Effects of cetyltrimethylammonium bromide on reactions catalyzed by maxizymes, a novel class of metalloenzymes

www.elsevier.nl/locate/jinorgbio Journal of Inorganic Biochemistry 78 (2000) 69–77

Effects of cetyltrimethylammonium bromide on reactions catalyzed by maxizymes, a novel class of metalloenzymes Aya Nakayama a,b, Masaki Warashina a,b, Tomoko Kuwabara a,b, Kazunari Taira a,c,* a

National Institute for Advanced Interdisciplinary Research, 1-1-4 Higashi, Tsukuba Science City 305-8562, Japan Institute of Applied Biochemistry, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba Science City 305-8572, Japan c Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Hongo, Tokyo 113-8656, Japan b

Received 2 September 1999; received in revised form 1 November 1999; accepted 5 November 1999

Abstract We demonstrated previously that some shortened forms of hammerhead ribozymes had high cleavage activity that was similar to that of the wild-type parental hammerhead ribozyme. Moreover, the active species appeared to form dimeric structures with a common stem II (in order to distinguish monomeric forms of conventional minizymes that have low activity from our novel dimers with high-level activity, the latter very active short ribozymes were designated ‘maxizymes’). The dimers can be homodimeric (with two identical binding sequences) or heterodimeric (with two different binding sequences). In the case of heterodimers, they are in equilibrium with inactive homodimers. In this study, we investigated the effects of cationic detergent, cetyltrimethylammonium bromide (CTAB), on reactions catalyzed by a variety of maxizymes. The slope of close to unity in profiles of pH versus rate demonstrated that the deprotonation was important in catalysis and that the rate-limiting chemical step was followed in these reactions. Addition of appropriate amounts of CTAB enhanced the activity of a variety of maxizymes. The activity of our least stable, least active maxizyme was enhanced 100-fold by CTAB. Thus, CTAB effectively enhanced the conversion of kinetically trapped inactive conformations to active forms. Moreover, we suggest that the activity and specificity of catalytic RNAs in vivo might be better estimated if their reactions are monitored in vitro in the presence of appropriate amounts of CTAB. q2000 Elsevier Science Inc. All rights reserved. Keywords: Metalloenzymes; Hammerhead ribozymes; Maxizymes; pH–rate profiles; Kinetics; Strand displacement; Facilitators

1. Introduction Catalytic RNAs include hammerhead, hairpin, and hepatitis delta virus (HDV) ribozymes; group I and II introns; the RNA subunit of RNase P; and ribosomal RNA [1–12]. Among these catalytic RNAs, the first two ribozymes to be discovered, by Cech et al. [1] and Altman and co-workers [2], respectively, were the group I intron and the RNA subunit of RNase P. Within five years of these discoveries, small ribozymes, such as hammerhead (Fig. 1(A)), hairpin and HDV ribozymes, were discovered in studies of the replication, via a rolling-circle mechanism, of certain viroids, satellite RNAs and an RNA virus [7–12]. The hammerhead ribozyme is the smallest of all these catalytic RNAs [13,14]. Over the past few years, the hammerhead ribozyme has been recognized as a metalloenzyme [7,9,15–28], although, under extreme conditions (in the presence of 1 to 4 M monovalent cations such as Liq, Naq, and NH4q), the hammerhead * Corresponding author. Tel.: q81-3-5841-8828; fax: q81-298-54-3019; e-mail: [email protected]

ribozyme does not require divalent metal ions for catalysis [29]. The trans-acting hammerhead ribozyme consists of an antisense section (stem I and stem III; Fig. 1(A)) and a catalytic domain with a flanking stem/loop II section [14]. Such RNA motifs can cleave RNA targets at specific sites (most effectively at GUC) [30–35]. Many attempts have been made to create ribozymes with improved features. In one case, for example, extra sequences were deleted from the stem/loop II region of the hammerhead ribozyme. Such deletions are acceptable since stem II is the only helix in the hammerhead ribozyme that is not involved in binding of the substrate and the stem/loop II region appeared initially not to be directly involved in catalysis [36] (Fig. 1). For the development of chemically synthesized ribozymes as potential therapeutic agents, it would certainly be advantageous to remove any surplus nucleotides that are not essential for catalytic activity. Removal of surplus nucleotides would obviously reduce the cost of synthesis, increase the overall yield of the desired polymer, and simplify purification. These considerations led to the production of short

0162-0134/00/$ - see front matter q2000 Elsevier Science Inc. All rights reserved. PII S 0 1 6 2 - 0 1 3 4 ( 9 9 ) 0 0 2 1 1 - 1

Monday Jan 31 02:45 PM

StyleTag -- Journal: JIB (Journal of Inorganic Biochemistry)

