Selective Strand Annealing and Selective Strand Exchange Promoted by the N-terminal Domain of Hepatitis Delta Antigen
2003; Elsevier BV; Volume: 278; Issue: 8 Linguagem: Inglês
10.1074/jbc.m207938200
ISSN1083-351X
AutoresZhi-Shun Huang, Wen-Huey Su, Jui-Ling Wang, Huey‐Nan Wu,
Tópico(s)Viral gastroenteritis research and epidemiology
ResumoWe have previously shown that the N-terminal domain of hepatitis delta virus (NdAg) has an RNA chaperone activityin vitro (Huang, Z. S., and Wu, H. N. (1998)J. Biol. Chem. 273, 26455–26461). Here we investigate further the basis of the stimulatory effect of NdAg on RNA structural rearrangement: mainly the formation and breakage of base pairs. Duplex dissociation, strand annealing, and exchange of complementary RNA oligonucleotides; the hybridization of yeast U4 and U6 small nuclear RNAs and of hammerhead ribozymes and cognate substrates; and the cis-cleavage reaction of hepatitis delta ribozymes were used to determine directly the role of NdAg in RNA-mediated processes. The results showed that NdAg could accelerate the annealing of complementary sequences in a selective fashion and promote strand exchange for the formation of a more extended duplex. These activities would prohibit NdAg from modifying the structure of a stable RNA, but allow NdAg to facilitate a trans-acting hammerhead ribozyme to find a more extensively matched target in cognate substrate. These and other results suggest that hepatitis delta antigen may have a biological role as an RNA chaperone, modulating the folding of viral RNA for replication and transcription. We have previously shown that the N-terminal domain of hepatitis delta virus (NdAg) has an RNA chaperone activityin vitro (Huang, Z. S., and Wu, H. N. (1998)J. Biol. Chem. 273, 26455–26461). Here we investigate further the basis of the stimulatory effect of NdAg on RNA structural rearrangement: mainly the formation and breakage of base pairs. Duplex dissociation, strand annealing, and exchange of complementary RNA oligonucleotides; the hybridization of yeast U4 and U6 small nuclear RNAs and of hammerhead ribozymes and cognate substrates; and the cis-cleavage reaction of hepatitis delta ribozymes were used to determine directly the role of NdAg in RNA-mediated processes. The results showed that NdAg could accelerate the annealing of complementary sequences in a selective fashion and promote strand exchange for the formation of a more extended duplex. These activities would prohibit NdAg from modifying the structure of a stable RNA, but allow NdAg to facilitate a trans-acting hammerhead ribozyme to find a more extensively matched target in cognate substrate. These and other results suggest that hepatitis delta antigen may have a biological role as an RNA chaperone, modulating the folding of viral RNA for replication and transcription. Hepatitis delta virus (HDV) 1The abbreviations used are: HDV, hepatitis delta virus; SSB, single-stranded DNA-binding protein; aa, amino acid(s); nt, nucleotide(s); oligo, oligonucleotide; snRNA, small nuclear RNA; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA is a satellite virus of hepatitis B virus. The genome of HDV comprises single-stranded circular RNA of ∼1700 nucleotides, and HDV RNA replicates through a symmetrical rolling circle mechanism (1Wang K.S. Choo Q.L. Weiner A.J. Ou J.H. Najarian R.A. Thayer R.M. Mullenbach G.T. Denniston K.L. Gerin J.L. Hoighton M. Nature. 1986; 323: 508-515Google Scholar). Hepatitis delta antigen is the only protein coded by HDV that is critical for viral replication (2Chao M. Hsieh S.Y. Taylor J.M. J. Virol. 1990; 64: 5066-5069Google Scholar) and virion assembly (3Glenn J.S. Watson J.A. Havel C.M. White J.M. Science. 