The loop B domain is physically separable from the ... - BioMedSearch

 1996 Oxford University Press

Nucleic Acids Research, 1996, Vol. 24, No. 14 2685–2689

The loop B domain is physically separable from the loop A domain in the hairpin ribozyme Chanseok Shin, Jin Nam Choi, Sang Ik Song, Jong Tae Song, Ji Hoon Ahn1, Jong Seob Lee1 and Yang Do Choi* Research Center for New BioMaterials in Agriculture and Department of Agricultural Chemistry, Seoul National University, Suwon 441-744, Korea and 1Department of Molecular Biology, Seoul National University, Seoul 151-742, Korea Received April 2, 1996; Revised and Accepted May 31, 1996

ABSTRACT In order to understand the catalysis mechanism of the hairpin ribozyme, mutant ribozymes were constructed. The distance between the loop A domain and the loop B domain was extended by inserting various lengths of nucleotide linkers at the hinge region in cis mutants, or the domains were separated physically in a trans mutant. All the mutant ribozymes, including the trans mutant, could cleave substrate RNA at the predicted site. A cis mutant with a single nucleotide insertion exhibited cleavage activity about twice as high as that of the wild-type (wt) ribozyme. The insertion of 2–5 nucleotides (nt) gradually reduced the activity to the level of the wt ribozyme. Insertion of a longer linker, up to 11 nt, resulted in the reduction of activity to one half of that of the wt ribozyme. The ribozyme with a single nucleotide insertion at the hinge region seems to form a more suitable conformation for catalysis by threedimensional fold-back of the loop B to loop A containing the cleavage site. The trans mutant, in which the A and B domains were physically separated, maintained a significant level of activity, suggesting that both domains are necessary for catalysis, but separable. These results demonstrate that interaction between the A and B domains results in catalysis. INTRODUCTION RNA catalysts, termed ribozymes, present possibilities for the alteration of gene expression. The hairpin ribozyme derived from the negative strand of a satellite RNA of tobacco ringspot virus [(–)sTRSV] catalyzes efficient cleavage of an external RNA substrate in trans (1,2). The reaction proceeds via a transphosphorylation mechanism, yielding products with 2′,3′-cyclic phosphate and 5′-hydroxyl termini at the -N↓GUC- sequence (3,4). This cleavage mechanism is shared by two other small RNA enzymes: the hammerhead and hepatitis delta ribozymes (reviewed in 5). The active conformation of the hairpin ribozyme is thought to consist of two non-base paired bulge loops, A and B, flanked by

* To

whom correspondence should be addressed

two helix stems each in the A and B domains respectively of the ribozyme–substrate complex (4,6–9). Mutagenesis, photocross-linking, chemical modification and in vitro selection experiments have revealed some of the essential features of the hairpin ribozyme (6–8,10–17). Nucleotide sequences in the non-base paired bulge loops A and B are highly conserved (6,7). The guanosine residue at the -N↓GUC- cleavage site seems to be absolutely conserved and it might interact with the opposite substrate binding strand (SBS) in the bulge loop A of the ribozyme–substrate complex (11,15–17). Bulge loops A and B might be coordinated by Mg2+ ion and the interaction between them seems to be responsible for the catalysis (8,13,14,18–21). However, the nature of the interaction and the function of the loop B in the catalytic cleavage of the substrate are not clear. As part of a strategy toward producing virus-resistant garlic plants, a hairpin-type ribozyme derived from (–)sTRSV which cleaves at the middle of RNA genome of the garlic latent virus was designed (22,23). In the wild-type (wt) (–)sTRSV-derived hairpin ribozyme, there was an adenosine ‘A’ residue at the hinge region between the A and B domains, which is weakly involved in the formation of the helix 3 structure (6,7). The ‘A’ residue seems to function as a flexible hinge to fold the two domains back together which results in the interaction between them. Enforcement of the interaction between the two domains by inserting various lengths of oligonucleotide or propanediol phosphate linkers at the opposite strand of the hinge region resulted in the increase of the ribozyme activity (19–21). To understand the spatial interaction between the two domains of the hairpin ribozyme, various lengths of nucleotide linker from 1 to 11 nucleotides (nt) were inserted between them in cis mutants in this study, which impart varying degrees of flexibility to the hinge. Furthermore, we separated the two domains physically in the trans mutant without losing too much activity. MATERIALS AND METHODS Enzymes and chemicals T7 RNA polymerase and RNase-free DNase were purchased from Promega. [α-32P]UTP and rNTPs were from Amersham International plc.

