Recognition of nucleoside triphosphates during RNA-catalyzed primer extension

In support of the idea that certain RNA molecules might be able to catalyze RNA replication, a ribozyme was previously generated that synthesizes shor t segments of RNA in a reaction modeled after that of proteinaceous RNA pol ymerases. Here, we describe substrate recognition by this polymerase ribozy me. Altering base or sugar moieties of the nucleoside triphosphate only mod erately affects its utilization, provided that the alterations do not disru pt 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 n ucleotides. Binding of the pyrophosphate leaving group appears weak, as evi denced by a very inefficient pyrophosphate-exchange reaction, the reverse o f the primer-extension reaction. Indeed, substitutions at the gamma -phosph ate can be tolerated, although poorly. Thio substitutions of oxygen atoms a t the reactive phosphate exert effects similar to those seen with cellular polymerases, leaving open the possibility of an active site analogous to th ose of protein enzymes. The polymerase ribozyme, derived from an efficient RNA ligase ribozyme, can achieve the very fast k(cat) of the parent ribozym e when the substrate of the polymerase (GTP) is replaced by an extended sub strate (pppGGA), in which the GA dinucleotide extension corresponds to the second and third nucleotides of the ligase. This suggests that the GA dinuc leotide, which had been deleted when converting the ligase into a polymeras e, plays an important role in orienting the 5'-terminal nucleoside. Polymer ase constructs that restore this missing orientation function should achiev e much more efficient and perhaps more accurate RNA polymerization.