Kinetic characterization of the first step of the ribozyme-catalyzed trans excision-splicing reaction P. Patrick Dotson II*, Joy Sinha* and Stephen M. Testa Department of Chemistry, University of Kentucky, Lexington, KY, USA We previously reported that a group I intron-derived ribozyme from Pneumocystis carinii can catalyze the excision of a targeted sequence from within an RNA transcript [1]. This reaction, called trans excision- splicing (TES), consists of two steps: substrate cleav- age (an intramolecular transesterification reaction) followed by exon ligation (Fig. 1). In the substrate- cleavage reaction, the phosphodiester backbone of an intermolecular substrate is cleaved via nucleophilic attack by the 3¢ terminal guanosine (G336), generat- ing 5¢ and 3¢ exon intermediates [1a]. In the exon-liga- tion step, the nucleophilic 5¢ exon intermediate attacks a phosphodiester backbone position within the 3¢ exon intermediate, simultaneously ligating the exons together and excising the internal segment. The substrate-cleavage reaction step is analogous to the 5¢ splice-site cleavage reaction in self-splicing [2], except that self-splicing utilizes an exogenous guanosine cofactor as the nucleophile. The TES substrate-clea- vage reaction is also directly analogous to the natu- rally occurring self-cyclization reaction, which results in the formation of full-length or truncated circular group I introns, in that they both utilize the 3¢ termi- nal guanosine of the intron (or ribozyme) as nucleo- philes [3–5]. Several studies have dissected the individual steps of RNA-catalyzed reactions through the establishment of kinetic frameworks [6–19]. This approach has been mechanistically informative and has greatly advanced our understanding of the chemical basis of RNA Keywords group I intron; ribozyme; RNA; self-splicing; trans excission-splicing Correspondence S. M. Testa, Department of Chemistry, University of Kentucky, Lexington, KY 40506, USA Fax: +1 859 323 1069 Tel: +1 859 257 7076 E-mail: testa@email.uky.edu *These authors contributed equally to this work (Received 3 March 2008, revised 7 April 2008, accepted 14 April 2008) doi:10.1111/j.1742-4658.2008.06464.x Group I introns catalyze the self-splicing reaction, and their derived ribo- zymes are frequently used as model systems for the study of RNA folding and catalysis, as well as for the development of non-native catalytic reactions. Utilizing a group I intron-derived ribozyme from Pneumocystis carinii, we previously reported a non-native reaction termed trans excision- splicing (TES). In this reaction, an internal segment of RNA is excised from an RNA substrate, resulting in the covalent reattachment of the flanking regions. TES proceeds through two consecutive phosphotranseste- rification reactions, which are similar to the reaction steps of self-splicing. One key difference is that TES utilizes the 3¢-terminal guanosine of the ribozyme as the first-step nucleophile, whereas self-splicing utilizes an exog- enous guanosine. To further aid in our understanding of ribozyme reac- tions, a kinetic framework for the first reaction step (substrate cleavage) was established. The results demonstrate that the substrate binds to the ribozyme at a rate expected for simple helix formation. In addition, the rate constant for the first step of the TES reaction is more than one order of magnitude lower than the analogous step in self-splicing. Results also suggest that a conformational change, likely similar to that in self-splicing, exists between the two reaction steps of TES. Finally, multiple turnover is curtailed because dissociation of the cleavage product is slower than the rate of chemistry. Abbreviations GBS, guanosine-binding site; RE1, recognition element 1; RE2, recognition element 2; RE3, recognition element 3; TES, trans excision-splicing. 3110 FEBS Journal 275 (2008) 3110–3122 ª 2008 The Authors Journal compilation ª 2008 FEBS catalysis. The fine details regarding the mechanism by which the first step of the TES reaction occurs is lar- gely unknown. In addition, little is known regarding the kinetics of 3¢ terminal guanosine-catalyzed reac- tions. Therefore, a minimal kinetic framework for this substrate-cleavage reaction was established (Fig. 2). There are multiple conclusions drawn from this kinetic framework as they relate to the TES reaction. The rate constant for the substrate-cleavage reaction is $ 60-fold lower than that reported for the first step of the self-splicing reaction using a Tetrahymena thermo- phila ribozyme, regardless of whether an intermolecular or intramolecular guanosine is being utilized as the first-step nucleophile [6,15]. The rate constant for the first step of the TES reaction is only fourfold greater than that for substrate dissociation. Furthermore, multiple turnover is curtailed because dissociation of the cleavage product is slower than the rate of cleavage. Lastly, the results indicate that a conformational change exists between the two steps of the TES reac- tion. Taken together, these results further demonstrate how group I intron-derived ribozymes exploit native recognition elements and catalytic sites to catalyze non-native, multi-step reactions. Results A kinetic scheme for the substrate-cleavage reaction, which is reaction step 1 in Fig. 1, is summarized in Fig. 2. One complication in studying the substrate- cleavage reaction is that the second reaction step of TES (exon–ligation) proceeds immediately after the first step [20]. To prevent the second reaction step while allowing the first, we previously utilized a sub- strate with a deoxyguanosine