1992; 256: 1331-1334Google Scholar), although the molecular mechanisms have not yet been elucidated. HDV RNA, of both genomic and antigenomic senses, contains a ribozyme domain that can adopt a pseudoknot-like structure and undergo cis-cleavage in vitro (4Kuo M.Y.P. Sharmeen L. Dinter-Gottlieb G. Taylor J.M. J. Virol. 1988; 62: 4439-4444Google Scholar, 5Wu H.N. Lin Y.J. Lin F.P. Makino S. Chang M.F. Lai M.M.C. Proc. Natil. Acad. Sci. U. S. A. 1989; 86: 1831-1835Google Scholar, 6Perrotta A.T. Been M.D. Nature. 1991; 350: 433-436Google Scholar, 7Rosenstein S.P. Been M.D. Nucleic Acids Res. 1991; 19: 5409-5416Google Scholar). The cis-cleaving activity is essential for generating monomeric size RNA molecules during viral replication (8Macnaughton T.B. Wang Y.J. Lai M.M.C. J. Virol. 1993; 67: 2228-2234Google Scholar, 9Jeng K.S. Daniel A. Lai M.M.C. J. Virol. 1996; 70: 2403-2410Google Scholar). Hepatitis delta antigen may enhance, although it is not required for, the processing of multimeric size HDV RNA in vivo (10Jeng K.S. Su P.Y. Lai M.M.C. J. Virol. 1996; 70: 4205-4209Google Scholar). Conceivably, hepatitis delta antigen by itself, or together with unidentified cellular factors, acts as an RNA chaperone that modulates the ribozyme activity of HDV RNA. RNA chaperones are defined as proteins that aid in the folding of RNA by preventing misfolding or by resolving misfolded structures (11Herschlag D. J. Biol. Chem. 1995; 270: 20871-20874Google Scholar). The RNA chaperone activities in vitro of several proteins that interact with RNA with broad specificity have been explored through their effect on ribozyme reactions. These proteins, including severalEscherichia coli ribosomal proteins (12Coetzee T. Herschlag D. Belfort M. Genes Dev. 1994; 8: 1575-1588Google Scholar), the C-terminal domain of heterogeneous nuclear ribonucleoprotein A1 protein (13Bertrand E.L. Rossi J.J. EMBO J. 1994; 13: 2904-2912Google Scholar), and the nucleocapsid protein of human immunodeficiency virus (14Tsuchihashi Z. Khosla M. Herschlag D. Science. 1993; 262: 99-102Google Scholar, 15Herschlag D. Khosla M. Tsuchihashi Z. Karpel R.L. EMBO J. 1994; 13: 2913-2924Google Scholar), can overcome the general limitations of ribozyme reactions and facilitate ribozyme catalysis. Proteins with RNA chaperone activity are thought to lower the activation energy necessary for breaking and reforming of base pairs, although the molecular mechanism underlying RNA chaperone activity is not known. In vitro, hepatitis delta antigen can modulate the cis-cleaving activities of HDV genomic RNA fragments and facilitate a trans-acting hammerhead ribozyme to find its target in RNAs of various sequences and lengths (16Huang Z.S. Wu H.N. J. Biol. Chem. 1998; 273: 26455-26461Google Scholar). Hepatitis delta antigen exerts its effect on these RNA-mediated processes by modifying the conformation of RNA molecules and by promoting strand annealing and strand dissociation. These properties of hepatitis delta antigen parallel many cellular proteins and viral proteins with RNA/nucleic acid chaperone activity (12Coetzee T. Herschlag D. Belfort M. Genes Dev. 1994; 8: 1575-1588Google Scholar, 13Bertrand E.L. Rossi J.J. EMBO J. 1994; 13: 2904-2912Google Scholar, 14Tsuchihashi Z. Khosla M. Herschlag D. Science. 