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Nuclease footprint analyses of the interactions between RNase P ribozyme and a model mRNA substrate
Introduction
Materials And Methods
DNA constructs and synthesis of ribozymes and RNA substrates
Kinetic analyses of the reactions catalyzed by the ribozymes
Structural analyses of the ribozymes
UV-crosslinking experiments
Results
Discussion
Interactions between M1GS ribozyme and a mRNA substrate
Comparison between the interactions of the ribozymes with a mRNA and a ptRNA substrate
Acknowledgements
References
Nuclease footprint analyses of the interactions between RNase P ribozyme and a model mRNA substrate
Received August 11, 1999; Accepted October 19, 1999
ABSTRACT RNase P ribozyme cleaves an RNA helix substrate which resembles the acceptor stem and T-stem structures of its natural tRNA substrate. By linking the ribozyme covalently to a sequence (guide sequence) complementary to a target RNA, the catalytic RNA can be converted into a sequence-specific ribozyme, M1GS RNA. We have previously shown that M1GS RNA can efficiently cleave the mRNA sequence encoding thymidine kinase (TK) of herpes simplex virus 1. In this study, a footprint procedure using different nucleases was carried out to map the regions of a M1GS ribozyme that potentially interact with the TK mRNA substrate. The ribozyme regions that are protected from nuclease degradation in the presence of the TK mRNA substrate include those that interact with the acceptor stem and T-stem, the 3[prime] terminal CCA sequence and the cleavage site of a tRNA substrate. However, some of the protected regions (e.g. P13 and P14) are unique and not among those protected in the presence of a tRNA substrate. Identification of the regions that interact with a mRNA substrate will allow us to study how M1GS RNA recognizes a mRNA substrate and facilitate the development of mRNA-cleaving ribozymes for gene-targeting applications.
INTRODUCTION
RNase P is a ribonucleoprotein complex responsible for the 5[prime] maturation of tRNAs (1,2). It catalyzes a hydrolysis reaction to remove a 5[prime] leader sequence from tRNA precursors (ptRNA) and several other small RNAs. In Escherichia coli, RNase P consists of a catalytic RNA subunit (M1 RNA) and a protein subunit (C5 protein) (1,2). In the presence of a high concentration of salt, such as 100 mM Mg2+, M1 RNA acts as a catalyst and cleaves ptRNAs in vitro in the absence of C5 protein (3). Extensive studies with both phylogenetic and biochemical analyses have established models for the secondary and three-dimensional structures of RNase P catalytic RNAs (4-8). These models provide a framework to identify the putative active site and substrate binding site, and to study the catalytic mechanism of RNase P catalytic RNAs.
The interactions between M1 RNA and a ptRNA substrate have been extensively studied by site-directed mutagenesis, kinetic analyses, UV crosslinking, chemical footprinting and interference experiments (6,8-19). These studies have revealed the regions of M1 RNA that are in close proximity to a ptRNA substrate (6,8). Moreover, these studies have led to the identification of the regions of M1 RNA that bind to the 3[prime] CCA sequence (17,19-21) and interact with a part of the T-stem of a ptRNA (10,22).
Systematic deletion analyses of a ptRNA molecule were also carried out to determine the minimal requirements for substrate recognition by M1 RNA and RNase P (23,24). These studies have revealed that a small model substrate, which contains a structure equivalent to the acceptor stem, the T-stem, the 3[prime] CCA sequence and the 5[prime] leader sequence of a ptRNA molecule, can be cleaved efficiently by M1 ribozyme (Fig. 1A). Accordingly, M1 catalytic RNA can cleave a mRNA sequence if the mRNA substrate forms a hybrid complex with its complementary sequence [external guide sequence (EGS)] (Fig. 1A) (23). Moreover, a mRNA-cleaving ribozyme, M1GS RNA, can be constructed by linking a guide sequence covalently to M1 RNA (Fig. 1A) (25,26). We have previously shown that a M1GS ribozyme cleaved the mRNA sequence encoding the thymidine kinase (TK) of herpes simplex virus 1 (HSV-1) (26). When the ribozyme was expressed in mammalian cells infected with HSV-1, both the viral TK mRNA and protein levels were reduced by 75% (26).
