Recognition of Nucleoside Triphosphates during RNA-Catalyzed Primer Extension

Abstract

In support of the idea that certain RNA molecules might be able to catalyze RNA replication, a ribozyme was previously generated that synthesizes short segments of RNA in a reaction modeled after that of proteinaceous RNA polymerases. Here, we describe substrate recognition by this polymerase ribozyme. Altering base or sugar moieties of the nucleoside triphosphate only moderately affects its utilization, provided that the alterations do not disrupt Watson-Crick pairing to the template. Correctly paired nucleotides have both a lower K m and a higher k cat , suggesting that differential binding and orientation each play roles in discriminating matched from mismatched nucleotides. Binding of the pyrophosphate leaving group appears weak, as evidenced by a very inefficient pyrophosphate-exchange reaction, the reverse of the primer-extension reaction. Indeed, substitutions at the γ-phosphate can be tolerated, although poorly. Thio substitutions of oxygen atoms at the reactive phosphate exert effects similar to those seen with cellular polymerases, leaving open the possibility of an active site analogous to those of protein enzymes. The polymerase ribozyme, derived from an efficient RNA ligase ribozyme, can achieve the very fast k cat of the parent ribozyme when the substrate of the polymerase (GTP) is replaced by an extended substrate (pppGGA), in which the GA dinucleotide extension corresponds to the second and third nucleotides of the ligase. This suggests that the GA dinucleotide, which had been deleted when converting the ligase into a polymerase, plays an important role in orienting the 5′-terminal nucleoside. Polymerase constructs that restore this missing orientation function should achieve much more efficient and perhaps more accurate RNA polymerization.The RNA world hypothesis proposes that early life relied on catalytic RNAs rather than protein enzymes for catalysis (1). Much of the appeal of this theory comes from the notion that ribozymes, which could have served as their own genes, would have been much simpler to duplicate than proteins (2-5). Hence, a vital component of the RNA world scenario is an RNA polymerase ribozyme responsible for replicating the essential ribozymes of early life (including itself). Whether there might be RNA sequences that can promote RNA polymerization with sufficient accuracy and efficiency for RNA self-replication is one of the fundamental unanswered questions related to the RNA world hypothesis (1, 6). Finding such a sequence would support the idea of the RNA world and provide a key component for the synthesis of simple living cells (7).The class I ligase ( Figure 1A) is a promising starting point for efforts to develop a self-replicating ribozyme. This ribozyme ligates two RNAs, generating a 3′,5′-phosphodiester linkage and releasing pyrophosphate, a reaction similar to that of RNA and DNA polymerases (8). Engineered forms of the ligase can polymerize short segments of RNA ( Figure 1C). In the presence of appropriate RNA templates and nucleoside triphosphates (NTP...

The three-dimensional architecture of the class I ligase ribozyme

“…Furthermore, these protein enzymes and the ligase show a similar stereospecific response to sulfur substitution at the reactive phosphate, which is consistent with the idea that one of these essential metal ions may be bound in the same position relative to substrate in both catalysts (Eckstein 1985;Glasner et al 2000). This finding leaves open the possibility that the ligase uses the same mechanism as that proposed for general protein-catalyzed phosphoryl transfer, and prompts questions of whether the ribozyme might also share structural features with analogous protein enzymes.…”

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

“…This is based on the observation that reaction time courses for the Ca 2+ -preincubation experiments fit a singleexponential curve, even at high pH, as expected for a homogeneous population of molecules that are correctly folded at the moment of Mg 2+ addition. The chemical step (R f ‚S f P + PP i ) is represented as irreversible because previous work has shown that the reverse reaction (pyrophosphorolysis with formation of a triphosphate) is 10 7 -times slower than the forward reaction (21).…”

Zeptomole detection of a viral nucleic acid using a target-activated ribozyme

“…Kinetic analysis of target-bound clone 21 half-ribozyme activity with the variant substrate RNA pairs indicates that substrate RNA complex recognition and utilization is not identical to that reported for the constitutively active Class I ligase Bergman et al 2000;Glasner et al 2000Glasner et al , 2002. Most strikingly, mutation of A3 only slightly decreased (A3G and A3C) or increased (A3U) activity, although this position is highly conserved in the Class I ligase.…”

“…Although in principle this approach is general to large ribozymes that use extensive Watson-Crick base pairs, we developed it using the extensively characterized Class I ligase motif of Bartel and colleagues Bergman et al 2000;Glasner et al 2000Glasner et al , 2002.…”