2686 Nucleic Acids Research, 1996, Vol. 24, No. 14 mutant was carried out as follows. A mixture of substrate (148 nt long) and SBS RNA (27 nt) was heated for 2 min at 95
C and cooled slowly to room temperature over a period of 30 min. The B domain RNA (54 nt) was denatured by heating at 95
C for 2 min and cooling. The mixture for cleavage reactions contained 40 mM Tris–HCl (pH 8.0), 4 mM MgCl2, 2 mM spermidine, 10 nM each of 32P-labeled substrate RNA and the loop B domain RNA, and 10 or 50 nM SBS RNA as indicated in a total volume of 5 µl. Reactions were carried out at 37
C and products were denatured by heating in loading mixture (50% formamide, 89 mM Tris-borate–EDTA buffer and 0.1% of xylene cyanol and bromophenol blue) and separated in 7% polyacrylamide–urea gel. The gel was dried and autoradiographed.

Figure 1. Structure of the substrate and hairpin ribozyme in the cis mutants. An arrow indicates the cleavage site of the substrate and an arrow head indicates the insertion site of nucleotide linker (N). Loops in the substrate binding and hairpin domains are identified by A and B. The sequences of nucleotide linkers inserted at the hinge region in mutants N1–N11 are shown. The length of extra sequences in the substrate are indicated. The transcription vector derived sequences are shown in lower case letters. The mutant N8 was not tested.

Kinetic analysis Ribozymes having various lengths of nucleotide linkers from 1 to 11 nt were maintained at 10 nM and the substrate concentration was varied from 10 to 320 nM. The reaction was carried out at 50
C for 1 h in 4 mM MgCl2, 2 mM spermidine and 40 mM Tris–HCl (pH 8.0).

Secondary structure analysis of RNA The secondary structures of RNA with the lowest possible free energy on the basis of Tm were calculated with the program PCFOLD which makes use of the secondary structure prediction algorithm developed by Zuker (24). Construction of ribozymes and substrate Construction of the transcription templates for the ribozyme and the substrate were described previously (22,25). The substrate construct was derived from the coat protein gene of the garlic latent virus, which gave a 148 nt long RNA transcript (23). DNA manipulations and cloning techniques were performed as described by Sambrook et al. (26). For cis mutant constructs, 26 bp synthetic double-stranded oligodeoxyribonucleotides (5′-ACCTTTGAAGAAGTCATTGTTAACTG-3′) containing SBS and a linker sequence with the HpaI site were annealed and ligated into pRIB as described (22). Linker sequences were designed so that no additional secondary structure formation was possible. The plasmid was digested with HpaI, treated with S1 nuclease and blunt-end ligated. Deletion mutants were identified by determining nucleotide sequences. Cis mutants having a linker varying in length from 1 to 11 nt, except 8 nt, were obtained (Fig. 1). For the trans mutant ribozyme, RNAs were transcribed separately from pRIB for the B domain and pBIND for SBS. Preparation of ribozyme and substrate RNA RNAs were prepared by in vitro transcription of the linearized plasmid with T7 RNA polymerase as suggested by the manufacturer. The labeled RNA was quantitated by Cerenkov counting. RNA products were analyzed by electrophoresis in 5–7% polyacrylamide–urea gels and autoradiography. Ribozyme cleavage reaction Ribozyme cleavage reactions with cis mutants were carried out as described previously (22). Cleavage reaction with the trans

RESULTS Effect of nucleotide linker insertion in cis mutants It is known that interaction between non-base paired bulge loop A in the substrate binding domain and the loop B domain of the hairpin ribozyme leads to the catalysis (Fig. 1; 5–7,13,14,19–21). The interaction could be modulated by the flexibility of the hinge between the two domains. To increase the flexibility of the hinge, various lengths of nucleotide linkers from 1 to 11 nt were inserted between SBS and the B domain in cis mutants (N1–N11; Fig. 1). The cis mutants with insertion of various length linkers were tested for cleavage activity. All of the mutant ribozymes (69–79 nt) could cleave the substrate RNA of 148 nt to the products of 44 and 104 nt long. A single nucleotide ‘A’ insertion resulted in the highest activity and then the activity gradually decreased to the level of the wt ribozyme as the length of the linker was increased to 5 nt (Fig. 2A and B). The activity decreased further with the increase of the length of nucleotide linker. The observed catalytic reaction constant (kcat) of the mutant with a single nucleotide insertion (N1) was about twice as high as that of the wt ribozyme. With the insertion of a linker >5 nt, kcat decreased to ∼1/2 of the wt ribozyme. The effect of the nucleotide sequence of the linker on the activity was tested with mutant ribozymes having 2 and 10 nt-long linkers (N2 versus N2′ and N10 versus N10′ in Fig. 1). However, no significant difference in the activity between them was observed (data not shown). The kinetic parameters of these mutant ribozymes N1 and N7 were obtained along with the wt ribozyme respectively (Table 1). The Km values of these two ribozymes were similar, whereas the kcat/Km value of N1 mutant was twice as high as that of the wt ribozyme. It suggests that the linker at the hinge does not affect the substrate binding affinity of the ribozyme but the catalytic efficiency. It is possible that the size, conformation or structural constraint of the linkers at the hinge affects the interaction between the loops A and B thus affecting the catalytic efficiency.