Article: 6307

70

A. Nakayama et al. / Journal of Inorganic Biochemistry 78 (2000) 69–77

Fig. 1. Secondary structures of (A) the wild-type hammerhead ribozyme (R32), (B) the maxizyme that is capable of forming a homodimer, and (C) the heterodimeric maxizyme. A schematic representation of the overall folding of the hammerhead ribozyme is shown in (A) on the right. Homodimeric maxizymes have two identical substrate-binding sites, whereas the MzL-MzR heterodimeric maxizyme can generate two different binding sites: one is complementary to the sequence of the substrate (S11) that we used in this study, and the other is complementary to a substrate with a different sequence (in this figure, an uncleavable pseudosubstrate is shown). S11 can be cleaved only after the formation of a dimeric maxizyme.

ribozymes (minizymes), namely, conventional hammerhead ribozymes with a deleted stem/loop II region [36–42]. However, the activities of most minizymes (except the recently selected one by Conaty et al. [43]) turned out to be two to three orders of magnitude lower than those of the parental hammerhead ribozymes, a result that led to the suggestion that minizymes might not be suitable as gene-inactivating agents [41]. We tried independently to create variants of hammerhead ribozymes with deletions in the stem/loop II region and,

Monday Jan 31 02:45 PM

fortunately, we found that some shortened forms of hammerhead ribozymes (Fig. 1(B)) had high cleavage activity that was similar to that of the wild-type parental hammerhead ribozyme (R32; Fig. 1(A)). Moreover, the active species appeared to form dimeric structures with a common stem II [44,45]. In the active short ribozymes, the linker sequences that replaced the stem/loop II region were palindromic so that two short ribozymes were capable of forming a dimeric structure with a common stem II (Fig. 1(B)). In order to distinguish monomeric forms of conventional minizymes that

StyleTag -- Journal: JIB (Journal of Inorganic Biochemistry)

Article: 6307

A. Nakayama et al. / Journal of Inorganic Biochemistry 78 (2000) 69–77

have low activity from our novel dimers with high-level activity, we chose the name ‘maxizymes’ for the very active short ribozymes that were capable of forming dimers [46–49]. Maxizymes can form not only a homodimer (Fig. 1(B)) but also a heterodimer (Fig. 1(C)), in the latter case one maxizyme left (MzL) and one maxizyme right (MzR) are the monomers that together formed one heterodimeric maxizyme. Such a heterodimeric maxizyme has two independent substrate-recognition arms and can cleave its substrates only when the individual maxizymes form a heterodimer (Fig. 1(C) and Fig. 2). Only in the dimeric configuration, but not in its monomeric form, the important G10.1–C11.1 base-pair can be maintained. Binding of a metal ion to the pro-Rp oxygen (P9 oxygen) of the phosphate moiety of nucleotide A9 and to N7 of the basepaired nucleotide G10.1 is critical for efficient catalysis. Indeed, almost all crystallographic and biochemical studies to date suggest that N7 of G10.1 and the pro-Rp oxygen of the phosphate moiety of A9 are ligated with a metal ion (the conserved P9 metal) [31,38,50–57].

71

Many so-called facilitators have recently been identified that significantly increase the rates of formation of RNA duplexes and of ribozyme-catalyzed reactions [58–69]. These facilitators include nuclear proteins, the HIV-1 nucleocapsid (NC) protein, and cationic detergents. It seems possible, therefore, that the capacity of ribozymes for the rapid and specific cleavage of RNAs in vivo might be enhanced by such facilitators if ribozymes or substrates in an inactive conformation could be converted to active forms in vivo. We demonstrate in this paper that appropriate amounts of CTAB effectively enhance the conversion of kinetically trapped inactive conformations to active forms. Moreover, we suggest that the activity and specificity of maxizymes in vivo might be estimated better if their reactions are monitored in vitro in the presence of appropriate amounts of CTAB. 2. Materials and methods 2.1. Synthesis of maxizymes and substrates Maxizymes and their short substrates (Figs. 1 and 2) were synthesized chemically by a DNA/RNA synthesizer (model

Fig. 2. Simultaneous cleavage of HIV-1 tat mRNA at two sites by a dimeric maxizyme. (A) The secondary structure of HIV-1 tat mRNA, as predicted by MulFold (Biocomputing Office, Biology Department, Indiana University, IN, USA). Cleavage site 1 (GUC triplet-1) and cleavage site 2 (GUC triplet-2) are indicated by arrows. (B) Simultaneous cleavage of a target mRNA at two sites by a dimeric maxizyme. The catalytic action of Mg2q ions is indicated by ‘Mg scissors’. (C) Secondary structures of dimeric maxizymes that target HIV-1 tat mRNA. The 2 bp dimeric maxizyme has two G–C pairs in the common stem II, while the 5 bp dimeric maxizyme has five G–C pairs in the common stem II. The short substrate of 19 nucleotides (S19), indicated by a long bracket, was used in this study.