1993; 262: 99-102Google Scholar, 15Herschlag D. Khosla M. Tsuchihashi Z. Karpel R.L. EMBO J. 1994; 13: 2913-2924Google Scholar). The functional domain of hepatitis delta antigen appears to locate at the N-terminal region that is rich in basic amino acids and contains the cryptic RNA binding domain, coiled-coil domain, and nuclear localization signal (16Huang Z.S. Wu H.N. J. Biol. Chem. 1998; 273: 26455-26461Google Scholar). The core of the functional domain overlaps with the coiled-coil domain, whereas the RNA binding domain that binds HDV RNA specifically is not required for the RNA chaperone activity (16Huang Z.S. Wu H.N. J. Biol. Chem. 1998; 273: 26455-26461Google Scholar). Here we investigated further the basis of the stimulatory effect of the N-terminal domain of hepatitis delta antigen, named as NdAg, on RNA structural rearrangement. The results confirm and extend our previous model. The results showed that NdAg could promote the annealing of a variety of complementary sequences and stabilize RNA duplexes. NdAg preferentially stimulated the formation of a more extended duplex among competing sequences by facilitating strand annealing and strand exchange in a selective fashion. Moreover, NdAg was able to facilitate a trans-acting hammerhead ribozyme to discriminate a completely matched from a non-completely matched target in a substrate RNA, but may not alter the structure of a stable RNA molecule. The selective strand annealing and selective strand exchange activity of NdAg may be important for modulating the folding of viral RNA for replication and transcription. The N-terminal region (residues 1–88) of hepatitis delta antigen, named as NdAg, was produced in E. coli BL21 (DE3) cells and purified by phosphocellulose column chromatography as described previously (16Huang Z.S. Wu H.N. J. Biol. Chem. 1998; 273: 26455-26461Google Scholar). Fractions containing NdAg were snap-frozen in liquid nitrogen and stored at −70 °C. The concentration of NdAg was determined by ninhydrin assay with lysine as standard. NdAg was diluted to the desired concentrations with protein dilution buffer (50 mm Hepes-NaOH (pH 7.9), 1 mNaCl, 1 mm EDTA (pH 8.0), and 20% glycerol) and used as 10× stock. T4 phage gene 32 protein (T4gp32) and E. colisingle-stranded DNA-binding protein (SSB) were purchased from AmershamBiosciences. RNA oligos 11, 14, 17, and 22 and DNA oligos A, B, and C were chemically synthesized. Short unlabeled nucleic acid oligos were used directly without purification. DNA oligos A and B were gel-purified. The concentration of each nucleic acid fragment was determined by its absorbance at 260 nm. Carrier-free 5′ end-labeled RNA oligo 22 and DNA oligo C were made using [γ-32P]ATP (Amersham Biosciences) and T4 polynucleotide kinase (New England Biolabs). These 5′ end-labeled nucleic acid fragments were purified from polyacrylamide, 7 m urea gels, and their concentrations were calculated from the radioactivity of each fragment and the specific activity of [γ-32P]ATP. HH16 and HH163 RNAs were synthesized by T3 RNA polymerase (Promega) with synthetic DNAs as templates. HJ12L RNA, Rz1.H2 RNA, KSS3 RNA, yeast U4 and U6 snRNAs, and P9 RNA were run-off transcripts of the T7 RNA polymerase (Promega) with restriction enzyme-linearized plasmid DNA as templates. These RNAs were internally labeled by incorporating [α-32P]CTP to in vitro transcription reactions and then purified from polyacrylamide, 7 m urea gels. The concentration of labeled RNA was calculated from the radioactivity of RNA and the specific activity of CTP. The cellular RNA of E. coli BL21 (DE3) cells was isolated by TRIzol reagent (Invitrogen) following the instructions from the manufacturer. φX174 virion ssDNA and φX174 replication form dsDNA were purchased from New England Biolabs. Nucleic acid concentrations were determined according to their absorbance at 260 nm. All nucleic acids were resuspended in TE buffer (10 mm Tris-HCl (pH 8.0) and 1 mm EDTA) and stored at −20 °C. Complementary RNA oligos, complementary DNA oligos, or U4 and U6 snRNAs were heated separately at 95 °C for 2 min, cooled to room temperature, and incubated at the reaction temperature for at least 5 min before use. In general, 10-μl reaction mixtures contained indicated amounts of nucleic acid fragments and NdAg or other protein in 1× reaction