at the second step reac- tion site, [r(5 ¢ -AUGACUdGCUC-3¢)], which prevents the second reaction step [1a]. We found that the observed rate constant for the substrate-cleavage reaction using the deoxyguanosine substrate (k obs = 3 ± 0.5Æmin )1 ) is comparable, within error, to the normal substrate (k obs = 3.7 ± 0.2Æmin )1 l; data not G (RE3) U A a 5′ U U A a u g a c u U A G G A U 5′ G c u c a u g a c u U A G (RE2) -3′ 5′-a -6 u -5 g -4 a -3 c -2 u -1 g 1 c 2 u 3 c 4 5′ augacucuc 3′ Product dissociation Ribozyme binding Substrate cleavage P1 (RE1) P1 (RE1) P10 (RE3) P10 (RE3) A G 5′ G C 5′ c u c c u c 3′ Ribozyme P1 (RE1) 1P1P (RE1) (RE2) u g a c u A G U G 5′ G C A U 3′ (10-mer substrate) Step 1 Ribozyme P1 (RE1) P1 (RE1) P10 (RE3) (RE2) A G U G 5′ C 3′ Ribozyme Step 2 Exon ligation (9-mer p roduct) G g 1 g 1 G G u c c g 1 u c c 3′ g 1 g 1 g 1 Fig. 1. Schematic of the two-step TES reaction. The rPC ribozyme is in uppercase lettering and, the 10-mer substrate is in lowercase lettering, and the ribozyme recognition elements recognition ele- ment RE1 and RE3 base pair with the substrate to form helices P1 and P10, respectively. Note that recognition elements RE1, RE2 and RE3 are so named because they correspond to the regions in self-splicing introns that bind the exon substrates. The sites of catalysis for the first step (substrate cleavage) and the second step (exon ligation) are shown with arrows, and the guanosine to be excised (G 1 ) is circled. The diagram shows only the recognition elements of the ribozyme. P. P. Dotson II et al. Kinetics of the trans excision-splicing reaction FEBS Journal 275 (2008) 3110–3122 ª 2008 The Authors Journal compilation ª 2008 FEBS 3111 shown). Note that the k obs value of the normal reac- tion is in reasonable agreement with the previously reported value of 4Æmin )1 [1]. Therefore, the deoxy- guanosine substrate reasonably mimics the normal substrate as a first reaction step substrate. Impor- tantly, this substrate inhibits the exon-ligation step, allowing us to isolate and analyze only the first reac- tion step. Observed rate constants for substrate cleavage, k obs and k 2 Experiments under ribozyme excess conditions were used to determine the pseudo-first-order rate constant for the substrate-cleavage reaction. Note that under these reaction conditions the ribozyme–product com- plex is denatured upon addition of stop buffer, and so product dissociation is not observable. Therefore, these experiments measure the rate of substrate cleavage from the ribozyme–substrate complex. The observed rate constants (k obs ) were measured in reactions containing various ribozyme concentrations (5–300 nm) and 1.3 nm of 5¢-end radiolabeled substrate (Fig. 3A,B). As seen in Fig. 3C, the observed rate con- stants at the higher ribozyme concentrations (100– 350 nm) are independent of ribozyme concentration, indicating that saturation of the ribozyme has been reached. Values of k 2 = 4.1 ± 0.5Æmin )1 and K M = 102 ± 0.4 nm were obtained by fitting the aver- age k obs values to the Michaelis–Menten equation. Herein, k 2 represents the maximum first-order rate of substrate cleavage under single turnover conditions. For lower ribozyme concentrations (5–40 nm) the k obs values for the substrate-cleavage reactions increase linearly with ribozyme concentration. This linear dependence reflects the apparent second-order rate constant, k 2 ⁄ K M , and the slope gives a value of (2.8 ± 0.5) · 10 7 Æm )1 Æmin )1 (Fig. 3C, inset). Note that the values obtained are similar to those reported previ- ously for group I intron-derived ribozymes (Table 1) [14,17,21]. Dependence of substrate cleavage on pH It has been reported that the rate of the substrate- cleavage step in Tetrahymena [22–25], Anabaena [14] and Azoarcus [17] group I introns, as well as reaction steps for some small ribozymes [26–28], show a log- linear increase in the reaction rate constant with increasing pH in the acid range (up to pH 7). This is consistent with a single deprotonation step that takes place prior to the actual cleavage reaction [29]. This is also consistent with the observed rate constant at a given pH being equivalent to the rate constant of the chemical step at that pH. This was investigated for the Pneumocystis ribozyme by measuring the pH depen- dence of the observed rate constant of the substrate- cleavage reaction. As seen in Fig. 4, the logarithm of the observed rate constant increases linearly with pH in the range 5–7 (slope = 0.5 ± 0.03), but not at higher pH values. In the case of the Tetrahymena group I intron-derived ribozyme, such non-linear behavior was attributed to a pH-dependent conforma- tional change occurring within the ribozyme [24,25]. This conformational change thus sets a limit on the observed rate constant of cleavage (k 2 ), even though the rate constant of chemistry (k c ) is expected to con- tinue to increase with increasing pH [24,25]. Appar- ently, for our substrate-cleavage reaction, the rate of the chemical step is being masked by a conformational change, and so k 2 is not equivalent to k c . The rate of chemistry (k c ), however, can be approximated by extrapolating the log-linear increase that occurs between pH 5 and 7 to higher pH values. In our case, k c is then approximately equal to 5.7 ± 1.1Æ min )1 at pH 7.5 (Fig. 4). Control experiments were run to determine whether the observed rate constants shown in Fig. 4 were being influenced by the specific buffers utilized in the respec- tive reactions. We found that the values obtained using Mes and Hepes at pH 6.8 were within 1 SD of each other. This was also