2687 Nucleic Acids Acids Research, Research,1994, 1996,Vol. Vol.22, 24,No. No.114 Nucleic

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Figure 3. Schematic presentation of the loop A duplex (left panel) and the loop B domain (right panel) in the trans ribozyme mutant. The length of extra sequences in the substrate are indicated. The transcription vector derived sequences are shown in lower case letters.

Figure 2. Effect of the linker nucleotide insertions on the hairpin ribozyme activity. (A) Relative activity of cis mutant ribozymes. Reactions were carried out in 10 mM Tris–HCl (pH 8.0), 4 mM MgCl2, 2 mM spermidine with 10 nM each of 32P-labeled substrate RNA and the ribozyme in a total volume of 5 µl at 50
C for 1 h and analyzed by 5% urea–polyacrylamide gel electrophoresis. Lane M, size marker (pUC19/EcoRI, HinfI); lane 1, wt hairpin ribozyme; lanes 2–6, the cis mutant with inserted linker of 1, 2, 4, 6 and 10 nt respectively. S, R, P1 and P2 indicate the substrate, ribozyme and reaction products respectively. (B) Comparison of kcat for cis mutant ribozymes. Values are means of four independent experiments and the standard deviation is shown by a bar.

domain RNAs transcribed separately in vitro were mixed together in the reaction buffer. Cleavage reaction occurred at the predicted site only if all three components were present (Fig. 4B). The trans mutant showed lower activity than that of the wt ribozyme but a significant fraction (3.1% in Fig. 4A) of the substrate was cleaved. When an equal molar concentration of the substrate and SBD RNA were added, the activity of the trans mutant was ∼1/10 of that of the wt ribozyme at 37
C (Fig. 4A, lane 4 versus lane 5). However, when the amount of SBS RNA was increased 5-fold compared with that of the substrate RNA, the cleavage reaction activity increased 2.5-fold (Fig. 4B, lane 4 versus lane 5); ∼1/4 of the wt ribozyme. The activity of the trans mutant ribozyme depended on the presence of Mg2+ and the optimum temperature was 37
C (data not shown) as did a typical hairpin ribozyme (2). These results demonstrate that the A and B domains are separable and the interaction between them is responsible for the catalysis of the ribozyme. This result is consistent with the recent report by Butcher et al. (27). DISCUSSION

Table 1. Kinetic parameters of ribozymes kcat (min–1)

Km (µM)

kcat/Km

wt

0.13

0.60

0.22

N1

0.24

0.60

0.40

N7

0.092

0.42

0.22

Physical separation of substrate binding and hairpin domains in a trans mutant To relieve structural constraint at the hinge between the substrate binding catalytic domain and the B domain, the two domains were separated physically (Fig. 3). The substrate, SBS, and the B

To study the catalysis mechanism of the hairpin ribozyme, cis mutant ribozymes in which various lengths of nucleotide linkers were inserted at the hinge between the A and B domains, and a trans mutant in which the two domains were separated physically from each other, were tested. Until now, the bulge loops A and B have been the focus of study into the catalytic mechanism of the hairpin ribozyme (4,8,11,15–17). Mutagenesis approaches have also been adopted to study mechanism of the hairpin ribozyme (6–8,10). Much attention has been paid to search for tertiary interactions between non-base paired bulge loops A and B (6,7,13,14,19–21). A bent structure at the hinge between the two domains which gives ‘paperclip structure’ seems to be the essential feature for the ribozyme activity (14,19–21). It was reported that ‘A’ at the hinge might be involved in the ribozyme fold-back for catalysis which results in the interaction between bulge loops (6,7).

