The effect of base mismatches in the substrate recognition helices of hammerhead ribozymes on binding and catalysis

Abstract

The ability of the hammerhead ribozyme to distinguish between matched and mismatched substrates was evaluated using two kinetically defined ribozymes that differed in the length and sequence of the substrate recognition helices. A mismatch in the innermost base pair of helix I affected k2, the chemical cleavage step, while more distal mismatches had no such effect. In contrast, mismatches in any of the four innermost base pairs of helix III affected k2. Chase experiments indicated that mismatches also increased the rate of substrate dissociation by at least 20-100-fold, as expected from the stabilities of RNA helices.

Hammerhead ribozyme kinetics

“…A: Consensus secondary structure of the hammerhead numbered according to (Hertel et al+, 1992)+ The essential core nucleotides are designated in bold (H ϭ A, U, C and N ϭ nucleotide)+ The three loops (L1-L3) vary in length and sequence depending on where the hammerhead motif is embedded+ Arrow represents the site of cleavage 39 of position 17+ B: Three bimolecular formats of the hammerhead designated by the helices through which the substrate binds the ribozyme+ used+ Steps that have been proposed include: (1) conversion of E{S to a short-lived active complex with the attacking 29 oxygen positioned in line with the scissile phosphodiester bond (Pley et al+, 1994;Scott et al+, 1995Scott et al+, , 1996; (2) a large conformational rearrangement that involves docking of the two domains of the catalytic core (Peracchi et al+, 1997); (3) a metal ion binding step (Long et al+, 1995); or (4) a conformational switch from an inactive E{S to an active E{S (Bassi et al+, 1995(Bassi et al+, , 1996+ It is well known that many RNA sequences can adopt multiple alternate structures that are as stable as the native structure (Herschlag, 1995;Uhlenbeck, 1995)+ The addition of a single alternate equilibrium involving one of the species of the minimal hammerhead kinetic pathway can alter the kinetics of cleavage in several different ways+ Both the rate of exchange and the overall equilibrium between the native and alternate structure can significantly alter the kinetic properties of the cleavage reaction+ To give just one example, consider a situation in which an alternate conformation of E{S, termed [E{S]9, forms off of the main pathway (Fig+ 3A)+ If the exchange rate is slow relative to the rate constant for cleavage (k 2 ) and the equilibrium constant results in, say, 40% of the complex being [E{S]9, the cleavage reaction will be biphasic with a fast rate, k 2 , up to 60% product, followed by a slow rate reflecting the conversion of [E{S]9 to E{S+ Very different behavior exists when the exchange rate is fast with respect to k 2 + As before, the amount of active E{S available for conversion to E{P1{P2 is reduced by the fraction of [E{S]9 formed at equilibrium, however, a single, slower rate of cleavage will be observed that equals (k conf 9/k conf )k 2 + Many other possible scenarios involving alternate structures can exist (Fig+ 3B,C,D) and these species are not always easy to detect+ The challenge is therefore to uncover these additional steps and to kinetically distinguish them from the steps of the minimal kinetic pathway+ The easiest hammerheads to work with are obviously those that do not have alternate conformations of the reaction species+ Several of these kinetically well-behaved or ideal hammerheads have been identified and steps can be taken to test whether sequences show such behavior (Fedor & Uhlenbeck, 1990, 1992Heus et al+, 1990;Hertel et al+, 1994;Clouetd'Orval & Uhlenbeck, 1996)+ Kinetically w...…”

