Person:

Zhang, Wen

Many high-resolution crystal structures have contributed to our understanding of the reaction pathway for catalysis by DNA and RNA polymerases, but the structural basis of nonenzymatic template-directed RNA replication has not been studied in comparable detail. Here we present crystallographic studies of the binding of ribonucleotide monomers to RNA primer–template complexes, with the goal of improving our understanding of the mechanism of nonenzymatic RNA copying, and of catalysis by polymerases. To explore how activated ribonucleotides recognize and bind to RNA templates, we synthesized an unreactive phosphonate-linked pyrazole analogue of guanosine 5′-phosphoro-2-methylimidazolide (2-MeImpG), a highly activated nucleotide that has been used extensively to study nonenzymatic primer extension. We cocrystallized this analogue with structurally rigidified RNA primer–template complexes carrying single or multiple monomer binding sites, and obtained high-resolution X-ray structures of these complexes. In addition to Watson–Crick base pairing, we repeatedly observed noncanonical guanine:cytidine base pairs in our crystal structures. In most structures, the phosphate and leaving group moieties of the monomers were highly disordered, while in others the distance from O3′ of the primer to the phosphorus of the incoming monomer was too great to allow for reaction. We suggest that these effects significantly influence the rate and fidelity of nonenzymatic RNA replication, and that even primitive ribozyme polymerases could enhance RNA replication by enforcing Watson–Crick base pairing between monomers and primer–template complexes, and by bringing the reactive functional groups into closer proximity.

The nonenzymatic replication of primordial RNA is thought to have been a critical step in the origin of life. However, despite decades of effort, the poor rate and fidelity of model template copying reactions have thus far prevented an experimental demonstration of nonenzymatic RNA replication. The overall rate and fidelity of template copying depend, in part, on the affinity of free ribonucleotides to the RNA primer–template complex. We have now used 1H NMR spectroscopy to directly measure the thermodynamic association constants, Kas, of the standard ribonucleotide monophosphates (rNMPs) to native RNA primer–template complexes. The binding affinities of rNMPs to duplexes with a complementary single-nucleotide overhang follow the order C > G > A > U. Notably, these monomers bind more strongly to RNA primer–template complexes than to the analogous DNA complexes. The relative binding affinities of the rNMPs for complementary RNA primer–template complexes are in good quantitative agreement with the predictions of a nearest-neighbor analysis. With respect to G:U wobble base-pairing, we find that the binding of rGMP to a primer–template complex with a 5′-U overhang is approximately 10-fold weaker than to the complementary 5′-C overhang. We also find that the binding of rGMP is only about 2-fold weaker than the binding of rAMP to 5′-U, consistent with the poor fidelity observed in the nonenzymatic copying of U residues in RNA templates. The accurate Ka measurements for ribonucleotides obtained in this study will be useful for designing higher fidelity, more effective RNA replication systems.

The importance of genome replication has inspired detailed crystallographic studies of enzymatic DNA/RNA polymerization. In contrast, the mechanism of nonenzymatic polymerization is less well understood, despite its critical role in the origin of life. Here we report the direct observation of nonenzymatic RNA primer extension through time-resolved crystallography. We soaked crystals of an RNA primer-template-dGMP complex with guanosine-5′-phosphoro-2-aminoimidazolide for increasing times. At early times we see the activated ribonucleotides bound to the template, followed by formation of the imidazolium-bridged dinucleotide intermediate. At later times, we see a new phosphodiester bond forming between the primer and the incoming nucleotide. The intermediate is pre-organized because of the constraints of base-pairing with the template and hydrogen bonding between the imidazole amino group and both flanking phosphates. Our results provide atomic-resolution insight into the mechanism of nonenzymatic primer extension, and set the stage for further structural dissection and optimization of the RNA copying process.