An Extra Nucleotide in the Consensus Catalytic Core of a Viroid Hammerhead Ribozyme
2001; Elsevier BV; Volume: 276; Issue: 37 Linguagem: Inglês
10.1074/jbc.m103867200
ISSN1083-351X
AutoresMarcos de la Peña, Ricardo Flores,
Tópico(s)Plant Virus Research Studies
ResumoHammerhead ribozymes catalyze self-cleavage of oligomeric RNAs generated in replication of certain viroid and viroid-like RNAs. Previous studies have defined a catalytic core conserved in most natural hammerheads, but it is still unknown why some present deviations from the consensus. We have addressed this issue in chrysanthemum chlorotic mottle viroid (CChMVd), whose (+) hammerhead has an extra A (A10) between the conserved A9 and the quasi-conserved G10.1. Effects of insertions at this position on hammerhead kinetics have not hitherto been examined. A10 caused a moderate decrease of thetrans-cleaving rate constant with respect to the CChMVd (+) hammerhead without this residue, whereas A10→C and A10→G substitutions had major detrimental effects, likely because they favor catalytically inactive foldings. By contrast, A10→U substitution induced a 3–4-fold increase of the rate constant, providing an explanation for the extra U10 present in two natural hammerheads. Because A10 also occupies a singular and indispensable position in the global CChMVd conformation, as revealed by bioassays, these results show that some hammerheads deviate from the consensus due to the involvement of certain residues in critical function(s) other than self-cleavage. Incorporation of the extra U10 into a model hammerhead also caused a similar increase in the rate constant, providing data for a deeper understanding of the hammerhead structural requirements and for designing more efficient ribozymes. Hammerhead ribozymes catalyze self-cleavage of oligomeric RNAs generated in replication of certain viroid and viroid-like RNAs. Previous studies have defined a catalytic core conserved in most natural hammerheads, but it is still unknown why some present deviations from the consensus. We have addressed this issue in chrysanthemum chlorotic mottle viroid (CChMVd), whose (+) hammerhead has an extra A (A10) between the conserved A9 and the quasi-conserved G10.1. Effects of insertions at this position on hammerhead kinetics have not hitherto been examined. A10 caused a moderate decrease of thetrans-cleaving rate constant with respect to the CChMVd (+) hammerhead without this residue, whereas A10→C and A10→G substitutions had major detrimental effects, likely because they favor catalytically inactive foldings. By contrast, A10→U substitution induced a 3–4-fold increase of the rate constant, providing an explanation for the extra U10 present in two natural hammerheads. Because A10 also occupies a singular and indispensable position in the global CChMVd conformation, as revealed by bioassays, these results show that some hammerheads deviate from the consensus due to the involvement of certain residues in critical function(s) other than self-cleavage. Incorporation of the extra U10 into a model hammerhead also caused a similar increase in the rate constant, providing data for a deeper understanding of the hammerhead structural requirements and for designing more efficient ribozymes. nucleotide(s) peach latent mosaic viroid chrysanthemum chlorotic mottle viroid satellite RNA of lucerne transient streak virus satellite RNA of Arabis mosaic virus polyacrylamide gel electrophoresis polymerase chain reaction Viroids, subviral circular RNAs of 247–401 nucleotides (nt),1 are the smallest autonomous replicons (1Diener T.O. FASEB J. 1991; 5: 2808-2813Crossref PubMed Scopus (61) Google Scholar, 2Flores R. Randles J.W. Bar-Joseph M. Diener T.O. van Regenmortel M.H.V. Fauquet C. Bishop D.H.L. Carstens E.B. Estes M.K. Lemon S.M. Maniloff J. Mayo M.A. McGeoch D.J. Pringle C.R. Wickner R.B. Virus Taxonomy: Seventh Report of the International Committee on Taxonomy of Viruses. Academic Press, San Diego, CA2000: 1009-1024Google Scholar). This minimal size imposes severe restrictions onto viroid genomes to accommodate a series of functions critical to their life cycle which include host selection, long distance and cell-to-cell movement, and targeting to specific subcellular organelles (nuclei or chloroplasts) where they replicate and accumulate. All these functions must result from the direct interaction of the viroid RNA, or some of its replicative intermediates, with cellular factors because the available evidence indicates that viroids do not code for proteins (3Davies J.W. Kaesberg P. Diener T.O. Virology. 