Monday Jan 31 02:45 PM

StyleTag -- Journal: JIB (Journal of Inorganic Biochemistry)

Article: 6307

72

A. Nakayama et al. / Journal of Inorganic Biochemistry 78 (2000) 69–77

394; Perkin-Elmer, Applied Biosystems (ABI), Foster City, CA). Reagents for RNA synthesis were purchased from Glen Research (Sterling, VA). Oligonucleotides were purified as described in the user bulletin from ABI (no. 53; 1989) with minor modifications. Further purification was performed by polyacrylamide gel electrophoresis, as described previously [19–22,44–49]. 2.2. Measurements of kinetic parameters Measurements of kinetic parameters of reactions catalyzed by maxizymes were made with 59-32P-labeled short substrates: S19 (59-CAG AAC AGU CAG ACU CAU C-39), which included the GUC triplet-2 of 272-meric HIV-1 tat mRNA (Fig. 2), was used for the reactions mediated by 2 bp and 5 bp dimeric maxizymes (Fig. 2(C)), and S11 (59-GCC GUC CCC CG-39) was used for the reactions mediated by homodimeric and heterodimeric maxizymes (Fig. 1(B) and (C)). The terms 2 bp and 5 bp dimeric maxizymes refer to dimeric maxizymes with two and five G–C pairs, respectively, in the common stem II. Reaction rates were measured, in 25 mM MgCl2, 50 mM Tris–HCl (pH 8.0; except for pH– rate profiles) and 10 mM NaCl under single-turnover conditions at 37 8C, in the presence or absence of 50 mM CTAB (Sigma, MO). Reactions were initiated by addition of appropriate amounts of MgCl2 after pre-incubation of the reaction mixture that contained all components apart from MgCl2 for several minutes at 37 8C. Reactions were stopped by removal of aliquots from the reaction mixture at appropriate intervals and mixing them with an equivalent volume of a solution that contained 100 mM EDTA, 9 M urea, 0.1% xylene cyanol, and 0.1% Bromophenol Blue. The substrate and the products of the reaction were separated by electrophoresis on a 20% polyacrylamide/7 M urea denaturing gel and were detected by autoradiography. The extent of cleavage was determined by quantitation of radioactivity in the bands of substrate and products with a Bio-Image analyzer (BAS2000; Fuji Film, Tokyo). Cleavage rates were obtained from the slopes of curves of the time courses of reactions at the initial stage.

3. Results and discussion 3.1. Kinetic analysis of reactions catalyzed by 2 bp and 5 bp dimeric maxizymes targeted to GUC triplet-2 of HIV-1 mRNA Our previous analysis indicated that increases in the length of the common stem II region were associated with increases in the activity of the heterodimeric maxizymes in vitro because maxizymes with larger numbers of base pairs in the common stem II were likely to form a larger proportion of active dimers [45,49]. Heterodimeric maxizymes that form two G–C pairs in the common stem II (2 bp maxizyme) and five G–C pairs in this region (5 bp maxizyme) were inves-