Figure 1. (A) Schematic representation of a natural ptRNA substrate, a small model substrate (EGS:mRNA) for ribonuclease P and M1 RNA from E.coli, and a complex formed between a M1GS RNA and its mRNA substrate (S). The structural components common to both precursor tRNA and the model substrate are highlighted. The site of cleavage by RNase P or M1 RNA is marked with an arrow. (B) Schematic representation of the substrates used in the study. The uridine positions of S5 that were incorporated with the photoactive 4-thio-uridine nucleotide are highlighted. The targeting sequences which bind to the guide sequence of M1-S5 are boxed. The regions upstream and downstream from the targeting sequence represent the 5[prime] leader sequence and the 3[prime] tail sequence, respectively.
RNA enzymes are being developed as promising gene-targeting reagents to specifically cleave RNA sequences of choice (27,28). For example, both hammerhead and hairpin ribozymes have been shown to inhibit HIV replication by cleaving viral mRNA sequences in infected cells (29,30). Targeted cleavage of a mRNA by RNase P ribozyme provides a unique approach to inactivate any RNA of known sequence expressed in vivo. Further studies to investigate the mechanism of how a M1GS RNA cleaves a mRNA substrate should provide insight into how to improve the catalytic efficiency and sequence specificity of the ribozyme. Recently, the M1GS ribozyme regions that are in close contact with a mRNA model substrate were mapped by UV-crosslinking studies (31). In the study reported here, we used a nuclease footprint procedure to analyze the interactions between a M1GS ribozyme and a TK mRNA model substrate. We have identified the RNase P ribozyme nucleotides that are protected from nuclease cleavage by the model mRNA substrate. These nucleotides are probably involved in binding the mRNA substrate. Furthermore, we were able to compare the regions protected by the mRNA substrate with those protected by a ptRNA. These results revealed the similarities and differences of the interactions between an RNase P ribozyme and a ptRNA or a model mRNA substrate.
MATERIALS AND METHODS
DNA constructs and synthesis of ribozymes and RNA substrates
Plasmid pFL117 contains the DNA sequence that codes for M1 RNA and is driven by the T7 RNA polymerase promoter (26). The DNA sequences that encode M1-S5 and M1ICP4 ribozymes were constructed by PCR using PvuII-digested pFL117 as the template, and oligonucleotides AF25 (5[prime]-GGAATTCTAATACGACTCACTATAG-3[prime]) as the 5[prime] primer and S5-GS (5[prime]-GTGGTGCCCGCGCCCGACTATGACCATG-3[prime]) and ICP4-GS (5[prime]-CCCGCTCGAGAAAAAATGGTGCATCGGCGATGG-CGAGCTATGACCATG-3[prime]) as the 3[prime] primers, respectively. Plasmid construct pS5 that contained the DNA sequence coding for substrate S5 has been described previously (31). The DNA sequences that code for RNA S5-5, S5-3 and S5-5-3 were constructed by PCR using pS5 as the template, AF25 as the 5[prime] primer and oligonucleotides oliS5-5 (5[prime]-CGCGGATCCGCGGAAAGGTCGGGCGCGGGCTATAGTGAGTCGTATTA-3[prime]), oliS5-3 (5[prime]-AGTCGGGCGCG-3[prime]) and oliS5-5-3 (5[prime]-AGTCGGGCGCGGGCTATAGTGAGTCGTATTA-3[prime]) as the 3[prime] primers, respectively. The DNA sequence that encodes substrate icp4 was constructed by PCR using pGEM3zf(+) as the template, oligonucleotide AF25 as the 5[prime] primer and sICP4 (5[prime]-CGG-GATCCGACGCCATCGCCGATGCGGGGCGATCCTATA-GTGAG-3[prime]) as the 3[prime] primer. The ribozymes and RNA substrates were synthesized in vitro from these DNA templates by T7 RNA polymerase. The substrates that contained the photoactive 4-thio-uridine were synthesized in the presence of 4-thio-uridine triphosphate (Amersham, Arlington Heights, IL) by T7 RNA polymerase.