buffer (40 mm Tris-HCl (pH 7.5), 0.1m NaCl, 0.02 mm EDTA, 2% glycerol). Reactions were performed at different temperatures; timings initiated by the addition of NdAg or other protein unless otherwise indicated. Reactions were terminated by transfer to ice, and the addition of 2 μl of ice-cold stop solution (50 mm EDTA (pH 8.0), 2.5% SDS, 25% glycerol, 0.01% xylene cyanol, and 0.01% bromphenol blue). Different nucleic acid species were resolved by electrophoresis through a native polyacrylamide gel in TBE buffer (89 mm Tris-HCl, 89 mm boric acid, and 1 mm EDTA) with or without 0.1% SDS at 4 °C. Gels were dried and examined by autoradiography. The pre-annealed RNA duplex was made by mixing 0.5 μm32P-labeled oligo 22 with 2 μm complement in TE buffer. The RNA solution was heated at 95 °C for 2 min, cooled slowly (∼1 h) to room temperature, and stored at −20 °C until used. The 32P-labeled pre-annealed RNA duplex was diluted in TE buffer and mixed with or without a competing RNA oligo, in the presence or absence of NdAg in 10 μl of reaction buffer at room temperature. Timing was initiated upon the addition of NdAg, and the reaction was stopped by the addition of a 0.2 reaction volume of ice-cold stop solution. All of the reaction mixtures were kept on ice before analysis. Electrophoresis of the RNA samples and the subsequent data analysis were performed as described for the annealing assay. Tubes containing the pre-annealed RNA duplex in 10 μl of reaction buffer were transferred to a PCR machine and incubated for 3 min at each indicated temperature. One tube was removed at 5 °C intervals through the temperature range indicated, and 2 μl of ice-cold stop solution was added immediately to terminate the reaction. All of the reaction mixtures were kept on ice before analysis. Electrophoresis of the RNA samples and the subsequent data analysis were performed as described for the annealing assay. Hepatitis delta ribozymes HJ12L and Rz1.H2 were heated at 95 °C for 2 min, cooled to room temperature, and incubated at 37 °C for 5 min before use. RNA was pre-incubated with NdAg in 1× reaction buffer for 30 min at 37 °C, and the cis-cleavage reaction of HJ12L or Rz1.H2 was initiated by the addition of MgCl2 to a final concentration of 12 mm. The reaction was performed at 37 °C for various periods of time. Further cis-cleavage was inhibited by the addition of an equal volume of quench solution containing 50 mm EDTA, 7 m urea, 0.005% xylene cyanol, and 0.005% bromphenol blue. RNAs in the ribozyme reaction mixture were analyzed on a 10% polyacrylamide, 7m urea gel. Hammerhead ribozyme and the KSS3 substrate RNA were separately heated at 95 °C for 2 min and then cooled to the reaction temperature for at least 5 min. The two RNAs were mixed and pre-incubated with NdAg in 1× reaction buffer for 30 min at 30 °C, and the trans-cleavage reaction was initiated by the addition of MgCl2 to a final concentration of 12 mm. The reaction was performed at 30 °C for 15 min, after which samples were treated with 1 mg/ml proteinase K at 37 °C for 25 min. RNAs in the ribozyme reaction mixture were analyzed on a 15% polyacrylamide, 7 m urea gel. The radioactivity of each RNA fragment was determined by quantification using a PhosphorImager (ImageQuant; AmershamBiosciences). The relative amount of each RNA fragment was determined by dividing the radioactivity of the RNA fragment with the number of C residues in the RNA molecule; thus, the fraction of RNA undergoing cleavage was calculated. Previously we had shown that the N-terminal domain of hepatitis delta antigen (NdAg) modulates the cis-cleaving activity of HDV genomic RNA fragments and activates the trans-cleavage reaction between hammerhead ribozymes and cognate substrates in vitro(16Huang Z.S. Wu