Monday Jan 31 02:45 PM

tigated in the presence or absence of CTAB that has been shown to enhance the activity of wild-type ribozymes by enhancing strand displacement [65–69]. Although we were concerned initially about the possibility that the strand displacement activity of CTAB might inhibit the dimerization process of the maxizymes, our preliminary results indicated that CTAB had a stimulatory rather than an inhibitory effect when the relatively long 272-meric HIV-1 tat mRNA was used as the substrate [49] (Fig. 2(A)). In this study, in order to quantitate the effect by CTAB, we used a short substrate of 19 nucleotides, S19, that corresponded to part of the HIV1 tat mRNA (Fig. 2(C)). The minimum reaction scheme for ribozymes can be described as shown in Fig. 3(A). First, the substrate (and Mg2q ions) binds to the ribozyme to form a Michaelis– Menten complex via formation of base pairs with stems I and III (kassoc). Then, a specific phosphodiester bond in the bound substrate (in this case, after GUC triplet-2) is cleaved by the action of Mg2q ions (kcleav; the ribozyme is recognized as a metalloenzyme [15–28]). This cleavage produces products with 29,39-cyclic phosphate and 59-hydroxyl groups. Finally, the cleaved fragments dissociate from the ribozyme (kdiss) and the liberated ribozyme is now available for a new series of catalytic events. The minimum reaction scheme for maxizymes is basically similar except for the additional dimerization process (Fig. 3(B)). We performed a kinetic study in the reaction mixtures that contained 50 mM Tris–HCl (pH 8.0), 10 mM NaCl, and 25 mM MgCl2, at 37 8C under singleturnover conditions [70,71], using a fixed concentration of the 2 bp or 5 bp maxizyme that was close to its respective apparent Km of 1.0 mM or 0.22 mM [45,49] and a fixed concentration of CTAB (50 mM) that was slightly above its critical micelle concentration (CMC). In these reactions, the short substrate (S19) was cleaved into a 10-meric 59-side product (labeled with P in Fig. 3) and a 9-meric 39-side product by the maxizymes. Effects of 50 mM CTAB on the maxizyme-catalyzed reactions are shown in Fig. 4: CTAB not only increased the yield of products (Fig. 4(A)) but it also enhanced the initial rate to a significant extent (Fig. 4(B)). In the case of 2 bp maxizyme (triangles), we recorded values of kobss0.001 miny1 and kobss0.1 miny1, respectively, in the absence and presence of CTAB. Corresponding values for the 5 bp maxizyme were kobss0.13 miny1 and kobss0.55 miny1. Thus, CTAB had a significant stimulatory effect especially in the case of 2 bp maxizyme: The rate of the reaction was increased 100fold by 50 mM CTAB. In the case of 2 bp maxizymes, the dimers are expected to generate a mixture of inactive (MzRPMzR)-dimers (the right dimer in blue in Fig. 5(A)), inactive (MzLPMzL)-dimers (the left dimer in red), and the desired active (MzRPMzL)-dimers (the dimer at the center). It is partly because of this mixed population of dimers that the activity of 2 bp maxizyme in the absence of CTAB is so low as compared with that of 5 bp maxizyme. In the case of 5 bp maxizymes, the sequence of the common stem II was designed such that the equilibrium concentration of the active

StyleTag -- Journal: JIB (Journal of Inorganic Biochemistry)

Article: 6307

A. Nakayama et al. / Journal of Inorganic Biochemistry 78 (2000) 69–77

73

Fig. 3. Reaction scheme of the cleavage mediated by (A) hammerhead ribozymes and (B) dimeric maxizymes. The upper panel shows the general reaction scheme for the parental hammerhead ribozyme and the lower panel shows that for the dimeric maxizymes. The dimeric maxizymes are more likely to exist in their inactive conformations.

complexes with perfect base pairing in the common stem II would be much higher than that of inactive complexes with only partial base pairing (Fig. 5(B)). It should be mentioned that, in the case of 2 bp maxizyme, the dimeric structure is stabilized not only by the formation of two G–C pairs at the common stem II but also by additional interactions that include two reversed-Hoogsteen G–A basepairs between G8–A13 and A9–G12, and a non-Watson–Crick A14–U7 base-pair that consists of one hydrogen bond, as indicated by dotted lines in Fig. 1. The extended stem II is stacked on the non-Watson–Crick base-pair, A15.1–U16.1, with resultant formation of a pseudo-A-form helix by stems II and III [52–54,72] (the right structure in Fig. 1(A)). 3.2. Effects of CTAB on the activities of homodimeric and heterodimeric maxizymes targeted to S11 In order to examine whether the CTAB-mediated activation was due solely to the conversion of mispaired dimeric maxizymes to correctly paired dimeric maxizymes (Fig. 5), we extended our study to include homodimeric maxizymes [44,47,49]. The homodimeric maxizyme we chose (Fig. 1(B)) cleaved the 11-meric substrate, S11 [27]. The corresponding heterodimeric maxizyme (Fig. 1(C)) was also

Monday Jan 31 02:45 PM

able to cleave the same S11 substrate, albeit at one site only. We did not expect the homodimeric maxizyme to be active in its monomeric configuration (the left structure in Fig. 1(B)) since binding of a conserved P9 metal ion to the proRp oxygen of the A9 phosphate and to the N7 of the basepaired nucleotide G10.1 is critical for efficient catalysis [31,38,50–57]. We at first used the same reaction conditions as described above, in the presence of 50 mM CTAB, with fixed concentrations of maxizymes that were close to the apparent Km values of 5.0 mM (Fig. 6(A) and (C)) and 0.5 mM (Fig. 6(B) and (D)), respectively [44,49], for the homodimer and heterodimer. In the case of the homodimer (Fig. 6(A) and (C)), we recorded values of kobss0.20 miny1 and kobss0.75 miny1, respectively, in the absence and presence of CTAB. Even in the case of the homodimeric maxizyme, CTAB increased the rate up to 3-fold. This finding is in accord with the previous conclusion [65–69] that CTAB enhances the annealing of a ribozyme to its substrate. It is also possible that, in this case, CTAB induced correct folding of intramolecularly misfolded maxizymes, as suggested in the case of 2 bp and 5 bp dimeric maxizymes (Fig. 5). With the heterodimeric maxizymes (Fig. 6(B) and (D)), the enhancement by 50 mM CTAB was much greater (36-fold) and we