Kinetic analyses of the reactions catalyzed by the ribozymes
The cleavage reactions of the substrates by ribozymes were carried out in either buffer M (50 mM Tris, pH 7.5, 100 mM NH4Cl, 100 mM MgCl2, 4% PEG) or buffer N (50 mM Tris, pH 7.5, 100 mM NH4Cl, 100 mM CaCl2, 4% PEG) as described previously (26,32). The cleavage products were separated on 8% denaturing gels, which were autoradiographed and/or dried and quantitated with a Molecular Dynamics STORM840 PhosphorImager. Assays to determine kinetic parameters under multiple-turnover conditions were performed as described previously (33,34). In brief, the cleavage of substrates was assayed at various concentrations of substrates, both above and below the Km for the enzyme. Aliquots were withdrawn from reaction mixtures at regular intervals and analyzed in denaturing polyacrylamide gels. Values of Km and kcat were obtained from Lineweaver-Burk double-reciprocal plots. The values of kinetic parameters determined in different experiments exhibited a variation of <25%.
To assay the catalytic activity of the crosslinks, the UV-crosslinked species were purified from denaturing gels, either first diluted 100-fold or directly incubated at 37°C in buffer M for different periods of time (from 5 min to 2 h). The cleavage products were separated in 8% polyacrylamide denaturing gels and quantitated with a Molecular Dynamics STORM840 PhosphorImager.
Structural analyses of the ribozymes
All ribozymes were synthesized by T7 RNA polymerase and purified in 4% polyacrylamide gels that contained 8 M urea. The 5[prime] termini of the ribozymes were radiolabeled with [[gamma]-32P]ATP and polynucleotide kinase while the 3[prime] ends were labeled in the presence of [32P]pCp and T4 RNA ligase. Prior to structural analysis, the ribozymes were incubated at 80°C for 2 min and then cooled slowly to room temperature.
RNase mapping was carried out in either buffer M or N, as described previously (33). In brief, RNases were diluted and incubated with radiolabled ribozymes. After different periods of time, the digestion reactions were stopped by adding 8 M urea. The alkaline treatment of ribozymes for preparation of a sequence ladder was performed as described elsewhere (33). The cleavage products were separated in denaturing polyacrylamide gels. All nucleases used in the study were purchased from either Pharmacia Biotech (Piscataway, NJ) or Gibco BRL (Grand Island, NY).
UV-crosslinking experiments
UV crosslinking was carried out as described previously (31,35,36). Radiolabeled RNA substrates (20-50 nM) were incubated with ribozymes (20-50 nM) in either buffer M or N for 5 min at 37°C. The reaction mixtures were then exposed to UV light (365 nm) for 15 min. RNA samples were recovered from the crosslinking reactions by ethanol precipitation. The crosslinked species were separated in denaturing gels and visualized by autoradiography. The conjugates were then excised from the gels and further purified.
RESULTS
RNA substrate S5, which contains a TK mRNA sequence, consisted of three sequence elements: (i) a 5[prime] leader sequence of 20 nt; (ii) a targeting sequence of 13 nt that base pairs with the guide sequence (GS) of a M1GS ribozyme; and (iii) a 3[prime] tail sequence of 11 nt (Figs 1A and B). Accordingly, the regions of the M1GS ribozyme (designated as M1-S5 RNA) that bind to S5 can be divided into three parts that interact specifically with each one of these three elements. In order to identify the regions of M1-S5 RNA that potentially interact with a particular part of S5 (e.g. the 5[prime] leader sequence), three substrates, S5-5, S5-3 and S5-5-3, were derived from S5 by deleting the 5[prime] leader sequence, the 3[prime] tail sequence, or both, respectively (Fig. 1).