H.N. J. Biol. Chem. 1998; 273: 26455-26461Google Scholar). Conceivably, NdAg exerts its effect on RNA-mediated processes by acting as an RNA chaperone, promoting RNA re-folding by facilitating the breakage and reforming of base pairs. To further investigate the basis of RNA chaperone activity, purified NdAg was assayed for the ability to promote the annealing of complementary sequences in RNA oligos and complicated RNA molecules in dilute solutions, and to accelerate strand exchange between an RNA duplex and a competing sequence. To assay the stimulatory effect of NdAg on strand annealing, complementary oligos of low concentrations were mixed, and duplex formation was monitored. In the case of RNA oligos 22 (0.025 nm) and 11 (0.125 nm) (Fig. 1 A), there was only negligible duplex formation in the absence of NdAg, but the extent and the rate of duplex formation were significantly elevated in the presence of 1 μm NdAg (Fig.2 A). Thus, NdAg promoted the annealing of complementary RNA oligos.Figure 2Effect of NdAg on strand annealing/hybrid formation. A, the annealing of RNA oligos. 0.025 nm32P-labeled oligo 22 and 0.125 nm oligo 11 were mixed with 0 or 10 mmMgCl2 in the absence or presence of 1 μm NdAg in 1× reaction buffer (40 mm Tris-HCl (pH 7.5), 0.1m NaCl, 0.02 mm EDTA, 2% glycerol) at room temperature for various periods. Samples were analyzed on an 18% polyacrylamide, 0.1% SDS gel at 4 °C. B, the annealing of DNA oligos. The reaction contains 0.5 nm32P-labeled oligo C alone, 0.5 nm32P-labeled oligo C and 0.4 nm oligo B, or 0.5 nm32P-labeled oligo C and 0.4 nmoligos A and B. The annealing reaction was performed in the absence or presence of 0.5 μm NdAg in 1× reaction buffer at 37 °C for 20 min. Samples were resolved on a 15% polyacrylamide, 0.1% SDS gel at 4 °C. C, the hybridization of internally labeled U4 and U6 snRNAs. 0.5 nm U4 snRNA and 2.5 nm U6 snRNA were incubated without any protein or with 50, 200, and 500 nm NdAg in 10 μl of 1× reaction buffer at 37 °C for 5 s, 30 min, 60 min, and 120 min. Mockrepresents the control experiment with the protein fraction obtained from the BL21(DE3) cells containing the empty pET15b vector. Samples were resolved on a 7% polyacrylamide, 0.1% SDS gel at 4 °C.Asterisks in these and subsequent figures indicate radioactive labeling.View Large Image Figure ViewerDownload (PPT) It is known that Mg2+ can neutralize the negative charge of phosphate groups and stabilize the structure of RNA molecules. To address the question of the effect of Mg2+ on the strand annealing activity of NdAg, we performed the strand annealing reaction in the presence of MgCl2 and/or NdAg. We found that MgCl2 of 10–40 mm alone facilitated the annealing of RNA oligos 22 and 11, although its stimulatory activity was significantly lower than that of NdAg. Nevertheless, MgCl2 at this concentration range reduced, although it did not eliminate, the strand annealing activity of 1 μm NdAg (Fig. 2 A and data not shown). Annealing assays were done on other complementary nucleic acid pairs of 11–56 nt; NdAg facilitated the annealing of all nucleic acid pairs tested, including DNA-DNA, DNA-RNA, and RNA-RNA pairs (data not shown). In the case of DNA oligos A (43 nt), B (56 nt), and C (18 nt) (oligos A and C are complementary to different regions of oligo B; Fig.1 B), NdAg greatly promoted the formation of the trimolecular duplex and both bimolecular duplexes at low DNA concentrations (∼0.5 nm) (B/C and B/CA duplex data are shown in Fig.2 B). Furthermore, NdAg accelerated the hybridization of more complicated RNAs in addition to simple complementary nucleic acid pairs. 