1974; 61: 281-286Crossref PubMed Scopus (63) Google Scholar, 4Hall T.C. Wepprich R.K. Davies J.W. Weathers L.G. Semancik J.S. Virology. 1974; 61: 486-492Crossref PubMed Scopus (48) Google Scholar, 5Gross H.J. Domdey H. Lossow C. Jank P. Raba M. Alberty H. Sänger H.L. Nature. 1978; 273: 203-208Crossref PubMed Scopus (375) Google Scholar). Genetic information, therefore, must be extremely compressed and even overlapping in viroids. Viroids replicate through a rolling-circle mechanism in which the infecting most abundant monomeric circular RNA, assumed by convention to have the (+) polarity, is successively transcribed into oligomeric (−) and (+) strands that are then excised into the linear monomeric forms and circularized to produce the progeny (6Branch A.D. Robertson H.D. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6381-6385Crossref PubMed Scopus (75) Google Scholar, 7Owens R.A. Diener T.O. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 113-117Crossref PubMed Scopus (61) Google Scholar). This is an RNA-based mechanism (8Grill L.K. Semancik J.S. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 896-900Crossref PubMed Scopus (65) Google Scholar), and depending on whether or not the (−) oligomeric intermediates are cleaved and ligated to their corresponding monomeric circular counterparts, which then serve as the initial template for the second half of the cycle, the mechanism is considered to be symmetric or asymmetric, respectively (9Branch A.D. Robertson H.D. Science. 1984; 223: 450-454Crossref PubMed Scopus (346) Google Scholar). Due to the lack of messenger activity of viroid RNAs, the whole replication process should be in principle catalyzed by host enzymes. However, in avocado sunblotch viroid (10Symons R.H. Nucleic Acids Res. 1981; 9: 6527-6537Crossref PubMed Scopus (114) Google Scholar, 11Hutchins C.J. Rathjen P.D. Forster A.C. Symons R.H. Nucleic Acids Res. 1986; 14: 3627-3640Crossref PubMed Scopus (439) Google Scholar), peach latent mosaic viroid, PLMVd (12Hernández C. Flores R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3711-3715Crossref PubMed Scopus (172) Google Scholar), and chrysanthemum chlorotic mottle viroid, CChMVd (13Navarro B. Flores R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11262-11267Crossref PubMed Scopus (135) Google Scholar), which together form the family Avsunviroidae (14Flores R. Daròs J.A. Hernández C. Adv. Virus Res. 2000; 55: 271-323Crossref PubMed Google Scholar), the cleavage step is autocatalytic and mediated by hammerhead structures that can be adopted by the strands of both polarities. Consequently, these three viroids are considered to replicate following the symmetric rolling-circle mechanism. In line with this view, the monomeric (−) circular RNA has been identified in avocado sunblotch viroid-infected avocado (15Hutchins C.J. Keese P. Visvader J.E. Rathjen P.D. McInnes J.L. Symons R.H. Plant Mol. Biol. 1985; 4: 293-304Crossref PubMed Scopus (97) Google Scholar, 16Daròs J.A. Marcos J.F. Hernández C. Flores R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12813-12817Crossref PubMed Scopus (128) Google Scholar, 17Navarro J.A. Daròs J.A. Flores R. Virology. 1999; 253: 77-85Crossref PubMed Scopus (47) Google Scholar) and in PLMVd-infected peach (18Bussière F. Lehoux J. Thompson D.A. Skrzeczkowski L.J. Perreault J.P. J. Virol. 1999; 73: 6353-6360Crossref PubMed Google Scholar). The rest of 25 viroid species, which make up the family Pospiviroidae (2Flores R. Randles J.W. Bar-Joseph M. Diener T.O. van Regenmortel M.H.V. Fauquet C. Bishop D.H.L. Carstens E.B. Estes M.K. Lemon S.M. Maniloff J. Mayo M.A. McGeoch D.J. Pringle C.R. Wickner R.B. Virus Taxonomy: Seventh Report of the International Committee on Taxonomy of Viruses. Academic Press, San Diego, CA2000: 1009-1024Google Scholar), are assumed to follow the asymmetric rolling-circle mechanism because the oligomeric forms are the predominant (−) strands accumulating in tissues infected by representative members of this family, whereas the monomeric (−) circular RNA has not been identified (9Branch A.D. Robertson H.D. Science. 