To determine the regions of the ribozymes that were protected by the mRNA model substrate, RNase P ribozymes were incubated either alone or with the substrates to allow binding, and then subjected to digestion by nucleases. Three different nucleases were used: RNase T1 which recognizes single-stranded RNA regions and only cleaves at guanosine positions; nuclease S1 which only cleaves at single-stranded regions; and RNase V (cobra venom RNase) which only reacts towards base-paired regions. To identify the substrate-specific rather than product-specific protections, some of the footprint analyses were also carried out under reaction conditions that prevent cleavage of the mRNA substrate. In order to reduce the rate of cleavage while preserving the interactions between the ribozyme and the substrate in an active ribozyme-substrate complex, two changes were introduced to the mRNA cleavage reactions: CaCl2 was used instead of MgCl2 as the source of divalent ions and the reactions were carried out at 20 instead of 37°C. It has been shown that divalent ions are essential for M1 catalytic activity (3) and M1 RNA cleaves a ptRNA substrate at least 30-fold slower in the presence of CaCl2 than in the presence of MgCl2 (37). Indeed, in the presence of CaCl2, cleavage of substrate S5 occurred and the rate of cleavage was at least 20-fold slower than that in the presence of MgCl2 (Fig. 2, lane 2). The reaction rate was further reduced at low temperatures (Fig. 2, lane 1). During the nuclease footprint analyses, no cleavage products were detected under these changed conditions (Fig. 2, lane 1) while substantial amounts of products were found under the optimal cleavage conditions (Fig. 2, lane 3). The overall secondary structure of the ribozymes in the cleavage buffer that contained CaCl2, mapped by nuclease cleavage, was very similar to that in the buffer that contained MgCl2 (data not shown).
Figure 2. Cleavage of the TK mRNA substrate S5 by M1GS RNA. Substrates (10 nM) were incubated alone (lanes 4 and 6), with 10 nM of M1-S5 (lanes 3 and 5) or with 50 nM of M1-S5 ribozyme (lanes 1 and 2). Cleavage reactions were carried out for 30 min either in the presence of S5 (lanes 1-4) or S5-thio (lanes 5 and 6), either in buffer M (50 mM Tris-HCl, pH 7.5, 100 mM NH4Cl, 100 mM MgCl2, 4% PEG) (lanes 3-6) or buffer N (50 mM Tris-HCl, pH 7.5, 100 mM NH4Cl, 100 mM CaCl2, 4% PEG) (lanes 1 and 2), and either at 20 (lane 1) or 37°C (lanes 2-6). Cleavage products were separated in 15% polyacrylamide gels containing 8 M urea.
The reaction mixtures that contained ribozymes in the presence and absence of the substrates were subjected to digestion by different nucleases of various concentrations in order to accurately determine the susceptibility of the ribozyme regions to digestion (Fig. 3, lanes 3-8). The cleavage products were separated in denaturing polyacrylamide gels and quantitated. Figure 3 shows an example of the nuclease footprint analyses to determine the nucleotides of M1GS RNA that are susceptible to cleavage by RNases T1 and V, and nuclease S1 in the presence and absence of S5. The amounts of the cleavage products at a particular position correlated with the susceptibility of this position to degradation by nucleases. If a position is located at a single-stranded region and is directly bound by the substrate, the nucleotide at this position is now expected to be less susceptible to cleavage by RNase T1 and nuclease S1, which cleave at single-stranded RNA regions. Meanwhile, the susceptibility of a position to cleavage by RNase V may be reduced if this position is bound by the substrate and located at a double-stranded region or involved in tertiary interactions. The levels of protection by the mRNA substrate were calculated by obtaining the ratio of the amounts of cleavage products in the absence of the mRNA substrate over those in the presence of the substrate. For comparison, the nucleotides of M1GS RNA that were susceptible to cleavage by RNase T1, nuclease S1 and RNase V were also determined in the presence of a ptRNA substrate, ptRNATyr. The results are summarized in Figure 4 and Table 1. The salient features of these results are as follows.
Figure 3. Nuclease footprint analyses of M1-S5 ribozyme in the presence and absence of substrate S5. One nanomole of 5[prime]-end labeled M1-S5 was digested in buffer N with RNase T1 (lanes 3 and 4), RNase V (lanes 5 and 6) and nuclease S1 (lanes 7 and 8) either in the absence of any substrate (lanes 3, 5 and 7) or in the presence of 5 nM of S5 (lanes 4, 6 and 8). The alkaline lysis (lane 1) and RNase T1 digestion (lane 2) of the ribozyme under denaturing conditions were carried out as described previously (33) and served as size markers.
Figure 4. Schematic representation of the nucleotides of M1-S5 that were protected from cleavage by RNase T1 and nuclease S1 in the presence of S5 and ptRNATyr (A), and S5-5, S5-3 and S5-5-3 (B). P, helix regions; J, junction regions between two helix sequences; L, loop regions (4-8). This is a summary of several independent experiments. Only the reproducible results are included. The regions that were found to be more susceptible to cleavage by RNase T1 and nuclease S1 are not shown.