true using Hepes and Epps at pH 7.5. Apparently, there is not a buffer-specific effect E E *S k 2 = 4.1 min –1 k 1 = 1 x 10 7 M –1 · min –1 k –1 = 0.9 min –1 k –3 = 0.09 min –1 k 3 = 3.5 x 10 3 M –1 ·min –1 E + P K d P = 69 nMK d S = 90 nM k c = 5.7 min –1 E P Fig. 2. Kinetic scheme for the substrate-cleavage reaction. E denotes the rPC ribozyme, S denotes the 10-mer substrate, and P denotes the 6-mer cleavage product. All rate and equilibrium constants were measured or calculated (boxed values) in this report. The scheme includes rate constants for substrate association (k 1 ) and dissociation (k )1 ), cleavage (k 2 ), and product association (k 3 ) and dissociation (k )3 ). Note that the observed rate constant for the cleavage step (k 2 ) is distinguishable from the actual rate constant for chemistry (k c ). The scheme also includes equilibrium constants for substrate (K d S ) and product (K d P ) dissociation. Kinetics of the trans excision-splicing reaction P. P. Dotson II et al. 3112 FEBS Journal 275 (2008) 3110–3122 ª 2008 The Authors Journal compilation ª 2008 FEBS on the observed rate constants (k obs ). Note that we have not examined the rates of substrate cleavage outside the pH range depicted because protonation or deprotonation of nucleotides is expected to cause general chemical denaturation of the ribozyme [30]. Rate constant for substrate dissociation, k ) 1 The upper limit of the rate constant for substrate dissociation was measured in a pulse–chase experiment (Fig. 5A). In this experiment, the time chosen for t 1 (30 s) was such that a significant fraction of substrate would remain unreacted. After the addition of the chase, which in this case is dilution with buffer, aliqu- ots were removed at designated times (defined as t 2 )up to 15 min. An otherwise identical reaction, but without the added chase, was carried out in parallel. The ribo- zyme–substrate complex will decay through substrate cleavage (k 2 ) and dissociation (k )1 ). Therefore, measur- ing the observed rate constant during the chase phase will reflect both substrate cleavage and dissociation. This is summarized by: k obs, chase = k 2 + k )1 [6,9]. Note that in this experiment k 2 = k obs, no-chase . The observed rate constants for the chase reaction (k obs, chase = 2.5 ± 0.04Æmin )1 ) and in the reaction without added chase (k obs, no-chase = 1.5 ± 0.01Æmin )1 ) were obtained from a single-exponential fit of product formation against t 2 (Fig. 5B). The substrate dissocia- tion rate constant (k )1 = 0.9 ± 0.04Æmin )1 ) was then determined using Eqn (2) (see Experimental proce- dures). Note that k )1 is comparable in value to the cleavage step (k 2 ), implying that the ribozyme– substrate complex does not reach equilibrium with free ribozyme prior to the cleavage step. Rate constant for substrate association, k 1 The kinetic data indicate that substrate dissociation is comparable in value to the cleavage step. This implies that the second-order rate constant for substrate cleavage, k 2 ⁄ K M , will be a combination of substrate association (k 1 ), dissociation ( k )1 ) and cleavage (k 2 ) steps. Thus, the second-order rate constant can be represented as k 2 ⁄ K M = k 1 k 2 ⁄ (k )1 + k 2 ) [31]. As discussed earlier, a value of 2.8 · 10 7 Æm )1 Æmin )1 was obtained for the second order rate constant k 2 ⁄ K M . Using this value of k 2 ⁄ K M and the values of k 2 and k )1 (4.1Æmin )1 and 0.9Æmin )1 respectively), the calcu- lated value of k 1 is 3.4 · 10 7 Æm )1 Æmin )1 . For confirmation, k 1 was directly measured in a pulse–chase experiment (Fig. 5A). In this case, various concentrations of ribozyme and radiolabeled substrate were combined for varying times, t l (15–120 s). During the pulse phase, t 1 , the concentrations of the ribozyme, substrate and ribozyme–substrate complex are predicted to approach equilibrium, where the rate of Time (min) 0.25 min 0.5 0.75 1 2 3 4 5 10 15 min Substrate Product % Product (+) Buffer Ribozyme (nM) k obs (min –1 ) 0 1 2 3 0 100 200 300 0 0.5 1 1.5 0 1020304050 Ribozyme (nM) k obs (min -1 )) A B C Fig. 3. Substrate-cleavage reactions. All reactions were conducted in H10Mg buffer. (A) Representative polyacrylamide gel with the 5¢-end labeled substrate and 166 n M rPC ribozyme. The positions of the substrate and the substrate-cleavage product on the gel are labeled. The lane marked (+) buffer contains a 15-min reaction in the absence of the ribozyme. (B) Representative plot of the sub- strate-cleavage reaction at ribozyme concentrations of 5 n M ( ), 10 n M (s), 20 nM (h), 40 nM (e), 166 nM (D) and 300 nM (d). Observed rate constants (k obs ) were obtained from these plots and are the average of two independent assays. All data points between the two independent assays have a standard deviation < 15%. (C) Non-linear least squares fit to the Michaelis–Menten equation of the average k obs values from (B) versus ribozyme concentration (0–350 n M). The plot resulted in a value of k 2 = 4.1 ± 0.5Æmin )1 and K M = 102 ± 0.4 nM respectively. These values are the average of the two independent assays. The inset shows a representative plot of the average k obs values from (B) versus ribozyme concentration (5–40 n M). The resulting k 2 ⁄ K M value (2.8 ± 0.5 · 10 7 ÆM )1 Æmin )1 )is the average of the two independent assays. P. P. Dotson II et al. Kinetics of the trans excision-splicing reaction FEBS Journal 275 (2008) 3110–3122 ª 2008 The Authors Journal compilation ª 2008 FEBS 3113 substrate association equals substrate dissociation [6,9]. For the chase phase, the mixtures were then incubated for a time t 2 = 15 min, which