‘Hairpin’ catalytic RNA model: evidence for helices and sequence requirement for substrate RNA

Abstract

We have identified the catalytic domain within the sequence of the negative strand of the satellite RNA of tobacco ringspot virus. Minimum energy RNA folding calculations predict a two dimensional model with four major helical regions which are supported by mutagenesis experiments. This model for the catalytic complex consists of a 50 base catalytic RNA and a 14 base substrate RNA folded together in a type of hairpin two dimensional structure. Part of the recognition region between the catalyst and substrate is two helices of 6 bases and 4 bases respectively. Catalytic activity remains when the bases in these two helices are changed but base pairing is maintained. Thus an appropriately engineered 'hairpin' catalyst is capable of cleaving heterologous RNA.

A quantitative study of the flexibility contributed to RNA structures by nicks and single-stranded gaps

“…The cleavage site of the hammerhead ribozyme occurs at a two-helix junction (Forster & Symons, 1987)+ Cleavage of the RNA substrate is favored over ligation of the products, suggesting that cleavage increases the extent of thermal motion in the hammerheadoligonucleotide complex, thereby making ligation entropically disfavored (Hertel et al+, 1994)+ Our results qualitatively support this interpretation, although tertiary interactions not present in the model duplexes are expected to modulate the frequency, extent, and direction of thermal motions in more complex RNA molecules+ Two-helix junctions also occur in the reactants for ligation in the hairpin ribozyme (Hampel et al+, 1990) and within P1 of group I introns after the first step of splicing (Cech et al+, 1994)+ Two helices separated by one or more single-stranded nucleotides are found in many other natural RNAs, including RNase P (James et al+, 1988) and the snRNAs U2 (Guthrie & Patterson, 1988) and U8 (Sollner-Webb et al+, 1993)+ Some of the aforementioned RNAs exist as ribonucleoprotein complexes and may display dynamic behavior different from that of the free RNA+ Comparing the conformational dynamics of free RNAs with their corresponding RNPs represents another application of the thiol-disulfide interchange chemistry to be pursued in future studies+…”

Group I Ribozymes: Substrate Recognition, Catalytic Strategies, and Comparative Mechanistic Analysis

“…These were instrumental in allowing detailed mechanistic studies of the transesterification reaction, as well as implementing the reaction in new ways, such as achieving template-dependent RNA ligation or efficient cleavage of single-stranded DNA. The principle of separating a self-reacting RNA into a catalytic moeity and a substrate portion has proven to be useful in a wide variety of RNAs (e.g., Jacquier and Rosbash 1986;Uhlenbeck 1987: Haseloff andGerlach 1988;Hampel et al 1990;Perrotta and Been 1992;Lorsch and Szostak 1994).…”

In vitro Analysis of Ribozyme-mediated Knockdown of an ADRP Associated Rhodopsin Mutation

“…The sequence of these arms can be manipulated to allow binding to a variety of RNA substrates. Loop A contains the BNGUC target sequence required for cleavage where B = G, C, or U and N is any nucleotide (Hampel et al, 1990). There are no conserved nucleotides in any of the helices, although a G in the substrate portion of helix 2 adjacent to loop A leads to an increased rate of cleavage (Joseph and Burke, 1993).…”

“…The target cleavage site of the hairpin is less flexible. Mutational analysis demonstrated that only the GUC triplet is cleaved efficiently (Hampel et al, 1990).…”

“…The design of the three hairpin ribozymes was different. HP1 is the hairpin ribozyme found in the negative strand of the Tobacco Ringspot virus (Hampel et al, 1990) while the HP2 has a helix 4 with three additional base pairs and a stable loop to stabilize its secondary structure and to increases its catalytic activity. The third hairpin ribozyme, HP3, also has the modifications found in HP2 with the addition of a stem loop between helix 2 and helix 3.…”