Table 1. Analyses of the protected regions of the ribozymes in the presence of different substrates
*Less susceptible (protected) to RNase T1 and nuclease S1 digestion.
#More susceptible to RNase T1 nuclease S1 digestion.
The numbers and letters in parentheses represent the nucleotide positions and domains of the ribozyme, respectively (4-8). P, helix regions; J, junction regions between two helix sequences; L, loop regions (4-8). This is a summary of several independent experiments and only the results that were reproducible are included.
(i) The patterns of the susceptibility of M1GS RNA to cleavage by RNase T1 and nuclease S1, and RNase V in the absence of the substrate generally agreed with the proposed secondary structures (4,5,7). This was evident as most of the nucleotides that were susceptible to cleavage by RNase T1 and nuclease S1 resided in the single-stranded regions while those susceptible to cleavage by RNase V were located in either the double-stranded regions or those that are involved in tertiary interactions (Figs 3 and 4A and data not shown). Most of these results were also consistent with the patterns of the susceptibility of M1 RNA to digestion by these nucleases and chemical modification as determined previously (17,18,32).
(ii) The nucleotides that were strongly protected by substrate S5 from cleavage by RNase T1 and/or nuclease S1 were located in regions of P3, J3/4, P10, J11/12, P12-14, J11/14, J5/15, J15/16, P17, J2/18 and J2/4 (Fig. 4A and Table 1). These positions constitute the regions that are potentially in contact with the substrate. The nucleotides located at P18 were more susceptible to cleavage by RNase T1 and nuclease S1, suggesting that these positions were probably involved in a conformational change and now became more susceptible to digestion by the nucleases.
(iii) When the 3[prime] cleavage product S5-5 was used, nucleotides that were strongly protected from cleavage by RNase T1 and/or nuclease S1 included those protected by substrate S5 except for the positions at P3, J11/12 and P12 (Fig. 4B and Table 1). Since substrate S5-5 was derived from S5 by deleting the 5[prime] leader sequence, the positions (i.e. P3, J11/12 and P12) that were protected by S5 but not by S5-5 may represent the regions that were potentially in contact with the 5[prime] leader sequence. Meanwhile, the regions (e.g. P13/P14 and J5/15) found to be protected by both S5 and S5-5 (Fig. 4B and Table 1) constitute the regions that may interact with the targeting sequence and the 3[prime] tail sequence which are commonly found in these two substrates (Fig. 1B).
The P13, P14 and J11/14 regions were strongly protected from cleavage by RNase T1 and/or nuclease S1 by S5 but not by S5-3 which was derived from S5 by deleting the 3[prime] tail sequence (Fig. 4B and Table 1). These observations suggested that these three protected regions may potentially interact with the 3[prime] tail sequence. This suggestion was further supported by the results with RNA substrate S5-5-3 which was derived from S5 by deleting both the 5[prime] leader and 3[prime] tail sequences (Fig. 1B). These three regions were found to be strongly protected from RNase T1 and nuclease S1 digestion by S5 and S5-5 but not by S5-5-3. Meanwhile, the regions (i.e. P3, J11/12 and P12) that potentially interact with the 5[prime] leader sequence were protected from RNase T1 and nuclease S1 digestion by S5 and S5-3 but not by S5-5-3 (Fig. 4 and Table 1).
(iv) Nucleotides that were strongly protected by substrate ptRNATyr from cleavage by RNase T1 and/or nuclease S1 include those at P3, P10, J11/12, J11/14, J5/15, J15/16, J2/18 and J2/4 (Fig. 4A and Table 1). These positions constitute the regions that are potentially in contact with the ptRNA substrate. Region P17 was more susceptible to cleavage by RNase T1 and nuclease S1, suggesting that these nucleotides are probably involved in a conformational change and are now more exposed to nuclease digestion. These results generally agree with the protection patterns of M1 RNA by tRNA from chemical modification and RNase digestion (17,18,32). Moreover, these data are generally consistent with the interactions proposed in the current models of the three-dimensional structure of M1 RNA (6,8).