ensures that essentially every substrate molecule that binds to the ribozyme during t l is converted to product. Therefore, the amount of product formed during the chase period is representative of the amount of ribozyme–substrate complex formed during t 1 . Note, however, that if k )1 $ k 2 , then both processes will be occurring during t 2 . The amount of product formed was plot against time t 1 (Fig. 6A). The k obs values reflect the rate of approach to equilibrium of the ribozyme–substrate complex formation, which is represented by k obs = k 1 [E]+k )1 [6,9]. The slope of the plot of k obs versus ribozyme concentration gives the rate of substrate association, k l = (1 ± 0.01) · 10 7 Æm )1 Æmin -l (Fig. 6B), which is in reasonable agreement (for ribozyme reac- tions) with the calculated value above. Reversibility of the substrate-cleavage reaction Under single-turnover conditions, the first-order rate constant (k 2 ) of the substrate-cleavage reaction is 4.1Æmin )1 (Fig. 2), with a typical end point of 70–80%. Over the period of 15–60 min, this end point does not change, indicating that either an internal equilibrium Table 1. Kinetic parameters for group I intron-derived ribozyme reactions. The k cat values correspond to the k 2 values reported throughout this text. Ribozyme origin k cat (min )1 ) k cat ⁄ K M (M )1 Æmin )1 ) K M S (lM) k c (min )1 ) Pneumocystis carinii a 4.1 2.8 · 10 7 0.102 5.7 Tetrahymena thermophila b 0.1 9 · 10 7 0.001 350 Anabaena PCC7120 c 4.0 2.9 · 10 5 15 4.0 Azoarcus sp. BH72 d 0.38 8.5 · 10 5 0.45 – a Substrate-cleavage reaction (endogenous guanosine-mediated) for the Pneumocystis ribozyme (rPC) with 10 m M MgCl 2 ,25mM Hepes (pH 7.5) and substrate (5¢-AUGACUdGCUC-3¢)at44°C. b Substrate- cleavage reaction (exogenous guanosine-mediated) of the Tetrahy- mena ribozyme (L21-ScaI) with 0.5 m M guanosine, 10 mM MgCl 2 , 50 m M Mes (pH 7) and substrate (5¢-G 2 CCCUCUAAAAA-3¢)at50°C [6]. c Substrate-cleavage reaction (exogenous guanosine-mediated) of the substrate (5¢-CUUAAAAA-3¢) using the Anabaena ribozyme (L-8 HH) with 2 m M guanosine, 15 mM MgCl 2 ,25mM Hepes (pH 7.5) at 32 °C [14]. d Substrate-cleavage reaction (exogenous guanosine-mediated) of Azoarcus ribozyme (L-10 HH) with 1 m M guanosine, 15 mM MgCl 2 ,25mM Hepes (pH 7.5) and substrate (5¢-CAUAAA-3¢)at30°C [17]. –1 –0.5 0 0.5 1 45678910 log k obs pH Fig. 4. pH dependence of the observed rate of substrate cleavage. Values for k obs were obtained from single-turnover reactions at 44 °C using 166 n M ribozyme, 1.3 nM 5¢-end labeled substrate in buffer containing 135 m M KCl and 10 mM MgCl 2 . The buffers used for this study were 50 m M Mes (pH 5.0–6.8), 50 mM Hepes (pH 7.0, 7.5) or 50 m M Epps (pH 8.0, 8.5). Each rate is the average of two independent measurements and has a standard deviation < 15%. The solid line represents the log-linear increase in the data set from pH of 5–7 (slope = 0.5 ± 0.03). The extrapolation of the line to pH 7.5 (depicted by dashed lines) gives a value of 0.75 ± 0.1 which corresponds to rate of chemistry (k c )of 5.7 ± 1.1Æmin )1 . Time (min) % Product during t 2 0 10 20 30 0246810121416 A B Fig. 5. Determination of the rate constant for substrate dissocia- tion, k )1 . (A) Scheme of the pulse–chase experiment, which was conducted in H10Mg buffer at 44 °C and 166 n M ribozyme. The chase was initiated by diluting the reaction mixture with H10Mg buffer. (B) Representative plot of cleaved substrate, after t 1 , versus time (t 2 ) with chase (closed circles) and without added chase (open circles). The resultant first-order rate constants obtained with (k obs, chase = 2.5 ± 0.04Æmin )1 ) and without (k obs, no-chase = 1.5 ± 0.01Æmin )1 ) the chase are the average of two independent assays. All data points between the two independent assays have a standard deviation < 10%. From this data, the rate of substrate dissociation, k )1 , is 0.9 ± 0.04Æmin )1 . Kinetics of the trans excision-splicing reaction P. P. Dotson II et al. 3114 FEBS Journal 275 (2008) 3110–3122 ª 2008 The Authors Journal compilation ª 2008 FEBS exists between ribozyme–substrate and ribozyme– product complexes or only 70–80% of the substrate is reactive. Such an internal equilibrium has previously been identified in a G-dependent substrate-cleavage reaction with Anabaena and Tetrahymena ribozymes [14,15]. Therefore, a pulse–chase experiment was con- ducted such that this equilibrium, if occurring, could be disturbed and thus detected [15]. In this assay, the substrate-cleavage reaction was allowed to proceed to completion and then an excess of unlabeled 5¢ exon mimic was added (Fig. 7A). Addition of a large excess of unlabeled 5¢ exon mimic is expected to prevent rebinding of any dissociated radiolabeled substrate or radiolabeled 5¢ exon reaction product. The result (Fig. 7B) shows that a substantial fraction of the bound radiolabeled product can be converted back to radiolabeled substrate, hence an internal equilibrium exists. Furthermore, the results imply that product dissociation is slower than or similar to substrate dis- sociation [15]. Equilibrium dissociation constant of the substrate-cleavage product, K d P and substrate, K d S A trace amount of 5¢-end radiolabeled substrate- cleavage product (the 6-mer) was incubated with vari- ous concentrations of ribozyme for 90 min at 44 °Cin H10Mg buffer, and the ribozyme–product complex was then partitioned from the unbound product on a native polyacrylamide gel [10]. The equilibrium dissociation constant of the 5¢ exon product (K d P =69±6nm) was then determined from a plot (Fig. 8) of the fraction product