2688 Nucleic Acids Research, 1996, Vol. 24, No. 14

Figure 4. Comparison of the relative activities of the trans mutant and the wt ribozyme. (A) Relative activity of the trans mutant ribozyme and the wt ribozyme was compared. Reaction mixture contains the wt ribozyme only (lane 1), the loop B domain and SBS of the trans mutant ribozyme (lane 2), the substrate RNA only (lane 3), 10 nM each of the wt ribozyme and substrate (lane 4) or 10 nM each of the trans mutant ribozyme, substrate and SBS (lane 5) respectively, in 40 mM Tris–HCl (pH 8.0), 4 mM MgCl2, 2 mM spermidine. Reaction was carried out at 37
C for 2 h and analyzed by 7% urea–polyacrylamide gel electrophoresis. Lane M, size marker (pUC19/EcoRI, HinfI). H, B, S, R, P1 and P2 indicate the B domain, SBS, the substrate, the wt ribozyme and reaction products respectively. (B) Effect of relative amount of the substrate and SBS in the trans mutant was tested. Reaction mixture contains the B domain and SBS (lane 1), the B domain and substrate (lane 2) or the substrate and SBS (lane 3), 10 nM each of the trans mutant ribozyme, substrate and SBS (lane 4), or the trans mutant ribozyme with five times more SBS (50 nM) compared with lane 4 (lane 5) respectively. Reactions were carried out at 37
C for 1 h and analyzed by 7% urea–polyacrylamide gel electrophoresis.

The fold-back structure could be coordinated by Mg2+ (8,13,14,18). It was reported that the hairpin ribozyme contains at least two cation binding sites essential for catalysis (14,18), but, one of these motifs could be involved in Mg2+-binding only when the B domains fold back (14). Point mutation analyses confirmed that most nucleotides at the bulge loops A and B of the hairpin ribozyme are highly conserved for cleavage reaction in addition to 5′-N↓GUC-3′ in the substrate (6–8,10). It has been proposed recently that the loop B in the hairpin domain could interact with

the cleavage site of the substrate, 5′-N↓GUC-3′, in the ground state (14). We tested the possibility of ribozyme fold-back by inserting nucleotide linkers at the hinge region between the A and B domains. The mutant ribozyme N1 which has a single nucleotide insertion at the hinge region showed the highest efficiency in cleaving the substrate. This mutant ribozyme could form a more suitable conformation for catalysis due to the fold-back of the loop B over the bulge loop A. As the length of linker increased to 5 nt, proper fold-back conformation might still be accommodated. When the linker length increased >5 nt, however, bulkiness of the linker seemed to be rather inhibitory to maintain the proper fold-back conformation, thereby making the cleavage reaction less efficient. Even though there was no significant effect by nucleotide sequence differences of linkers, we cannot exclude the possiblity that longer nucleotide linkers may bring up unfavorable secondary structure for the ribozyme. The phosphate backbone of a single ‘A’ at the hinge of the wt ribozyme does not seem to be flexible enough to allow efficient interaction between the bulge loops A and B. When those two domains were pushed back by connecting the opposite strand with nucleotide or propanediol phosphate linkers, increases in the catalytic activity were noticed (19,21). Even though the joint was disconnected to relieve the structural stress at the site, the ribozyme was still active as far as where the opposite strand is connected by an oligocytidylate linker which holds the two domains together (20). Ribozyme activity of their reverselyjoined ribozyme also increased with the length of oligocytidylate linker. These results are consistent with our observation that the increase in flexibility of the joint, due to the addition of a 1–5 nt linker, resulted in higher activity. The concept of modular assembly in RNA molecules could be supported by these results (27,28). The structural motif present in loop B which is common to eukaryotic 5S rRNA and the sarcin/ricin loop of 26S rRNA, might dock with loop A in the hairpin ribozyme. In docking of the two loop domains, spatial arrangement for productive interaction was controlled with various efficiencies by the inserted linker in this study. This modular assembly concept was supported by the activity of the trans mutant. To relieve the structural constraint at the hinge region further, the A and B domains were physically separated, which allowed free interaction between the two domains. In the trans mutant, we showed that the A domain and the B domain were necessary for catalysis. Our results demonstrate, however, that two domains were separable physically into two molecules without losing too much activity. The observed reaction velocity of the trans mutant was 1/10 of that of the wt ribozyme as shown in Figure 4. It has been reported by Butcher et al. (27) in which kcat of the reconstituted trans ribozyme was ∼1/2–1/10 of the corresponding intact wt ribozyme depending on the relative concentration of SBS and the loop B domain to the substrate. The Km value for the reconstituted reaction increased 103–104 compared with that of the intact wt ribozyme. These results support the possible interaction between the loops A and B. From a photo-cross-linking study, it was suggested that domain B could fold into a similar tertiary structure in the presence or absence of domain A (12). This result suggests that the trans mutant ribozyme studied in this work may function by the same mechanism as the wt hairpin ribozyme. Further studies by mutagenesis with the trans mutant could reveal the regions responsible for the interaction between loops A and B and thus catalysis mechanism.