In order to exclude the possibility that the observed protection was due to non-specific binding of the substrates to the ribozyme (e.g. annealing of the substrate to the ribozyme regions other than the GS), two sets of experiments were carried out. First, footprint analyses were performed in the absence and presence of S5 to determine the protection patterns of ribozymes M1 RNA and M1ICP4. M1 RNA does not contain a guide sequence while M1ICP4 contains a guide sequence that targets another HSV-1 mRNA, ICP4 mRNA (Trang,P., Kilani,A., Kawa,D. and Liu,F., unpublished data). Second, the susceptibility of the regions of M1-S5 ribozyme to RNase T1 and nuclease S1 digestion in the presence of substrate icp4, that contained the HSV-1 ICP4 mRNA sequence, was determined. No specific cleavage-protection on the regions of the ribozymes in these experiments was observed (data not shown). These results suggested that the observed cleavage-protection patterns of M1-S5 RNA in the presence of S5, S5-5, S5-3 and S5-5-3 were specifically derived from the ribozyme-substrate complexes formed by base-pairing interactions of the substrate and the guide sequence of the ribozyme. These patterns were not from the complexes formed through non-specific binding between the substrates and other regions of the ribozyme.
It is possible that the protections detected at some of the nucleotides were due to misfolding of the ribozyme and might not reflect the interactions within an active ribozyme-substrate complex. To assess these possibilities, photoactive 4-thio-uridine nucleotides were introduced in substrate S5. Upon UV irradiation, 4-thio-uridine crosslinks to the nearby regions within a distance of 5-10 Å (38). This photoactive agent has been extensively used in UV-crosslinking studies to investigate RNA-RNA and RNA-protein interactions (35,36,38). The RNA substrate that contained 4-thio-uridine (designated as S5-thio) was synthesized in vitro in the presence of 4-thio-UTP and the photoactive group was incorporated at the 5[prime] region of the 3[prime] tail sequence (Fig. 1B). The cleavage of S5-thio by M1-S5 also yielded two products that comigrated with those from cleavage of S5 (Fig. 2, lanes 5 and 6) (31). Further characterization of the cleavage products indicated that the cleavage of S5-thio occurs at the same location as that of S5 (31). Kinetic analyses were carried out under multiple-turnover conditions to determine the cleavage rate of these substrates by the ribozymes. The values of kcat and Km for the reactions with substrate S5-thio were very similar to those with substrate S5 (Table 2). Thus, the presence of the photoactive group did not significantly affect the cleavage of the substrate by M1-S5.
Table 2. Multiple-turnover kinetic parameters of substrates in reactions catalyzed by M1-S5 ribozyme in buffer M (50 mM Tris, pH 7.5, 100 mM NH4Cl, 100 mM MgCl2, 4% PEG)
The values shown are the averages derived from triplicate experiments.
The reaction mixtures of ribozymes complexed with S5-thio were irradiated with UV light. If the ribozyme-substrate complexes were folded into an active conformation during the nuclease footprint analyses, the crosslinked conjugates were expected to represent the active complexes. The substrates in the conjugates should be cleaved by the enzyme within the same complex when the complexes were incubated under M1GS RNA optimal cleavage conditions (e.g. 100 mM MgCl2). To determine whether this was the case, 3[prime] end-labeled S5-thio was crosslinked with M1-S5. The crosslinked products were purified and then incubated for 2 h in the presence of 100 mM MgCl2 to allow cleavage to proceed. The cleavage products were separated in denaturing gels (Fig. 5). The 5[prime] cleavage product of S5-thio was released from the complex while the 3[prime] product, which included the photoactive groups, was retained in the complex (Fig. 5, lanes 1-5). More than 95% of substrates in these complexes were cleaved after a 2 h incubation (lane 5), suggesting that the majority of the substrates were crosslinked to the active conformation of the ribozymes. Similar results were also observed when the crosslinked complexes were diluted 100-fold before incubation under the in vitro optimal cleavage conditions (data not shown), supporting the notions that the cleavage of the substrates occurred within the crosslinked complexes and were not catalyzed by ribozymes from other adjacent crosslinked complexes.
Figure 5. Catalytic activity of the crosslinked complexes. 5[prime] labeled S5-thio was crosslinked with M1-S5. The purified crosslinked conjugates were either first incubated in buffer M (lane 5) or directly loaded on denaturing gels (lane 4). The cleavage products comigrated with the corresponding products generated from cleavage of S5-thio (lane 3). The radiolabeled ribozyme was shown in lane 1 while the substrate was shown in lane 2.