bound versus ribozyme concentration [32,33]. For the equilibrium dissociation constant of the substrate, K d S , an estimated value can be obtained from the equation K d S =(k )1 ⁄ k 1 )= 90 nm [19]. Time (min) % Substrate 0 20 40 60 80 100 0 50 100 150 A B Fig. 7. Substrate-cleavage products undergo the reverse reaction. (A) Scheme of the pulse–chase experiment, which was conducted using 166 n M ribozyme and a trace amount of 5¢-end labeled sub- strate in H10Mg buffer at 44 °C. The reaction was allowed to pro- ceed for 15 min (t 1 ), followed by the addition of excess unlabeled 5¢ exon product as the chase. (B) A plot of the disappearance of substrate in a normal reaction (no chase, closed circles) and reap- pearance of the substrate in the presence of chase (open circles). Each point on the plot is the average of two independent experi- ments, and have a SD of < 15%. Note that the error bars present on the graph are too small to be statistically relevant. Time (t 1 ) (min) % Product Ribozyme (nM) k obs (min –1 ) 0 20 40 60 0120.5 1.5 0 1 2 3 0 50 100 150 200 250 A B Fig. 6. Determination of the rate constant for substrate associa- tion, k 1 . (A) Representative plot of pulse–chase experiments in H10Mg buffer at 44 °C with five different ribozyme concentrations: 30 n M (s), 50 nM ( ), 100 nM (e), 150 nM (r) and 200 nM (d). All data points between the two independent assays have a standard deviation < 10%. (B) Representative plot of the k obs values against ribozyme concentration. The line is fit to the equation k obs = k 1 [E]+k )1 and the substrate association rate (k 1 = 1 ± 0.01 · 10 7 ÆM )1 Æmin -l ) was calculated from the slope. Note that the error bars present on the graph are too small to be statisti- cally relevant. P. P. Dotson II et al. Kinetics of the trans excision-splicing reaction FEBS Journal 275 (2008) 3110–3122 ª 2008 The Authors Journal compilation ª 2008 FEBS 3115 Rate constant for dissociation of the 5¢ exon product, k ) 3 The product dissociation rate constant (k )3 ) was deter- mined using a pulse–chase assay (Fig. 9A), combined with native PAGE. In this assay, an excess of ribo- zyme was mixed with 1.3 nm 5¢-end labeled 5¢ exon mimic, which was then incubated in H10Mg buffer containing 3.4% glycerol at 44 °C for 30 min. An excess amount of unlabeled 5¢ product was then added to initiate the chase, and aliquots were removed at des- ignated times. These aliquots were directly loaded onto a running native polyacrylamide gel to isolate the bound and unbound fractions. For quantification, the amount of product not bound after the chase was sub- tracted from that at time t 1 , which yields the amount of product dissociated due to the chase. The rate of product dissociation (k )3 = 0.09 ± 0.05Æmin )1 ) was then obtained from fitting Eqn (1) to a single expo- nential function (Fig. 9B). Apparently, product diss- ociation is slower than substrate dissociation, which has previously been shown for a Tetrahymena ribo- zyme [6]. Discussion In this report, a kinetic framework for the first step of the TES reaction was obtained. Although the TES reaction is not known to occur in nature, the full- length circularization reaction, which does occur natu- rally, has mechanistic similarities [3–5]. Perhaps most importantly, both reactions utilize a 3¢ terminal guano- sine as a nucleophile to attack the 5¢ splice site (sub- strate-cleavage site). Furthermore, neither reaction requires an exogenous guanosine cofactor, which is standard for self-splicing reactions. Finally, neither reaction is dependent on the formation of helix P10 for the 5¢ splice site cleavage reaction (see Fig. 1). Note that in these studies, deoxyribose-containing substrates were used to isolate the first reaction step (substrate cleavage) by preventing the second reaction step (exon ligation). In addition, the product of the first reaction step is actually the intermediate in the full TES reaction. Substrate binding The rate constant for the substrate binding the Pneu- mocystis ribozyme, k 1 , is far below the diffusional limit of 10 11 Æm )1 Æmin )1 for the collision of small molecules [34]. Thus, unlike classical enzymes which react near diffusion-controlled limits [31,35–37], the Pneumocystis Time (min) % Unbound product 0 5 10 15 20 0 102030405060 A B Fig. 9. Determination of the rate constant for dissociation of the 5¢ exon product, k )3 . (A) Scheme of the pulse–chase experiment con- ducted with rPC ribozyme and 5¢-end labeled 5¢ exon mimic in H10Mg buffer containing 3.4% glycerol at 44 °C. In this reaction t 1 = 30 min. Excess unlabeled 5¢ exon mimic was added to initiate the chase, and product dissociation was followed by native band- shift gel electrophoresis. (B) Representative plot of the fraction of unbound product versus chase time, t 2 . The rate of product dissoci- ation, k )3 , is 0.09 ± 0.05Æmin )1 , which is the average of two inde- pendent assays with each data point having a standard deviation typically < 20%. Ribozyme (nM) % Product bound 0 20 40 60 0 50 100 150 200 250 Fig. 8. Determination of the equilibrium dissociation constant of the substrate-cleavage product, K d P . In the reaction, various con- centrations of ribozyme were mixed with trace amounts of 5¢-end radiolabeled substrate-cleavage product in H10Mg buffer containing 3.4% glycerol. Shown is a representative plot of the percent sub- strate-cleavage product bound to the ribozyme versus ribozyme concentration. The resultant value of K d P is 69 ± 6 nM is the aver- age of two independent assays with each data point having a stan- dard deviation < 15%. Kinetics of the trans