Kinetics of intermolecular cleavage by hammerhead ribozymes

Abstract

The hammerhead catalytic RNA effects cleavage of the phosphodiester backbone of RNA through a transesterification mechanism that generates products with 2'-3'-cyclic phosphate and 5'-hydroxyl termini. A minimal kinetic mechanism for the intermolecular hammerhead cleavage reaction includes substrate binding, cleavage, and product release. Elemental rate constants for these steps were measured with six hammerhead sequences. Changes in substrate length and sequence had little effect on the rate of the cleavage step, but dramatic differences were observed in the substrate dissociation and product release steps that require helix-coil transitions. Rates of substrate binding and product dissociation correlated well with predictions based on the behavior of simple RNA duplexes, but substrate dissociation rates were significantly faster than expected. Ribozyme and substrate alterations that eliminated catalytic activity increased the stability of the hammerhead complex. These results suggest that substrate destabilization may play a role in hammerhead catalysis.

Hammerhead ribozyme kinetics

“…. k 2 , the E{S complex is in rapid equilibrium with free E and S and therefore the reaction behaves as a MichaelisMenten enzyme and the saturation experiment can be analyzed by an Eadie-Hofstee plot+ In this case, K M ϭ (k Ϫ1 ϩ k 2 )/k 1 reduces to K d ϭ k Ϫ1 /k 1 + Because, for a regime-one hammerhead, the rates of product release are always faster than k Ϫ1 , k cat ϭ k 2 in such a substrate saturation experiment+ In at least one case where it was carefully tested, the values of k max and K d obtained from a ribozyme saturation experiment agreed well with k cat and K M determined by a substrate saturation experiment (Fedor & Uhlenbeck, 1992)+ It is important to point out that, for hammerheads in the second kinetic regime, either a ribozyme or a substrate saturation experiment does not give K d or, indeed, any information about E{S stability, although the data will often superficially resemble a binding curve+ This is because the cleavage rate at subsaturating ribozyme does not reflect the fraction of ribozyme bound to substrate at equilibrium, but rather reflects the fraction of ribozyme that binds substrate during the incubation time chosen and therefore is governed by k 1 + E{S stability for hammerheads in the second regime can, however, be estimated by using the overall reaction equilibrium (Hertel et al+, 1994)+ Three example outcomes for a ribozyme saturation experiment are illustrated in Figure 6+ The first example is for a well-behaved hammerhead that does not have any alternate conformations on its reaction pathway+ The k max is 0+95 min Ϫ1 , representing the rate of chemistry, and the K d is 50 nM+ The second example represents a ribozyme with a K d of 60 nM, similar to that of the first hammerhead, but its k cat is slow at 0+3 min Ϫ1 + This type of behavior may either reflect an alternate conformation of E{S on the reaction pathway (Fig+ 3D), or an alternate conformation of S that binds E, forming an inactive E{S9 complex+ Example three represents a hammerhead with a K d of 620 nM and a k max of 0+85 min Ϫ1 + This hammerhead may have very stable alternate conformations or aggregates of the ribozyme, E9, that reduce the amount of active E available to form active E{S (Fig+ 3B)+ Thus, increasing the ribozyme concentration eventually provides enough active ribozyme to reach the maximal rate+ Alternatively, the increase in K d may be due to slow assembly of the ribozyme-substrate complex because conversion of inactive S9 to active S is required for binding (Fig+ 3C)+ Hertel and coworkers recently compared the experimentally determined free energies for E{S stability for nine well-behaved hammerheads with the free energy of the corresponding uninterrupted helix I-III as calculated using the well-established rules for RNA duplex stability (Turner et al+, 1988;Serra & Turner, 1995)+ A good correlation between the predicted and mesured free energies was observed when a free energy of ϩ3+1 kcal/mol was assigned to the essential core nucleotides and stem loop II, which interrupt the perfect I-III helix (Hertel et al+, 1994; K+J+ Hertel, T+K+ StageZimmermann, G+ Ammons, & O+C+ Uhlenbeck, in prep+)+ This analysis permits the ⌬G8 and thus the K d for the E{S complex of any hammerhead sequence to be estimated by subtracting the experimentally determined core energy from the calculated helix energy+…”

“…A minimal kinetic pathway has been established for the hammerhead cleavage reaction containing four main species, the ribozyme (E), substrate (S), ribozymesubstrate complex (E{S), and ribozyme-product complex (E{P1{P2) (Fig+ 2) (Fedor & Uhlenbeck, 1992)+ In the I/III format, free ribozyme and substrate bind through helices I and III to form E{S+ In the presence of magnesium or other divalent metal ions, the E{S complex can either cleave, producing E{P1{P2, or dissociate to free E and S+ From E{P1{P2, the reaction either ligates back to E{S or proceeds with dissociation of each of the products from the ribozyme+ Each of these steps is defined by an elemental rate constant (Fig+ 2) (Fedor & Uhlenbeck, 1992;Hertel et al+, 1994)+ It is possible that additional steps exist on the pathway that are too fast to be detected by the experimental methods currently FIGURE 1. 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 ...…”

“…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+…”

“…For those hammerheads in the first kinetic regime, the K d for E{S can be obtained from a ribozyme saturation experiment where the rate of cleavage is measured over a range of ribozyme concentrations from lownanomolar to mid-micromolar (Fedor & Uhlenbeck, 1992)+ The substrate concentration should be kept at least 10-fold lower than that of the lowest ribozyme concentration so that the free ribozyme concentration approximates the total ribozyme concentration+ Either the annealing or nonannealing protocol can be used for this experiment because there is a rapid pre-equilibrium between E, S, and E{S+ The observed rate of cleavage, k obs , is plotted as a function of ribozyme concentration and fit to a binding equation to determine the rate at saturating ribozyme (k max ) and K d + For hammerheads in the first kinetic regime, the value of K d can also be determined in a substrate saturation experiment where the rate of cleavage is measured over a range of substrate concentrations and a low ribozyme concentration (Fedor & Uhlenbeck, 1990)+ In this case, many moles of substrate are cleaved by each ribozyme in a "multiple-turnover" reaction+ Because k Ϫ1 . .…”