the RNA-catalyzed synthesis of an active ribozyme from an RNA template.

Ice as a protocellular medium for RNA replication.

A crucial transition in the origin of life was the emergence of an informational polymer capable of self-replication and its compartmentalization within protocellular structures. We show that the physicochemical properties of ice, a simple medium widespread on a temperate early Earth, could have mediated this transition prior to the advent of membraneous protocells. Ice not only promotes the activity of an RNA polymerase ribozyme but also protects it from hydrolytic degradation, enabling the synthesis of exceptionally long replication products. Ice furthermore relieves the dependence of RNA replication on prebiotically implausible substrate concentrations, while providing quasicellular compartmentalization within the intricate microstructure of the eutectic phase. Eutectic ice phases had previously been shown to promote the de novo synthesis of nucleotide precursors, as well as the condensation of activated nucleotides into random RNA oligomers. Our results support a wider role for ice as a predisposed environment, promoting all the steps from prebiotic synthesis to the emergence of RNA self-replication and precellular Darwinian evolution.