excision-splicing reaction P. P. Dotson II et al. 3116 FEBS Journal 275 (2008) 3110–3122 ª 2008 The Authors Journal compilation ª 2008 FEBS ribozyme is not under diffusion control. This value, however, is within the range (10 7 –10 9 Æm )1 Æmin )1 ) expected for the formation of RNA duplexes [38–42], as seen with other ribozymes [6,8,13,18,19,43]. Thus, the rate of assembly of the Pneumocystis ribozyme– substrate complex appears to be limited by the process of helix formation. Nevertheless, because k 2 ⁄ K M (k 2 = 4.1Æmin )1 and K M = 102 nm respectively) approaches the rate of substrate association, catalysis can be expected to occur about as fast as base-pairing between the ribozyme and substrate. This is typical of ribozymes that bind their substrates through double helices [6,13,16,19,44]. Substrate cleavage The observed rate constant for the substrate-cleavage reaction, k 2 , under single turnover conditions is 4.1Æmin )1 . Although the true rate constant for the actual chemical step is being masked, probably by a local conformational change that occurs after substrate binding and before the actual chemical step, this rate is approximately four times faster than the rate con- stant for substrate dissociation (k )1 = 0.9Æmin )1 ). Therefore, although the substrate is more likely to react than it is to dissociate, the similar order of magnitude suggests that a non-trivial fraction of the substrate will dissociate before the substrate-cleavage reaction occurs. The ‘catalytic power’ of an RNA-cleaving ribozyme can be estimated by comparing the observed rate constant of a catalyzed reaction with that of an equivalent uncatalyzed reaction. Under simulated physiological conditions, the uncatalyzed rate constant of the phosphotransesterification reaction (k noncat )is estimated to be 10 )9 Æmin )1 [6,45]. Thus, a rate of 4.1Æmin )1 for the substrate-cleavage reaction repre- sents a catalytic rate enhancement (k 2 ⁄ k noncat )of $ 10 9 -fold. This rate enhancement also corresponds to $ 13 kcalÆmol )1 of transition-state stabilization according to the following equation: DG° = )RT ln (k 2 ⁄ k noncat ), as discussed [6]. It was previously reported that a Tetrahymena ribo- zyme can also catalyze a 3¢ terminal guanosine-medi- ated substrate-cleavage reaction [3,4,15,46]. In one such study [15], the 3¢ terminal guanosine catalyzed reaction was reported to behave similar to the exoge- nous guanosine catalyzed reaction, for which k c = 350Æmin )1 [6]. In comparison, the P. carinii endogenous reaction is $ 60-fold slower (k c = 5.7 min )1 ; Table 1). This substantial difference might be due to the Tetrahymena ribozyme being faster than the Pneumocystis ribozyme, the difference in reaction conditions, or that the intramolecular guanosine nucle- ophile in the Pneumocystis ribozyme, although bound to the guanosine-binding site (GBS), is not bound in an ideal orientation. Indeed, this last idea may be sup- ported in that proper alignment of the intramolecular guanosine nucleophile with respect to the Pneumocystis ribozyme could be hindered by the absence of a P9.0 helix interaction, which is predicted to align the intra- molecular guanosine into the GBS [15]. The observed rate constant of substrate cleavage shows pH independence between pH 7 and 8.5, implying that in this range the rate of chemistry associated with substrate cleavage is masked by a conformational change. The simplest interpretation of this result is that the rate of substrate cleavage is not equivalent to the rate of chemistry, and that the rate of chemistry (extrapolated to be k c = 5.7Æmin )1 ) is faster than the rate of substrate cleavage (mea- sured to be k 2 = 4.1Æmin )1 ). Note that the nature of the conformational change is unknown with respect to the substrate-cleavage reaction, including any spe- cific rate constants associated with it, and so it is not included as a separate step in the reaction scheme (Fig. 2). Product dissociation For the fraction of substrates that do undergo the reaction, the resultant products dissociate from the ribozyme relatively slowly on the time scale of the reaction. Furthermore, dissociation of the 5¢ exon product is slower than the cleavage step (by $ 75- fold), which significantly impedes the ribozyme from catalyzing multiple turnover reactions. Of course, the 5¢ exon product of the cleavage reaction is an inter- mediate in the complete TES reaction, and so slow product dissociation is beneficial for the TES reac- tion as a whole. In addition, the product off-rate, k )3 , is 20-fold slower than the substrate off-rate, k )1 . It was also found in a Tetrahymena ribozyme [6,47] that the product off-rate is slower than the substrate off-rate, although in Tetrahymena there is only a twofold difference. Apparently, there are additional or more stable interactions that the ribozyme uses to bind the product relative to the ribozyme binding the substrate. This is perhaps due to destabilization of substrate binding via positioning of the 3¢-bridg- ing phosphoryl oxygen at the cleavage site next to a required Mg ion in the ground state [47]. As the negative charge develops on the 3¢ oxygen upon entering the transition state, this interaction will become more favorable. This transition state stabil- ization is thought to be an important stabiliza- P. P. Dotson II et al. Kinetics of the trans excision-splicing reaction FEBS Journal 275 (2008) 3110–3122 ª 2008 The Authors Journal compilation ª 2008 FEBS 3117 tion ⁄ destabilization factor in ribozyme-substrate bind- ing [47]. A conformation change exists between the two steps of the TES reaction The