StyleTag -- Journal: JIB (Journal of Inorganic Biochemistry)

Article: 6307

74

A. Nakayama et al. / Journal of Inorganic Biochemistry 78 (2000) 69–77

Fig. 4. Effects of CTAB on cleavage catalyzed by the 2 bp and 5 bp dimeric maxizymes. (A) Time courses of cleavage reactions catalyzed by the 2 bp (triangles) and 5 bp (circles) dimeric maxizymes in the presence (open symbols) and absence (closed symbols) of 50 mM CTAB. (B) The initial rates of the reactions catalyzed by the 2 bp and 5 bp dimeric maxizymes in the presence and absence of 50 mM CTAB. Pt is the relative amount of product at any time t [74].

recorded values of kobss0.0045 miny1 and kobss0.16 miny1, respectively, in the absence and presence of CTAB. We then investigated the effects of 50 mM CTAB, using the same reaction conditions as described above, at different concentrations of homodimeric (0.1 to 25 mM) and heterodimeric (0.1 to 10 mM) maxizymes (Fig. 6(E) and (F)). Interestingly, we observed bell-shaped profiles for both homodimeric and heterodimeric maxizymes. At high concentrations of maxizymes, the stimulatory effect of CTAB diminished. Nevertheless, at lower concentrations, CTAB enhanced heterodimeric maxizyme-catalyzed reactions significantly more than homodimeric maxizyme-catalyzed reactions. Finally, we varied the concentrations of CTAB under identical reaction conditions to those in the experiments shown in Fig. 6(A)–(D) where a fixed concentration of the homodimeric maxizyme was used that was close to its apparent Km of 5 mM and of the heterodimeric maxizyme that was close to its apparent Km of 0.5 mM. Results of these experiments are shown in Fig. 6(G) and (H). Although lower concentrations of CTAB enhanced the maxizyme-catalyzed reactions, the stimulatory effect of CTAB leveled off at higher concentrations. At high concentrations ()200 mM), CTAB instead inhibited maxizyme-catalyzed reactions. Interestingly, the CMC of CTAB appears to coincide with the optimum stimulatory concentration of CTAB. These data indicate that stimulatory effects were observed only when appropriate amounts of CTAB (near CMC) were used in maxizyme-catalyzed reactions. Under these stimula-

Fig. 5. Stimulation by CTAB of the conversion of inactive dimers to active dimers. (A) Active and inactive dimeric forms of 2 bp dimeric maxizymes. A large fraction of the population of dimers is expected to be in an inactive form. (B) The active and inactive dimeric forms of the 5 bp dimeric maxizymes. The formation of active forms is favored because perfect base pairing occurs only in the case of active complexes.

Monday Jan 31 02:45 PM

StyleTag -- Journal: JIB (Journal of Inorganic Biochemistry)

Article: 6307

A. Nakayama et al. / Journal of Inorganic Biochemistry 78 (2000) 69–77

75

Fig. 6. Effects of CTAB on cleavage catalyzed by the homodimeric maxizyme and the heterodimeric maxizyme. (A) Time courses of cleavage reactions catalyzed by the homodimeric maxizyme in the presence and absence of 50 mM CTAB. (B) Time courses of cleavage reactions catalyzed by the heterodimeric maxizyme in the presence and absence of 50 mM CTAB. (C) The initial rates of reactions catalyzed by the homodimeric maxizyme in the presence and absence of 50 mM CTAB. (D) The initial rates of the reactions catalyzed by the heterodimeric maxizyme in the presence and absence of 50 mM CTAB. For definition of Pt, see legend to Fig. 4. (E) Dependence of apparent rate constants (kobs) on the concentration of the homodimeric maxizyme in the presence of 50 mM CTAB. (F) Dependence of apparent rate constants (kobs) on the concentration of the heterodimeric maxizyme in the presence of 50 mM CTAB. (G) Effects of varying amounts of CTAB on cleavage catalyzed by the 5 mM homodimeric maxizyme. (H) Effects of varying amounts of CTAB on cleavage catalyzed by the 0.5 mM heterodimeric maxizyme.