50–500 nm NdAg facilitated 0.5 nm U4 snRNA and 2.5 nm U6 snRNA to form U4/U6 hybrid that potentially contains two intermolecular base pairing regions (Figs. 1 C and2 C). All of the aforementioned simple complementary nucleic acid pairs hybridize spontaneously; however, U4 snRNA and U6 snRNA did not hybridize spontaneously at even up to 25 nm in the absence of NdAg (data not shown). This suggests NdAg influences the ability of complex RNAs to interact. Therefore, in addition to short complementary oligos, NdAg also promotes the annealing of the complementary sequences reside in longer or more complicated RNAs. To test whether non-homologous nucleotide sequences perturb the acceleration of strand annealing or hybrid formation stimulated by NdAg, we analyzed the hybridization of a constant amount of U4 snRNA (0.5 nm161-nt RNA, 80.5 nm nucleotide) and U6 snRNA (2.5 nm 112-nt RNA, 280 nm nucleotide)) in the presence of increasing amounts of a non-homologous P9 RNA (a 160-nt run-off transcript of pET15b vector) at 100, 250, or 500 nmNdAg. As shown in Fig. 3 A, for a 360 nm nucleotide concentration of U4 snRNA (0.5 nm RNA, 80.5 nm nucleotide) and U6 snRNA (2.5 nm RNA, 280 nm nucleotide), U4/U6 hybridization was not inhibited until the molar ratio of total nucleotide (the sum of two snRNAs and P9 RNA) to NdAg exceeded 5:1, and at higher molar ratios (>5:1), U4/U6 hybrid did not form. The result suggests that there is a finite amount of NdAg available to facilitate duplex hybridization with a limiting ratio of one NdAg to five nucleotides. To confirm this finding, we performed strand annealing reactions of DNA oligos A and B at low concentrations (total nucleotide was ∼5 nm) in the presence of a relatively high concentration of NdAg (1 μm) and increasing concentrations of three kinds of nucleic acid: E. coli cellular RNA, φX174 ssDNA and φX174 dsDNA. As shown in Fig. 3 B, B/A duplex formation was not affected by the E. coli cellular RNA or φX174 ssDNA until the total nucleotide concentration exceeded 5 μm, whereas the presence of φX174 dsDNA did not fully inhibit the annealing of oligos A and B at even 12 μm total nucleotide. These results support the notion that the binding site size of an NdAg monomer is ∼5 nucleotides, and suggests that complementary strand annealing can occur in the presence of a 100–1000-fold excess of non-homologous sequence as long as the amount of NdAg is sufficient to coat the non-homologous sequences and the complementary nucleic acid pair. The acceleration of strand annealing/hybrid formation associated with NdAg is not specific for any particular type of nucleic acid. Hence, NdAg interacts with nucleic acid with broad specificity. We tested whether two other general nucleic acid-binding proteins share similar strand annealing properties with NdAg. We found that E. coliSSB (single-stranded nucleic acid-binding protein) and T4 phage gp32 protein at a concentration range of 1–10 μm failed to promote U4/U6 hybrid formation or to stimulate DNA or RNA duplex formation (data not shown). In addition, the stimulatory effect of 50 nm NdAg on U4/U6 hybrid formation was not perturbed in the presence of a 20-fold excess of T4 phage gp32 protein, a 50-fold excess of E. coli SSB, or a 200-fold excess of bovine serum albumin (Fig. 4). Thus, NdAg can facilitate strand annealing/hybrid formation in the presence of these two nucleic acid-binding proteins. These results indicate that the stimulation of strand annealing is not a general property of nucleic acid-binding proteins. We next examined the stimulatory effect of NdAg on the annealing of RNA oligos of different lengths to a common sequence. RNA oligos 11, 17, and 22 were used for this study (Fig. 1 A). Both oligos 11 and 17 are complementary to oligo 22. Oligo 17 has the same sequence as oligo 11 but also has a 2-nt extension at the 5′ terminus and a 3-nt extension at the 3′ terminus. Hence, oligo 17 can form a 17-base