Importance of specific purine amino and hydroxyl groups for efficient cleavage by a hammerhead ribozyme.

Abstract

Eight modified ribozymes of 19 residues have been prepared with individual purine amino or hydroxyl groups excised. The modified ribozymes were chemically synthesized with the substitution of a single 2'-deoxyadenosine, 2'-deoxyguanosine, inosine, or purine riboside for residues G10, A"l, G3, or A". Five of the modified ribozymes cleaved the 24-mer substrate with little change in rate as monitored by simple first-order kinetics. However, deletion of the 2-amino group at G10 (replacement with inosine) or deletion of either of the 2'-hydroxyls at G10 or G13 (replacement with 2'-deoxyguanosine) resulted in ribozymes with a drastic decrease in cleavage efficiency. Increasing the concentration of the Mg2+ cofactor from 10 mM to 50 mM significantly enhanced cleavage efficiency by these three derivatives. Steady-state kinetic assays for these three ribozymes indicated that the modifications result in both an increase in K. and a decrease in kct,. These results suggest that the exocyclic amino group at G1' and the hydroxyls at G"' and G13 are important both for ribozymesubstrate binding and for the Mg2+-catalyzed cleavage reaction.RNA-processing reactions that involve the cleavage of phosphodiester bonds are critical steps for the production of many mature RNAs from corresponding precursors and appear to be important in the replication of several plant satellite RNAs (for reviews see refs. 1-3). Autolytic self-cleavage regions are present in certain virusoid RNAs and occur in a common structural domain termed a "hammerhead" (4). The consensus structure of a self-cleaving hammerhead contains 13 conserved nucleotides held together by three helical regions (4, 5). Nine of the conserved nucleotides occur in nonhelical regions and are critical for the observed cleavage reaction. Sequence variations in the helical regions can alter the rate of the reaction (6), but only the A-U and possibly the C-G base pair at the base of stem III, at the 5' side of the cleavage site, appear to be critical for activity (7). In addition to the hammerhead structural domain, a metal cofactor is required for the observed processing reaction. Mg2" is usually employed, but Mn2+ appears to function equally well in some cases (8). Cleavage occurs at the phosphodiester bond 3' to the residue located at the bend between two of the helices (this is most commonly cytidine) (6).The in vivo reaction takes place in a unimolecular complex, but cleavage can be observed in vitro from complexes formed from two (or even three) oligonucleotides (9-12), as long as the conserved nucleotide residues and the hammerhead structure are maintained. Hammerheads composed of two RNA fragments can exist in three distinct complexes differing in the location of the respective 3' and 5' termini and the hairpin loop. All three complexes exhibit the self-cleavage reaction but the rates of cleavage (as measured by tl/2 values) can differ significantly (13). The RNA cleavage reaction, catalyzed by Mg2+ (or Mn2+), proceeds as a transesterification ofthe 2'-hydroxyl with the...

Inter-domain cross-linking and molecular modelling of the hairpin ribozyme

“…Further, we can ®nd no evidence for the base to¯ip into a syn con®guration as suggested previously (Chowrira & Burke, 1991). At this point it is worth noting that in the hammerhead ribozyme removal of the N2 amino groups of guanine G 12 , involved in a sheared G ÁA pair (and also to some extent G 8 ) leads to severe reduction in catalytic activity despite the fact that they are far away from the cleaved phosphodiester (Fu & McLaughlin, 1992;Slim & Gait, 1992;Tuschl et al, 1993).…”

Hammerhead ribozyme kinetics

“…where F 0 is the fraction of product at zero time (which should be zero unless breakdown has occurred), F`is the fraction of product at the endpoint of the reaction, and k is the rate of cleavage (Fersht, 1985)+ For proper analysis, the data set should include several time points in the linear phase of the reaction as well as additional time points to establish the endpoint of the reaction+ A good fit to the equation thoughout the time course ensures that only a single rate constant is appropriate (Fig+ 4A)+ Early hammerhead kinetic papers often analyzed cleavage data by plotting the natural log of the fraction of substrate as a function of time and obtaining a rate constant from the slope of the early, linear part of the curve (Ruffner et al+, 1989;Fedor & Uhlenbeck, 1990, 1992Fu & McLaughlin, 1992;Hendrix et al+, 1995)+ Although this approach yields approximately the correct rate, it either ignores the endpoint altogether or attempts to correct the rate using the experimental endpoint+ Because it introduces error into the measurement, this method is not recommended+…”

Hammerhead ribozyme design and application

“…Mutagenesis studies and incorporation of base analogues have established several highly important or essential positions and their essential functional groups. The importance of the 2%-hydroxyl groups of all the residues in the conserved core has been investigated, as well as the exo-and endocyclic nitrogens of the purines, and the O 6 keto group of the guanines [34][35][36][37][38][39][40][41]. The 2%-hydroxyl groups of G5, G8 and A9 in the ribozyme and of U16.1 and N17 in the substrate (a 2%-hydroxyl at this position acts as the nucleophile in the reaction and is therefore essential) have been found to be important for full catalytic activity (reviewed in ref.…”