Monday Jan 31 02:45 PM

StyleTag -- Journal: JIB (Journal of Inorganic Biochemistry)

Article: 6307

76

A. Nakayama et al. / Journal of Inorganic Biochemistry 78 (2000) 69–77

tory conditions, activities of maxizymes in vitro in the presence of CTAB [49] correlated with their activities in vivo [48]. Moreover, our separate experiment indicates that, although it is not possible to predict the activity and specificity of some maxizymes from their computer-predicted structures nor from their kinetic behaviors in vitro, it is significant that not only their activity but also their specificity in vivo are likely to be estimated from their kinetics in vitro when reactions are carried out in the presence of CTAB [73]. These findings suggest that, even though the activities and specificities in vitro of ribozymes in general do not necessarily reflect their activities in vivo, it might be possible to predict their activities and specificities in vivo by monitoring their activities and specificities in vitro in the presence of appropriate amounts of CTAB. 3.3. Conversion of kinetically trapped inactive conformations to active forms by appropriate amounts of CTAB Even though the homodimer and the heterodimer had the same number of G–C pairs in the common stem II and even though they were directed at the same target (Fig. 6), in other words, they were directed at the same substrate-binding site, it was apparent that there were different extents of enhancement in their activities by 50 mM CTAB. The greater enhancement by CTAB of activities of the heterodimeric maxizyme indicates that higher concentrations of inactive complexes, such as MzRPMzR and MzLPMzL (Fig. 5), were present in the absence of CTAB and were converted to active complexes (MzRPMzL) by CTAB. Therefore, CTAB appeared to have enhanced the association step (kassoc in Fig. 3(B)) of the reaction by converting kinetically trapped inactive conformations to active forms: Note that, in experiments shown in Fig. 6 (except for (E) and (F)), we followed the reactions under so-called kcat/Km conditions and CTAB is known to inhibit the chemical cleavage step (kcleav) [66,68]. This interpretation is at least consistent with the pH–rate profiles shown in Fig. 7. In ribozyme-catalyzed reactions, it is already established that deprotonation of 29-OH, by the action of a catalytic metal ion, of the nucleophile at the cleavage site is required in the formation of catalytically active species [7,9,15–28]. We examined the dependence of apparent kobs values on pH (the relationship between pH and logarithm of the rate; ‘pH–log rate profile’) under the same conditions described in Fig. 6(A)–(D) except for the pH (Fig. 7). The existence of a linear relationship between pH and log(kobs) demonstrates that the chemical step is the ratelimiting step, at least in part. The action of CTAB resembled that of an RNA chaperon [66], apparently inducing maxizymes that had misfolded to refold into an active conformation. Therefore, at each point in Fig. 7, the level of activity reflects the concentration of correctly folded maxizymes (the active complex shown in Fig. 3(B)) with a deprotonated 29-oxygen nucleophile at the cleavage site.

Monday Jan 31 02:45 PM

Fig. 7. The pH–log rate profiles of reactions catalyzed by (A) the homodimeric maxizyme and (B) the heterodimeric maxizyme. Deprotonation of the 29-OH, by the action of a catalytic metal ion, of the nucleophile at the cleavage site is required in the formation of catalytically active species [7,9,15–28].

4. Conclusions The strand displacement activity of CTAB appears to enhance the conversion of inactive misfolded maxizymes to active appropriately folded forms. In vivo, various facilitators exist that enhance strand displacement reactions similarly to CTAB, and they are expected to have stimulatory effects on the activities of dimeric maxizymes. Maxizymes with a short stem II such as 2 bp maxizymes, which tend to form inactive structures in vitro, in the absence of CTAB, were found to have significant activity in the presence of appropriate amounts of CTAB. In fact, the corresponding 2 bp maxizymes in HeLa cells under the control of a pol III promoter showed high-level activity that was at least as high as that of the parental hammerhead ribozyme [48]. These observations further strengthen the conclusion reached in the present study that various kinds of facilitator in vivo that function similarly to CTAB might enhance the activity of dimeric maxizymes. Moreover, our findings suggest that, even though the activities and specificities in vitro of ribozymes in general do not necessarily reflect their activities in vivo, it might be possible to predict not only their activities but also specificities in vivo by monitoring their activities and specificities in vitro in the presence of appropriate amounts (near CMC) of CTAB. Lastly, we should add that all maxizymes that have been constructed so far had higher activities in vivo than the corresponding parental hammerhead ribozymes [46–48,73].

StyleTag -- Journal: JIB (Journal of Inorganic Biochemistry)

