Template-directed primer extension catalyzed by the Tetrahymena ribozyme.

Abstract

The Tetrahymena ribozyme has been shown to catalyze an RNA polymerase-like reaction in which an RNA primer is extended by the sequential addition of pN nucleotides derived from GpN dinucleotides, where N = A, C, or U. Here, we show that this reaction is influenced by the presence of a template; bases that can form Watson-Crick base pairs with a template add as much as 25-fold more efficiently than mismatched bases. A mutant enzyme with an altered guanosine binding site can catalyze template-directed primer extension with all four bases when supplied with dinucleotides of the form 2-aminopurine-pN.Our interest in the possible role of RNA replicases in the origin and early evolution of life (2,5,8,12,16,24) led us to examine the extent to which a template can direct the incorporation of mononucleotides in a primer extension reaction catalyzed by the Tetrahymena ribozyme (1). The primer extension reaction involves nucleophilic attack by the 3' hydroxyl group of an oligonucleotide primer on the phosphate of a GpN (where N = A, C, or U) dinucleotide. This reaction extends the primer by one nucleotide and releases the guanosine nucleoside ( Fig. la). Each addition is analogous to the second step of self-splicing (6), in which the primer represents the 5' exon and is extended by a singlenucleotide 3' exon (17). This primer extension reaction is also analogous to protein-catalyzed RNA and DNA polymerization in that the 3' end of the growing chain is extended by successive additions of mononucleotides, with guanosine as the leaving group instead of pyrophosphate.Template-influenced primer extension. The original primerextension system studied by Been and Cech (1) did not include a uniquely defined template base that could interact with the incoming nucleotide. We tested the possibility that such a template base might influence the primer extension reaction by adding a template region just 5' of the primerbinding site, as illustrated in Fig. la. In our scheme, the primer can anneal with the primer-binding site in only one way; since the primer cannot slide along the template, it is possible to examine the influence of a single template base.Four ribozymes that differed in the template base were incubated with dinucleotide and labeled primer, with enzyme in large excess over primer (Fig. lb). All four ribozymes begin with pppG, followed by the indicated template base, followed by the primer-binding site, followed by the catalytic core (bases G27 to G405 in reference 7) of the wild-type Tetrahymena intron. These ribozymes were generated by runoff transcription of plasmid templates that were linearized at an EcoRI site inserted immediately after the position corresponding to G405 of the wild-type intron. Plasmids were constructed by polymerase chain reaction mutagenesis (21), and transcription and purification of ribozymes were as previously described (19). Primer was transcribed from a single-stranded oligonucleotide template * Corresponding author.(20). Dinucleotide initiation (22) with 4 mM GpC and abortive cycling (18) w...

Templating efficiency of naked DNA

“…The reactivity landscape of RNA may not be as smooth as that of DNA. Primer extension on RNA templates, catalyzed by ribozyme polymerases, can be strongly sequence dependent (42), though the order or reactivity is generally similar to that found here (43). Quantitative date like those provided here may help to better isolate substrate reactivity from catalysis.…”

Characterization of the B6.61 polymerase ribozyme accessory domain

“…While a number of strategies to create such a polymerase have been explored, none has been as successful as those making use of the Class I ligase ribozyme as a core catalytic component (Bartel et al 1991;Bartel and Szostak 1993;Johnston et al 2001;McGinness et al 2002;Lawrence and Bartel 2005). This ribozyme, originally isolated from a high diversity random sequence pool of RNA, is one of the fastest known ligase ribozymes (Bergman et al 2000).…”

New ligase-derived RNA polymerase ribozymes

“…Some progress has been made using natural self-splicing introns as a starting point (Been and Cech 1988;Doudna and Szostak 1989;Bartel et al 1991;Chowrira et al 1993;Doudna et al 1993;, while other approaches have focused on an artificial ribozyme called the Class I RNA ligase (Bartel and Szostak 1993;Ekland et al 1995). Derivatives of this ligase are able to extend an RNA primer by several nucleotides, but they require specific base-pairing to the RNA template strand (Ekland and Bartel 1996;.…”