RNA-Catalyzed RNA Polymerization: Accurate and General RNA-Templated Primer Extension

Abstract

The RNA world hypothesis regarding the early evolution of life relies on the premise that some RNA sequences can catalyze RNA replication. In support of this conjecture, we describe here an RNA molecule that catalyzes the type of polymerization needed for RNA replication. The ribozyme uses nucleoside triphosphates and the coding information of an RNA template to extend an RNA primer by the successive addition of up to 14 nucleotides-more than a complete turn of an RNA helix. Its polymerization activity is general in terms of the sequence and the length of the primer and template RNAs, provided that the 3Ј terminus of the primer pairs with the template. Its polymerization is also quite accurate: when primers extended by 11 nucleotides were cloned and sequenced, 1088 of 1100 sequenced nucleotides matched the template.The RNA world hypothesis states that early life forms lacked protein enzymes and depended instead on enzymes composed of RNA (1). Much of the appeal of this hypothesis comes from the realization that ribozymes would have been far easier to duplicate than proteinaceous enzymes (2-5). Whereas coded protein replication requires numerous macromolecular components [including mRNA, transfer RNAs (tRNAs), aminoacyl-tRNA synthetases, and the ribosome], replication of a ribozyme requires only a single macromolecular activity: an RNA-dependent RNA polymerase that synthesizes first a complement, and then a copy of the ribozyme. If this RNA polymerase were itself a ribozyme, then a simple ensemble of molecules might be capable of self-replication and eventually, in the course of evolution, give rise to the protein-nucleic acid world of contemporary biology. Finding a ribozyme that can efficiently catalyze general RNA polymerization would support the idea of the RNA world (1, 6, 7) and would provide a key component for the laboratory synthesis of minimal life forms based on RNA (8, 9).

Metal Ion Requirements for Structure and Catalysis of an RNA Ligase Ribozyme

“…While there is no evidence supporting the specific metal contacts required by the two-metal-ion mechanism for the ligase reaction, there are several parallels between reactions catalyzed by the ligase ribozyme and proteinaceous polymerases that are consistent with this mechanism. The ligase, which forms the catalytic core of an RNA polymerase ribozyme, has the same stereochemical preference for sulfur substitution at the R-phosphate as polymerases composed of protein (20,21), and both the ligase and polymerases made of protein can tolerate only Mg 2+ or Mn 2+ at their active sites (30). In addition, kinetic and structural analysis in the presence of Mg 2+ and Ca 2+ suggested that the ligase may require two metal ions for catalysis.…”

Metabolically Coupled Replicator Systems: Overview of an RNA-world model concept of prebiotic evolution on mineral surfaces

“…It seems very reasonable to assume that some random RNA 175 molecules -or an assembly of a few different ones -generated on the mineral surface might have 176 had a weak RNA-replicase activity. This would have been sufficient to ignite the selection process 177 for a gradual increase of replicase activity (Attwater et al 2010; Johnson et al 2001). The snag with 178 this straightforward reasoning is empirical: even though RNA replicase ribozymes are actively 179 searched for in many laboratories (Johnson et al 2001 differentially, then the number of potentially coexistent replicators is equal to the number of 202 monomer pairs (two in this case: A+U and C+G, (Szilágyi et al 2013)).…”

“…This would have been sufficient to ignite the selection process 177 for a gradual increase of replicase activity (Attwater et al 2010; Johnson et al 2001). The snag with 178 this straightforward reasoning is empirical: even though RNA replicase ribozymes are actively 179 searched for in many laboratories (Johnson et al 2001 differentially, then the number of potentially coexistent replicators is equal to the number of 202 monomer pairs (two in this case: A+U and C+G, (Szilágyi et al 2013)). Since the differential use of 203 monomers by the replicators -i.e., a marked difference in their A+U and C+G demand -would 204 represent a severe constraint on their function (replicase activity), it seems reasonable to assume 205 that the monomer pool constitutes a single resource, which implies a single winner.…”

Evolutionary patterns of non-coding RNAs

“…In this RNA World scenario [117,116], the translation of RNA into proteins and, finally, the usage of DNA [110] as information storage device are later innovations. The wide range of catalytic activities that can be realized by relatively small ribozymes [22,177,188,191,236,411] as well as the usage of RNA catalysis at crucial points of the information metabolism of modern cells [186,86,289] provides support for the RNA World hypothesis. Plausible ribozyme catalyzed pathways for a late-stage ribo-organism [191], the role and evolution of co-enzymes [180], and a rather detailed model of the steps leading from the RNA world to modern cellular architectures [333] have been the subject of detailed investigations.…”