Article: 6307

A. Nakayama et al. / Journal of Inorganic Biochemistry 78 (2000) 69–77

References [1] T.R. Cech, A.J. Zaug, P.J. Grabowski, Cell 27 (1981) 487–496. [2] C. Guerrier-Takada, K. Gardiner, T. Marsh, N. Pace, S. Altman, Cell 35 (1983) 849–857. [3] R.F. Gesteland, J.F. Atkins (Eds.), The RNA World, Cold Spring Harbor Laboratory Press, New York, 1993. [4] S. Altman, Adv. Enzymol. 62 (1989) 1–36. [5] N.R. Pace, J.W. Brown, J. Bacteriol. 177 (1995) 1919–1928. [6] T.R. Cech, Angew. Chem., Int. Ed. Engl. 29 (1989) 759–768. [7] J. Bratty, P. Chartrand, G. Ferbeyre, R. Cedergren, Biochim. Biophys. Acta 1216 (1993) 345–359. [8] R.H. Symons, Annu. Rev. Biochem. 61 (1992) 641–671. [9] K.R. Birikh, P.A. Heaton, F. Eckstein, Eur. J. Biochem. 245 (1997) 1–16. [10] J.M. Buzayan, W.L. Gerlach, G. Bruening, Nature 323 (1986) 349– 353. [11] L. Sharmeen, M.Y.-P. Kuo, G. Dinter-Gottlieb, J. Taylor, J. Virol. 62 (1988) 2674–2679. [12] H.-N. Wu, Y.-J. Lin, F.-P. Lin, S. Makino, M.-F. Chang, M.M.C. Lai, Proc. Natl. Acad. Sci. USA 86 (1989) 1831–1835. [13] O.C. Uhlenbeck, Nature 328 (1987) 596–600. [14] J. Haseloff, W.L. Gerlach, Nature 334 (1988) 585–591. [15] S.C. Dahm, W.B. Derrick, O.C. Uhlenbeck, Biochemistry 32 (1993) 13040–13045. [16] M. Yarus, FASEB J. 7 (1993) 31–39. [17] A.M. Pyle, Science 261 (1993) 709–714. [18] T. Uchimaru, M. Uebayasi, K. Tanabe, K. Taira, FASEB J. 7 (1993) 137–142. [19] M. Uebayasi, T. Uchimaru, T. Koguma, S. Sawata, T. Shimayama, K. Taira, J. Org. Chem. 59 (1994) 7414–7420. [20] S. Sawata, M. Komiyama, K. Taira, J. Am. Chem. Soc. 117 (1995) 2357–2358. [21] D.-M. Zhou, N. Usman, F.E. Wincott, J. Matulic-Adamic, M. Orita, L.-H. Zhang, M. Komiyama, P.K.R. Kumar, K. Taira, J. Am. Chem. Soc. 118 (1996) 5862–5866. [22] P.K.R. Kumar, D.-M. Zhou, K. Yoshinari, K. Taira, in: F. Eckstein, D.M.J. Lilley (Eds.), Nucleic Acids and Molecular Biology, vol. 10, Catalytic RNA, Springer, Berlin, 1996, pp. 217–230. [23] B.W. Pontius, W.B. Lott, P.H. von Hippel, Proc. Natl. Acad. Sci. USA 94 (1997) 2290–2294. [24] D.-M. Zhou, L.-H. Zhang, K. Taira, Proc. Natl. Acad. Sci. USA 94 (1997) 14343–14348. ¨ [25] S. Kuusela, H. Lonnberg, Curr. Top. Solution Chem. 2 (1997) 29– 47. [26] W.B. Lott, B.W. Pontius, P.H. von Hippel, Proc. Natl. Acad. Sci. USA 95 (1998) 542–547. [27] D.-M. Zhou, K. Taira, Chem. Rev. 98 (1998) 991–1026. [28] D.M.J. Lilley, Curr. Opinion Struct. Biol. 9 (1999) 330–338. [29] J.B. Murray, A.A. Seyhan, N.G. Walter, J.M. Burk, W.G. Scott, Chem. Biol. 5 (1998) 587–595. [30] M. Koizumi, S. Iwai, E. Ohtsuka, FEBS Lett. 239 (1988) 285–288. [31] D.E. Ruffner, G.D. Stormo, O.C. Uhlenbeck, Biochemistry 29 (1990) 10695–10702. [32] C.C. Sheldon, R.H. Symons, Nucleic Acids Res. 17 (1989) 5679– 5685. [33] R. Perriman, A. Delves, W.L. Gerlach, Gene 113 (1992) 157–163. [34] T. Shimayama, S. Nishikawa, K. Taira, Biochemistry 34 (1995) 3649–3654. [35] M. Zoumadakis, M. Tabler, Nucleic Acids Res. 23 (1995) 1192– 1196. [36] M.J. McCall, P. Hendry, P.A. Jennings, Proc. Natl. Acad. Sci. USA 89 (1992) 5710–5714. [37] J. Goodchild, V. Kohli, Arch. Biochem. Biophys. 284 (1991) 386– 391.