substrate guanosine to be excised (G 1 ) and its 2¢-OH group are required for the second step of TES [48], similar to (if not the same as) the role of the xG in the second step of self-splicing [48–56]. This suggests that the guanosine to be excised is likely binding to the GBS of the ribozyme for the exon-ligation step of TES. In the substrate-cleavage step, however, the 3¢ terminal guanosine (G336) of the ribozyme (Fig. 1) is binding to that same GBS. Therefore, for the TES reaction, there is likely a local conformational change between the two reaction steps that sees G 1 displace the ribozyme’s 3¢ terminal guanosine for binding into the GBS (see Fig. 1). The local conformational change that occurs in TES is likely similar to the local conformational change that occurs in self-splicing, with the displace- ment of the intermolecular guanosine by the xGof the intron [57–60]. Nevertheless, because TES uses an intramolecular nucleophile and self-splicing uses an intermolecular nucleophile, the local conforma- tional changes between the two steps of each reac- tion can not be identical. Implications for TES applications TES substrates, once bound, are four times more likely to undergo the substrate-cleavage reaction than they are to dissociate. Therefore, to make more effective TES ribozymes, one could decrease the rate of sub- strate dissociation relative to that for the substrate- cleavage reaction. Potential strategies for achieving this are to increase the strength of helix P1, either through target selection or elongation of helix P1. Note, how- ever, that this strategy could result in a decrease in the substrate cleavage rate. Results also suggest that the Pneumocystis ribozyme catalyzes the substrate-cleavage reaction (catalyzed by either an intermolecular or intramolecular guanosine) $ 60-fold slower than the Tetrahymena ribozyme. Therefore, it appears that there is room for improve- ment in terms of the rate of reaction. This would be beneficial not so much in terms of the rate of the over- all reaction, as the cleavage reaction is not the limiting step (binding is slower), but in terms of decreasing the amount of substrate that dissociates from the ribozyme before reactivity, effectively increasing the yield of the reaction. Experimental procedures Oligonucleotide synthesis and purification RNA oligonucleotides were obtained from Dharmacon (Lafayette, CO, USA), deprotected following the manufac- turer’s protocol, and stored in sterile water. Unlabeled RNAs were used without further purification. The substrate RNAs were 5¢-end radiolabeled with T4 polynucleotide kinase (New England Biolabs, Beverly, MA, USA) and [ 32 P]ATP[cP] (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and gel purified on a 20% nondenaturing polyacrylamide gel [33]. Transcription The ribozyme precursor plasmid was generated as described previously [33]. Prior to run-off transcription, the ribozyme plasmid was linearized with XbaI and purified using a QIA- quick PCR Purification kit (Qiagen, Valencia, CA, USA). The ribozyme, rPC, was then synthesized by run-off tran- scription and isolated as described previously [1]. After- wards, the ribozyme was precipitated with 2-propanol, with ethanol, dissolved in sterile water, and quantified using a Beckman DU-650 UV-Vis spectrophotometer (Beckman Coulter Inc., Fullerton, CA, USA) at 260 nm. Measurement of observed substrate cleavage rate constants (k obs and k 2 ) The first-order rate constant for substrate cleavage, k obs , was measured under single-turnover conditions, in which case the release of product would not affect the observed rate constants. Most reactions were conducted at 44 °Cin H10Mg buffer, which consists of 50 mm Hepes (25 mm Na + ), 135 mm KCl and 10 mm MgCl 2 at pH 7.5. These reaction conditions appear to be optimal for the TES reac- tion [1]. For the pH-dependence studies, Hepes (pH 7.5) was replaced with Mes (pH 5.0–6.8), Hepes (pH 6.8–7.5) or Epps (pH 7.5–8.5). Reactions were initiated by adding 5 lL of an 8 nm solution of 5¢-end radiolabeled substrate [r(5¢-AUGACUdGCUC-3¢)] in the appropriate buffer (at 44 °C) to a 25 lL solution of various concentrations of ribozyme (6–360 nm) in the same buffer (also at 44 °C). Note that the ribozyme solution was preincubated at 60 °C for 5 min and then allowed to slow cool to 44 °C to facilitate folding of the ribozyme prior to the addition of the radio- labeled substrate. Aliquots (3 lL) were removed at specified times and quenched with an equal volume of 2 · stop buffer (10 m urea, 0.1 · TBE, 3 mm EDTA). The substrate and products were denatured at 90 °C for 1 min and then sepa- rated on a 12.5% denaturing polyacrylamide gel. The bands were visualized on a Molecular Dynamics Storm 860 Phosphorimager and quantified using imagequant software (Molecular Dynamics, GE Healthcare, Piscataway, NJ, Kinetics of the trans excision-splicing reaction P. P. Dotson II et al. 3118 FEBS Journal 275 (2008) 3110–3122 ª 2008 The Authors Journal compilation ª 2008 FEBS USA). Data were fit using the kaleidagraph curve-fitting program (Synergy Software, Reading, PA, USA). The final concentration of the radiolabeled substrate in all reactions is 1.3 nm. A typical reaction utilized H10Mg buffer and a final ribozyme concentration of 166 nm. Pseudo-first-order rate constants for the appearance of products were fit using the following single exponential equation: ½P t ¼½P 1 ð1 À e Àkt Þð1Þ In Eqn (1) [P] t and [P] ¥ are the percentages of product formed at time t and at the end point, respectively, and k is the first-order rate constant. Measurement of the substrate