DISCUSSION
In this study, we have identified the nucleotides of RNase P ribozymes that are protected by different regions of a mRNA substrate. The change of protection patterns of the ribozymes in the presence of a mRNA substrate can be attributed to either direct interaction of the ribozyme to a mRNA sequence or a conformational change induced upon binding of the substrate. It is possible that some of the protections detected in the presence of the substrates are either results of non-specific binding of the mRNA substrates to the ribozymes or represent minor misfolded inactive conformations that are in rapid equilibrium with the native structure. However, several lines of evidence strongly suggest that this is not the case. First, no specific protection patterns of the ribozymes were observed when the M1-S5 ribozyme was incubated with substrate icp4 that contained the HSV-1 ICP4 mRNA sequence or when substrate S5 was incubated with M1 RNA or M1ICP4 ribozyme which targeted the ICP4 mRNA sequence. Second, >95% of the substrates were in the active ribozyme-substrate complexes under the conditions used in the nuclease footprint analyses, as shown in the UV-crosslinking experiments. Thus, the majority of the protection patterns represent the interactions between the RNase P ribozyme and the mRNA model substrate in an active ribozyme-substrate complex.
Interactions between M1GS ribozyme and a mRNA substrate
The regions that potentially interact with the targeting sequence (i.e. substrate S5-5-3) include those at 64-65 (J3/4), 120-121 (P10), 246-248 (J5/15), 258-259 (J15/16), 284-285 (P17), 291-292 (J15/16), 330-331 (J2/18) and 350 (J2/4). Most of these regions were also protected by ptRNATyr (Fig. 4A and Table 1). The J3/J4, P10 and J2/4 regions were shown to be in close proximity to the acceptor stem and T-stem of a tRNA (6,14,22). Meanwhile, regions J5/15 and J2/18 are in close contact to the +1 position of a tRNA and a mRNA substrate (12,31) while region J15/16 is a part of the binding site to the 3[prime] terminal CCA sequence of a tRNA (17,19). Thus, most of the regions found to be protected by substrate S5-5-3 are also in close proximity to the acceptor stem and T-stem structure, the cleavage site and the 3[prime] CCA sequence of a tRNA. Our results are consistent with the notion that the ribozyme uses its binding motifs to these structures and sequences of a tRNA to interact with the targeting region of a mRNA model substrate. The P4 helix is a part of the binding site to the acceptor stem of a tRNA (6,8) and has recently been shown to be in close proximity to the targeting sequence of a mRNA model substrate by UV-crosslinking studies (31). The lack of a cleavage-protection pattern at the P4 helix in our study can be explained as this helix region is located in the catalytic core of the ribozyme and is not accessible for nuclease cleavage. This interpretation is consistent with the observations that little cleavage by all the three nucleases was detected in these regions (32) (Figs 3 and 4A).
The importance of the 5[prime] leader sequence and 3[prime] tail sequence of a mRNA substrate for substrate binding by RNase P ribozyme has been indicated by kinetic analyses. Removal of the 5[prime] leader sequence and 3[prime] tail sequence resulted in the increase of the value of Km (Nepomuceno,E., Liou,K., Kilani,A. and Liu,F., unpublished data). The regions that potentially interact with the 5[prime] leader sequence include those at P3, J11/12 and P12, while those that potentially interact with the 3[prime] tail sequence are at P13/P14 and J11/14 (Table 1). These results are consistent with recent observations that photoactive 4-thio-uridine nucleotides incorporated in the 5[prime] leader sequence and 3[prime] tail sequence of a mRNA model substrate crosslinked to these regions, respectively (31). These findings support the interpretations that these regions are in direct contact with the 5[prime] leader sequence and 3[prime] tail sequence, respectively. It remains possible, however, that conformational rearrangements change the cleavage pattern. Indeed, local conformational changes perhaps provide an explanation for the enhanced protection of some positions upon binding of the mRNA model substrate. These positions may represent the regions that undergo local rearrangement upon binding of the substrate while the rest of the ribozyme regions remain conformationally unaffected.