Monday Jan 31 02:45 PM

77

[38] T. Tuschl, F. Eckstein, Proc. Natl. Acad. Sci. USA 90 (1993) 6991– 6994. [39] J.B. Thomson, T. Thschl, F. Eckstein, Nucleic Acids Res. 21 (1993) 5600–5603. [40] D. Fu, F. Benseler, L.W. McLaughlin, J. Am. Chem. Soc. 116 (1994) 4591–4598. [41] D.M. Long, O.C. Uhlenbeck, Proc. Natl. Acad. Sci. USA 91 (1994) 6977–6981. [42] P. Hendry, M.J. McCall, F.S. Santiago, P.A. Jennings, Nucleic Acids Res. 23 (1995) 3922–3927. [43] J. Conaty, P. Hendry, T. Lockett, Nucleic Acids Res. 27 (1999) 2400– 2407. [44] S.V. Amontov, K. Taira, J. Am. Chem. Soc. 118 (1996) 1624–1628. [45] T. Kuwabara, S.V. Amontov, M. Warashina, J. Ohkawa, K. Taira, Nucleic Acids Res. 24 (1996) 2302–2310. [46] T. Kuwabara, M. Warashina, T. Tanabe, K. Tani, T. Asano, K. Taira, Mol. Cell 2 (1998) 617–627. [47] T. Kuwabara, M. Warashina, M. Orita, S. Koseki, J. Ohkawa, K. Taira, Nature Biotechnol. 16 (1998) 961–965. [48] T. Kuwabara, M. Warashina, A. Nakayama, J. Ohkawa, K. Taira, Proc. Natl. Acad. Sci. USA 96 (1999) 1886–1891. [49] A. Nakayama, T. Kuwabara, M. Warashina, K. Taira, FEBS Lett. 448 (1999) 67–74. [50] J.M. Buzayan, H. van Tol, P.A. Feldstein, G. Breuning, Nucleic Acids Res. 18 (1990) 4447–4451. [51] D.E. Ruffner, O.C. Uhlenbeck, Nucleic Acids Res. 18 (1990) 6025– 6029. [52] H.W. Pley, K.M. Flaherty, D.B. McKay, Nature 372 (1994) 68–74. [53] W.G. Scott, J.T. Finch, A. Klug, Cell 81 (1995) 991–1002. [54] W.G. Scott, J.B. Murray, J.R.P. Arnold, B.L. Stoddard, A. Klug, Science 274 (1996) 2065–2069. [55] A. Peracchi, L. Beigelman, N. Usman, D. Herschlag, Proc. Natl. Acad. Sci. USA 93 (1996) 11522–11527. [56] A. Peracchi, L. Beigelman, E.C. Scott, O.C. Uhlenbeck, D. Herschlag, J. Biol. Chem. 272 (1997) 26822–26826. [57] J.B. Murray, D.P. Terwey, L. Maloney, A. Karpeisky, N. Usman, L. Beigelman, W.G. Scott, Cell 92 (1998) 665–673. [58] B.W. Pontius, P. Berg, Proc. Natl. Acad. Sci. USA 87 (1990) 8403– 8407. [59] A. Kumar, S.H. Wilson, Biochemistry 29 (1990) 10717–10722. [60] Z. Tsuchihashi, M. Khosla, D. Herschlag, Science 262 (1993) 99– 102. [61] P. Oberosler, P. Hloch, U. Ramsperger, H. Stahl, EMBO J. 12 (1993) 2389–2396. [62] D.S. Portman, G. Dreyfess, EMBO J. 13 (1994) 213–221. [63] E.L. Bertrand, J.J. Rossi, EMBO J. 13 (1994) 2904–2912. [64] M. Altmann, B. Wittman, N. Methot, N. Sonenberg, H. Trachsel, EMBO J. 14 (1995) 3820–3827. [65] B.W. Pontius, P. Berg, Proc. Natl. Acad. Sci. USA 88 (1991) 8237– 8241. [66] D. Herschlag, M. Khosla, Z. Tsuchihashi, R.L. Karpel, EMBO J. 13 (1994) 2913–2924. [67] M. Homann, W. Nedbal, G. Sczakiel, Nucleic Acids Res. 24 (1996) 4395–4400. [68] W. Nedbal, G. Sczakiel, Biochem. Soc. Trans. 24 (1996) 615–618. [69] W. Nedbal, M. Homann, G. Sczakiel, Biochemistry 36 (1997) 13552– 13557. [70] Y. Takagi, K. Taira, FEBS Lett. 361 (1995) 273–276. [71] M. Warashina, Y. Takagi, S. Sawata, D.M. Zhou, T. Kuwabara, K. Taira, J. Org. Chem. 62 (1997) 9138–9147. [72] A.L. Feig, W.G. Scott, O.C. Uhlenbeck, Science 279 (1998) 81–84. [73] M. Hamada, T. Kuwabara, M. Warashina, A. Nakayama, K. Taira, FEBS Lett. 461 (1999) 77–85. [74] A. Cornish-Bowden (Ed.), Fundamentals of Enzyme Kinetics, Portland Press, London, 1995.

StyleTag -- Journal: JIB (Journal of Inorganic Biochemistry)

Article: 6307