dissociation rate constant (k ) 1 ) Pulse–chase experiments [6,61] were used to measure the rate constant for substrate dissociation, k )1 . In these experi- ments, 10 lL of 200 nm ribozyme in H10Mg buffer was combined with 2 lLof8nm 5¢-end radiolabeled substrate in H10Mg buffer for t 1 = 30 s. The ribozyme solution was preincubated at 60 °C for 5 min and then slow cooled to 44 °C before addition of the substrate, which was also at 44 °C. The chase phase was then initiated by removing 5 lL of the reaction mixture and diluting the reaction mix- ture with 25 lL of H10Mg buffer (at 44 °C) so that [E]<K M . During the chase period, t 2 , dissociation of labeled substrate from the ribozyme is essentially irrevers- ible. Aliquots were removed at various times during the chase phase and the reaction was quenched by adding an equal volume of 2 · stop buffer. An otherwise identical reaction, but without adding the chase (which in this case is buffer), was carried out in parallel. The first-order observed rate constants k obs, chase and k obs, no-chase were obtained from a single-exponential fit of this data using Eqn (1) (as a function of t 2 ). The observed rate constant for substrate dissociation (k )1 ) was then calculated (Eqn 2) as the differ- ence between the two measured observed rate constants: k À1 ¼ k obs;chase À k obs;noÀchase ð2Þ Measurement of the substrate association rate constant (k 1 ) The rate constant for substrate binding, k 1 , was measured using a series of pulse–chase experiments. In each reaction, 5 lL of a ribozyme stock (from 36 to 240 nm) in H10Mg buffer was combined with 1 lLof8nm 5¢-end labeled sub- strate and allowed to react in a total volume of 6 lL. The ribozyme solution was preincubated at 60 °C for 5 min and then slow cooled to 44 °C before the addition of the sub- strate, which was also at 44 ° C. For each ribozyme concen- tration, several chase reactions were initiated. In each chase, 1 lL of the original reaction mixture was removed and diluted fivefold with H10Mg buffer at 44 °C, t 1 ,at times ranging from 15 to 120 s. The addition of chase ren- ders the dissociation of the substrate essentially irreversible. The chase reaction, t 2 , was then allowed to proceed for 15 min, at which point the substrate-cleavage reaction was essentially complete. The reaction was quenched with an equal volume of 2 · stop buffer. The percent product formed during the chase period was plotted against time t 1 . Observed rate constants (k obs ) were obtained by fitting the data to Eqn (1). This observed rate constant measures the rate of approach to equilibrium where substrate association is equal to substrate dissociation. Hence, the rate of sub- strate association was obtained [6,13] by plotting k obs against ribozyme concentration and fitting to the equation: k obs ¼ k 1 ½Eþk À1 : Measurement of the dissociation constant, K d P of the ribozyme–product complex The equilibrium dissociation constant K d P of the 5¢ exon mimic binding to the ribozyme was determined using native PAGE [8,12,33,62]. In this assay, several concentrations of ribozyme, ranging from 1.5 to 300 nm, were preannealed in 5 lL total volume containing 3.4% glycerol and H10Mg buffer for 5 min at 60 °C. After the solutions slow cooled to 44 °C, 2.5 lL of a stock of 0.5 nm radiolabeled 5¢ exon mimic in H10Mg buffer at 44 °C was added. The mixture was incubated at 44 °C for at least 90 min. To maintain the integrity of the bound species during gel electrophoresis, the gel and the running buffer were made of H10Mg buffer and were prewarmed to 44 °C before the samples were loaded. The bound and unbound 5¢ exon mimics were sepa- rated from each other by running 6 lL of each reaction on a 10% nondenaturing polyacrylamide gel. The gel was placed on chromatography paper (Whatman 3MM CHR) and dried under vacuum for 30 min at 70 °C. The bands were visualized on a Molecular Dynamics Storm 860 Phos- phorimager and quantified using imagequant software (Molecular Dynamics). Data were fit using the kaleida- graph curve-fitting program (Synergy Software) using the equation: h = [ribozyme] u ⁄ ([ribozyme] u + K d ) [32,33]. In this equation, K d is the equilibrium dissociation constant of the 5¢ exon mimic, h is the fraction of 5¢ exon mimic bound to the ribozyme, and [ribozyme] u is the concentration of unbound ribozyme in the reaction. Measurement of rate constant of substrate-cleavage product dissociation (k ) 3 ) The dissociation rate constant of the 5¢ exon intermediate (k )3 ), was measured by a pulse–chase protocol, followed by analysis of the ribozyme ⁄ product complex using native PAGE. In a typical experimental to measure k )3 , a solution of 300 nm ribozyme in 10 lL H10Mg buffer containing 3.4% glycerol was preincubated for 5 min at 60 °C and P. P. Dotson II et al. Kinetics of the trans excision-splicing reaction FEBS Journal 275 (2008) 3110–3122 ª 2008 The Authors Journal compilation ª 2008 FEBS 3119 [...]...Kinetics of the trans excision-splicing reaction P P Dotson II et al then allowed to slow cool to 44 °C Then 5 lL of 0.5 nm 5¢-end labeled 5¢ exon intermediate was added and the reaction mixture was incubated at 44 °C for 30 min to allow complete binding A chase reaction was then initiated by the addition of 40 lL of 5.4 lm unlabeled 5¢ exon intermediate in reaction buffer to follow the practically... 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Patrick Dotson II*, Joy Sinha* and Stephen. isolate the first reaction step (substrate cleavage) by preventing the second reaction step (exon ligation). In addition, the product of the first reaction step