pair duplex with oligo 22, whereas oligo 11 can form an 11-base pair duplex with oligo 22. In the presence of 1 μm NdAg, the kinetics of annealing of oligos 22 and 17, and oligos 22 and 11 were quite similar, and the duplex formation reaction of both pairs of oligos reached an equilibrium within 10 min at 30, 37, and 42 °C (data not shown). The melting temperatures of the 22/11 duplex and 22/17 duplex were ∼50 and ∼65 °C, respectively, under annealing reaction conditions (Fig. 5 A). The competitive strand annealing reaction was performed in the presence of 0.025 nm32P-labeled oligo 22 and 0.5 nm oligos 11 and 17 at temperatures below theTm of each duplex. In the absence of NdAg, only a small fraction of oligos formed duplexes at 30, 37, or 42 °C. Moreover, the amount of 22/11 duplex was slightly higher than that of 22/17 duplex at each temperature, and both duplexes were stable at 42 °C (Fig. 5 B). In the presence of 1 μmNdAg, all of oligo 22 participated in duplex formation. However, oligo 22 annealed more favorably to oligo 17 at 30 and 37 °C, and exclusively to oligo 17 at 42 °C (Fig. 5 B). Moreover, the relative amount of 22/17 and 22/11 duplexes at each incubation temperature was not changed when the reaction time varied between 10 and 180 min (data not shown). Thus, NdAg promotes the formation of a more extended duplex among competing sequences, with the activity higher at 42 °C than 30 and 37 °C. Therefore, hypothetically NdAg facilitates strand annealing or stimulates duplex dissociation in a selective manner. We next monitored the ability of NdAg to promote strand exchange between an RNA duplex and a competing RNA oligo (Fig.6). The pre-annealed32P-labeled 22/11 duplex (0.25 nm) was mixed with a 100-fold excess of oligo 17 (25 nm). In the absence of NdAg, the pre-annealed duplex gradually dissociated over a 30-min period, with the released 32P-labeled oligo 22 concurrently forming a duplex with oligo 17. Consequently, the32P-labeled 22/11 duplex was converted to the32P-labeled 22/17 duplex (Fig. 6 A). In the presence of NdAg, the conversion of 22/11 duplex to 22/17 duplex occurred at an elevated rate at least during the first 10 min of the reaction (Fig. 6 A). Thus, in this experiment, NdAg facilitated strand exchange. However, a strikingly different result was obtained when the assay was performed using two other combinations of pre-annealed RNA duplex and a competing RNA oligo in the presence of NdAg. The data in Fig. 6 (B and C) show that strand exchange between a pre-annealed 22/14 duplex (0.25 nm) and oligo 11 (25 nm) as well as between a pre-annealed 22/17 duplex (0.25 nm) and oligo 11 (25 nm) was not observed in the presence of 1 μmNdAg; each pre-annealed duplex was stabilized by NdAg instead. In the absence of NdAg, there was a build-up of 22/11 duplex after 10 min, whereas the conversion of the pre-annealed duplex to 22/11 duplex was not completed. Because 22/14 duplex and 22/17 duplex contain more base pairs than 22/11 duplex, NdAg appears to promote strand exchange only when the formation of a more extended duplex occurs. These results suggest that NdAg promotes strand exchange in a selective fashion, and excludes the only role of NdAg in the strand exchange process as being the promotion of the dissociation of the pre-annealed duplex. Furthermore, the strand exchange reaction was also performed in the presence of MgCl2 with or without NdAg. We found that 1 μm NdAg speeded up the strand exchange reaction between 22/11 duplex and RNA oligo 17 in the presence of 0–5 mmMgCl2 but failed to facilitate the conversion of 22/11 duplex to 22/17 duplex in the presence of 10 or 20 mmMgCl2 (Fig. 6 D). The studies with the synthetic nucleic acid oligos disclose that NdAg promotes strand annealing and strand exchange