Comparison between the interactions of the ribozymes with a mRNA and a ptRNA substrate
Our results suggest that the ribozyme utilizes its binding domains to the acceptor stem and T-stem of a tRNA to interact with the targeting sequence of the mRNA substrate. Moreover, our data suggests that regions P3, J11/12 and P12 potentially interact with the 5[prime] leader sequence. The 5[prime] leader sequence of a ptRNA has been shown to have extensive interactions with RNase P ribozyme and RNase P holoenzyme (13,17,36,39-42). However, the exact location of the binding site of the ribozyme to the 5[prime] leader sequence of a ptRNA substrate remains elusive. Nucleotides C92, G332 and A333 were shown to be in close contact with the nucleotides of the leader sequence that are close to the cleavage site (13,17,36,42). Meanwhile, P3 has recently been shown to be in close proximity to the active site and A136 of J11/12 is important for binding of ptRNA (6,43). However, little is known about whether the P3 and J11/12 regions are in close contact with the 5[prime] leader sequence of a ptRNA. These two regions were also protected by ptRNATyr (Fig. 4A and Table 1). It will be interesting to determine whether P3 and J11/12 also participate in binding of the 5[prime] leader sequence of a ptRNA.
Little is known about the binding site of M1GS ribozyme to the 3[prime] tail sequence of a mRNA substrate. Our results suggest that the P13/P14 and J11/14 regions were protected by the 3[prime] tail sequence from nuclease digestion. In contrast, most of these positions were not protected by ptRNATyr (Fig. 4A and Table 1). These observations suggest that these regions only interact with the mRNA substrate but not ptRNATyr. Recent UV-crosslinking studies indicated that these regions are in close proximity to the 3[prime] tail sequence of a TK mRNA model substrate (31). However, the P13-P14 regions are not believed to be in close proximity to a ptRNA substrate (6,8). No crosslinks were found between the P13/P14 regions and regions of the ribozyme and ptRNA when M1 RNA was folded either in the presence of a ptRNA or in the absence of a substrate. Thus, the ribozyme appears to interact with the mRNA substrate by utilizing binding motifs or conformations (e.g. P13/P14) different from those used to interact with a ptRNA. Such a mechanism for an enzyme to utilize different binding motifs or conformations to interact with various substrates is commonly used by protein enzymes and might also be generally used by RNA catalysts (1,44-46). This suggestion is also consistent with the notion that M1 RNA utilizes multiple binding sites or conformations to interact with different natural substrates such as ptRNA and p4.5S RNA (36,46,47).
Identification of the potential binding regions of the ribozyme to a mRNA substrate serves as a starting point to delineate the mechanism of how M1GS ribozyme recognizes the mRNA substrate and achieves sequence specificity. We believe that the ribozyme regions that potentially interact with substrate S5-5-3 include the general binding site to various targeting sequences that base pair with their guide sequences. Moreover, it is reasonable to suggest that P3, J11/12, P12, P13/P14 and J11/14 regions might also include the universal binding sites for the 5[prime] leader and 3[prime] tail regions of different target mRNA sequences. These hypotheses were consistent with our results of UV-crosslinking studies that most of these protected ribozyme positions are in close proximity to the different regions (e.g. 5[prime] leader sequence) of a mRNA substrate (31). Further studies to identify the functional groups within these regions of the ribozyme that interact with the mRNA substrate will reveal the nature of the interactions between the ribozyme and the substrate. These studies will provide insight into how the ribozyme achieves sequence specificity and will facilitate the development of ribozymes that exhibit optimal substrate binding and cleavage activity.
ACKNOWLEDGEMENTS
We are grateful to Ahmed Kilani and Kwa Liou for the reagents, their helpful discussions and sharing their unpublished results. F.L. is a Pew Scholar in Biomedical Sciences and a recipient of Regent's Junior Faculty Fellowship of University of California and a Basil O'Connor Starter Scholar Research Award from March of Dimes Birth Defects Foundation. This research has been supported by grants from Universitywide AIDS research program and NIH.
REFERENCES
*To whom correspondence should be addressed at: School of Public Health, University of California, 140 Warren Hall, Berkeley, CA 94720, USA. Tel: +1 510 643 2436; Fax: +1 510 642 6350; Email: liu_fy{at}uclink4.berkeley.edu
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