“…The hammerhead ribozyme can fall into one of two kinetic regimes that are defined by the relationship between the dissociation rate, k Ϫ1 , and the rate constant for cleavage, k 2 (Herschlag, 1991)+ The first regime is when k Ϫ1 is much faster than k 2 such that the E{S complex is in rapid equilibrium with free E and S+ The second regime is when k Ϫ1 is very slow compared to k 2 so that no pre-equilibrium occurs and every substrate that binds the ribozyme goes on to cleave+ Because, as discussed above, k 2 is a constant for all hammerheads (in a given buffer), the major factor that determines the kinetic regime of a given hammerhead is the value of k Ϫ1 , which is related to the stability of helices I and III+ To determine which regime a given hammerhead falls into and to obtain information on k Ϫ1 , a pulse-chase experiment is performed+ The protocol for this experiment, described in more detail elsewhere (Fedor & Uhlenbeck, 1992;Werner & Uhlenbeck, 1995;Clouetd'Orval & Uhlenbeck, 1996), involves combining an excess, saturating concentration of ribozyme with tracelabeled substrate and allowing the reaction to proceed for a certain time, t 1 , long enough such that all of the substrate is bound to the ribozyme (typically less than 1 min)+ After time t 1 , the reaction is chased either by addition of a large excess of nonradioactive substrate or by dilution with reaction buffer, and the time course of the cleavage reaction is monitored+ As a result of the chase, any labeled substrate that falls off the ribozyme during the chase period is unable to rebind and therefore is not cleaved+ Several times of incubation prior to the chase (t 1 ) should be tried to verify that the result obtained is independent of this parameter+ The dilution method is preferred over the nonradioactive substrate chase because the latter can lead to an abnormally fast substrate dissociation rate (Werner & Uhlenbeck, 1995 This pulse-chase experiment effectively defines the two kinetic regimes for the hammerhead ribozyme+ If no additional cleavage is seen after the chase is added, k Ϫ1 . .…”

“…. k 2 and the first regime holds true (Fig+ 5, triangles)+ If cleavage is not affected by the chase, k Ϫ1 , , k 2 and the second regime fits (Fig+ 5, squares)+ Certain hammerheads have a rate of substrate dissociation on the order of the rate of chemistry, k Ϫ1 Х k 2 so that cleavage is seen during the chase period (Fig+ 5, diamonds)+ In this relatively unusual case, k Ϫ1 can be determined directly from the chase experiment by one of two methods (Fedor & Uhlenbeck, 1992)+ For some hammerhead sequences, the kinetic regime can be changed by altering the pH of the reaction and thereby changing k 2 (Werner & Uhlenbeck, 1995;Clouet-d'Orval & Uhlenbeck, 1996)+ Because k Ϫ1 is not expected to depend on pH, changing the pH effectively alters the relative values of the two rate constants and potentially creates regime-one conditions+ Determining the kinetic regime for a hammerhead by the pulse-chase experiment defines the strategy to obtain k 1 and k Ϫ1 , the elemental rate constants for the binding step+ For hammerheads in the first regime, k 1 and k Ϫ1 cannot be determined directly, although K d ϭ k Ϫ1 /k 1 is easily obtained (see next section)+ For hammerheads in the second regime, k 1 can be obtained by measuring the rate of cleavage at subsaturating ribozyme concentration where binding is rate-limiting+ Under these conditions, k obs ϭ k 1 [R]+ The value of k 1 has only been measured for a limited number of hammerheads, but a value between 10 7 and 10 8 M Ϫ1 min Ϫ1 is usually obtained (Fedor & Uhlenbeck, 1992;Hertel et al+, 1994)+ This value is similar to k 1 values reported for simple RNA duplexes in high concentrations of monovalent cation (Pörschke & Eigen, 1971;Pörschke et al+, 1973;Nelson & Tinoco, 1982)+ However, k 1 values of DNA helices in buffers containing MgCl 2 are often faster (Williams et al+, 1989), suggesting that faster hammerhead association rates may be possible+…”

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

“…These mutations allow cleavage of the mutant mRNA but not the wild-type mRNA because the mismatched base pairs disrupt the formation of the catalytic core of the ribozyme (Grasby et al, 1995). Werner and Uhlenbeck (Werner and Uhlenbeck, 1995) have shown that a mismatch in any of the first four innermost nucleotides of helix III of a hammerhead ribozyme prevented cleavage. Only a mismatch in the innermost nucleotide of helix I prevented cleavage.…”

A hitchhiker's guide to antisense and nonantisense biochemical pathways

“…93 Interactions between viral proteins and viral RNAs are popular ificity of the hammerhead cleavage reaction were recently explored. 109 Substrates with mismatches close to the site of targets of RNA decoys.…”