2689 Nucleic Acids Acids Research, Research,1994, 1996,Vol. Vol.22, 24,No. No.114 Nucleic ACKNOWLEDGEMENTS The present investigation was supported by a grant from the Genetic Engineering Program of the Ministry of Education and in part by a grant from the Ministry of Science and Technology of Korea. REFERENCES 1 Feldstein, P. A., Buzayan, J. M. and Bruening, G. (1989) Gene 82, 53–61. 2 Hampel, A. and Tritz, R. (1989) Biochemistry 28, 4929–4933. 3 Buzayan, J. M., Gerlach, W. L., Bruening, G., Keese, P. and Gould, A. R. (1986) Virology 151, 186–199. 4 Chowrira, B. M., Berzal-Herranz, A. and Burke, J. M. (1991) Nature 354, 320–322. 5 Symons, R. H. (1992) Annu. Rev. Biochem. 61, 647–671. 6 Anderson, P., Monforte, J., Tritz, R., Nesbitt, S., Hearst, J. and Hampel, A. (1994) Nucleic Acids Res. 22, 1096–1100. 7 Berzal-Herranz, A., Joseph, S., Chowrira, B. M., Butcher, S. E. and Burke, J. M. (1993) EMBO J. 12, 2567–2573. 8 Chowrira, B. M., Berzal-Herranz, A., Keller, C. F. and Burke, J. M. (1993) J. Biol. Chem. 268, 19458–19462. 9 Hampel, A., Tritz, R., Hicks, M. and Cruz, P. (1990) Nucleic Acids Res. 18, 299–304. 10 Schmidt, S., Beigelman, L., Karpeisky, A., Usman, N., Sorensen, U. S. and Gait, M. J. (1996) Nucleic Acids Res. 24, 573–581. 11 Berzal-Herranz, A., Joseph, S. and Burke, J. M. (1992) Genes Dev. 6, 129–134.

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12 Butcher, S. E. and Burke, J. M. (1994) Biochemistry 33, 992–999. 13 Butcher, S. E. and Burke, J. M. (1994) J. Mol. Biol. 244, 52–63. 14 Grasby, J. A., Mersmann, K., Singh, M. and Gait, M. J. (1995) Biochemistry 34, 4068–4076. 15 Joseph, S., Berzal-Herranz, A., Chowrira, B. M., Butcher, S. E. and Burke, J. M. (1993) Genes Dev. 7, 130–138. 16 Vitorino Dos Santos, D. V., Vianna, A-L., Fourrey, J-L. and Favre, A. (1993) Nucleic Acids Res. 21, 201–207. 17 Vitorino Dos Santos, D. V., Fourrey, J-L. and Favre, A. (1993) Biochem. Biophys. Res. Commun. 190, 377–385. 18 Chowrira, B. M., Berzal-Herranz, A. and Burke, J. M. (1993) Biochemistry 32, 1088–1095. 19 Komatsu, Y., Koizumi, M., Nakamura, H. and Ohtsuka, E. (1994) J. Am. Chem. Soc. 116, 3692–3696. 20 Komatsu, Y., Kanzaki, I., Koizumi, M. and Ohtsuka, E. (1995) J. Mol. Biol. 252, 296–304. 21 Feldstein, P. A. and Bruening, G. (1993) Nucleic Acids Res. 21, 1991–1998. 22 Shin, C., Choi, J. N., Song, S. I., Song, J. T., Ahn, J. H., Lee, J. S. and Choi, Y. D. (1996) Mol. Cells 6, 209–214. 23 Choi, J. N., Song, J. T., Song, S. I., Ahn, J. H., Choi, Y. D. and Lee, J. S. (1995) Agric. Chem, Biotech. 38, 49–54. 24 Zuker, M. (1989) Methods Enzymol. 180, 262–288. 25 Kikuchi, Y. and Sasaki, N. (1991) Nucleic Acids Res. 19, 6751–6755. 26 Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harber, NY. 27 Butcher, S. E., Heckman, J. E. and Burke, J. M. (1995) J. Biol. Chem. 270, 29648–29651. 28 Szewczak, A. A. and Moore, P. B. (1995) J. Mol. Biol. 247, 81–98.