in a selective fashion, with this property allowing NdAg to stimulate the formation of the most stable duplex among complementary sequences. This finding led us to speculate that NdAg could assist a contiguous sequence in adopting a stable structure, which may not be biologically active, and that NdAg would not alter the structure of a stable RNA. Here we investigated these speculations by studying the effect of NdAg on an RNA that contains the autolytic domain of the HDV genome and a cis-acting hepatitis delta ribozyme mutant that has a more stable structure than its wild type counterpart. Because hepatitis delta ribozyme has to adopt a specific catalytic structure, the alteration of autocatalytic activity upon NdAg treatment reflects ribozyme structure change. HJ12L contains the autolytic domain of the HDV genome, and Rz1.H2 is a hepatitis delta ribozyme mutant that has had its helix 1 substituted, the helix 2 extended from 5 to 8 base pairs, and the helix 4 replaced by a stable hairpin loop (Fig.7 A). Rz1.H2 cis-cleaved efficiently and rapidly in the absence of NdAg, whereas HJ12L was much less active under the same conditions (Fig. 7 B). The extended helix 2 rather than the alterations in helixes 1 and 4 of Rz1.H2 may account for the elevated cis-cleaving activity under native conditions, because we showed previously that the elongation of helix 2 enhances the resistance to formamide and stabilized the catalytic core of hepatitis delta ribozymes (17Lee C.B. Lai Y.C. Ping Y.H. Huang Z.S. Lin J.Y. Wu H.N. Biochemistry. 1996; 35: 12303-12312Google Scholar). Moreover, in the experiment shown in Fig. 7 B, we found that pre-mixing with NdAg prior to the initiation of cis-cleavage did not alter the cis-cleavage reaction of Rz1.H2, whereas the same treatment attenuated the cis-cleaving activity of HJ12L. The results indicate that NdAg modified the structure of HJ12L but not Rz1.H2 and that the interaction with NdAg does not alter the catalysis of Rz1.H2. The finding supports the hypothesis that NdAg does not modify the structure of a stable RNA. We then used the trans-cleavage reactions of a hammerhead ribozyme HH16 to further study the RNA chaperone activity of NdAg. The trans-cleavage reaction of a hammerhead ribozyme involves at least two steps. First, the ribozyme anneals to its target in a substrate RNA for the assembly of a hammerhead catalytic domain; then, cleavage occurs at a specific location by the catalysis of divalent cations (18Hertel K.L. Herschlag D. Uhlenbeck O.C. Biochemistry. 1994; 33: 3374-3385Google Scholar). KSS3 is a 107-nt RNA containing three targets for the hammerhead ribozyme HH16 (Fig.8 A). The first target has a C to A substitution; hence, upon binding to HH16, the helix I of the reconstituted hammerhead catalytic domain contains an AG mismatched pair. The second and the third targets are completely matched to HH16; each can form 16 base pairs with HH16. HH163 is a mutant of HH16 and has a G to U substitution to compensate for the mutation of the first ribozyme target of KSS3 (i.e. no mismatch; Fig.8 A). We showed previously that NdAg promotes the trans-cleavage reaction of HH16 and all its targets in KSS3 under ribozyme excess conditions, with NdAg appearing to exert its effect by elevating the mutual accessibility of two RNAs (16Huang Z.S. Wu H.N. J. Biol. Chem. 1998; 273: 26455-26461Google Scholar). Here, we found that >95% of KSS3 was cleaved by HH16 and HH163 when 1 nmKSS3 and 10 nm of each ribozyme were pre-incubated with 1 μm NdAg prior to the initiation of trans-cleavage (Fig.8 B). Furthermore, the major products of the two ribozymes were the same; they were the 49-, 23/22-, and 13-nt RNAs, with none of them containing any ribozym
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