1984; 223: 450-454Crossref PubMed Scopus (346) Google Scholar, 19Branch A.D. Benenfeld B.J. Robertson H.D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9128-9132Crossref PubMed Scopus (83) Google Scholar, 20Feldstein P.A. Hu Y. Owens R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6560-6565Crossref PubMed Scopus (51) Google Scholar). Cleavage of the oligomeric (+) RNA intermediates in family Pospiviroidae is generally believed to require a host ribonuclease (21Tsagris M. Tabler M. Mühlbach H.P. Sänger H.L. EMBO J. 1987; 6: 2173-2183Crossref PubMed Google Scholar, 22Baumstark T. Schröder A.R.W. Riesner D. EMBO J. 1997; 16: 599-610Crossref PubMed Scopus (123) Google Scholar), although the possibility that the cleavage step is RNA-catalyzed in all cases has been also advanced (23Liu Y.-H. Symons R.H. RNA. 1998; 4: 418-429Crossref PubMed Scopus (19) Google Scholar). The hammerhead ribozyme is a small RNA motif able to self-cleave at a specific phosphodiester bond in the presence of a divalent metal ion, generally Mg2+, and under mild temperature and pH conditions, producing 2′,3′-cyclic phosphate and 5′-hydroxyl termini (11Hutchins C.J. Rathjen P.D. Forster A.C. Symons R.H. Nucleic Acids Res. 1986; 14: 3627-3640Crossref PubMed Scopus (439) Google Scholar, 24Prody G.A. Bakos J.T. Buzayan J.M. Schneider I.R. Bruening G. Science. 1986; 231: 1577-1580Crossref PubMed Scopus (450) Google Scholar, 25Forster A.C. Symons R.H. Cell. 1987; 49: 211-220Abstract Full Text PDF PubMed Scopus (564) Google Scholar). Structural dissection of the 23 natural hammerhead structures reported so far (for a review, see Ref. 26Flores R. Hernández C. De la Peña M. Vera A. Daròs J.A. Methods Enzymol. 2001; 341: 540-552Crossref PubMed Scopus (46) Google Scholar) shows a central core composed of 11 strictly conserved nucleotides flanked by three double-helix regions (I, II, and III) with loose sequence requirements except positions 10.1 and 11.1, which in most cases form a G-C pair, and positions 15.2 and 16.2, which in most cases form a C-G pair (Fig.1 A). Site-directed mutagenesis has revealed that the conserved residues play a critical role in determining the rate constant of cleavage (27Ruffner D.E. Stormo G.D. Uhlenbeck O.C. Biochemistry. 1990; 29: 10695-10702Crossref PubMed Scopus (448) Google Scholar), and analysis by x-ray crystallography has uncovered a complex array of noncanonical interactions between the residues forming the central core (28Pley H. Flaherty K.M. McKay D.B. Nature. 1994; 372: 68-74Crossref PubMed Scopus (928) Google Scholar, 29Scott W.G. Finch J.T. Klug A. Cell. 1995; 81: 991-1002Abstract Full Text PDF PubMed Scopus (690) Google Scholar), prominent among which are three non-Watson-Crick pairs (involving A9 and G12, G8 and A13, and U7 and A14) that extend helix II (Fig. 1 A). However, deviations from the consensus hammerhead core have been observed in some natural hammerhead structures. This is the case of the CChMVd (+) hammerhead structure, which is peculiar in having an extra A inserted between the strictly conserved A9 and the highly conserved G10.1 residues (13Navarro B. Flores R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11262-11267Crossref PubMed Scopus (135) Google Scholar). These two residues are contiguous in all known natural hammerhead structures except in those of (+) strands of satellite RNAs of lucerne transient streak virus (sLTSV) (25Forster A.C. Symons R.H. Cell. 1987; 49: 211-220Abstract Full Text PDF PubMed Scopus (564) Google Scholar) and Arabis mosaic virus (sArMV) (30Kaper J.M. Tousignant M.E. Steger G. Biochem. Biophys. Res. Commun. 1988; 154: 318-325Crossref PubMed Scopus (44) Google Scholar), in which an extra U exists at the same position (Fig.1 B), and in those of (+) strand of satellite RNA of cereal yellow dwarf virus-RPV (31Miller W.A. Hercus T. Waterhouse P.M. Gerlach W.L. Virology. 1991; 183: 711-720Crossref PubMed Scopus (47) Google Scholar, 32Song S.I. Silver S.L. Aulik M.A. Rasochova L. Mohan B.R. Miller W.A. J. Mol. Biol. 1999; 293: 781-793Crossref PubMed Scopus (17) Google Scholar) and (−) strand of the carnation small viroid-like RNA (33Hernández C. Daròs J.A. Elena S.F. Moya A. Flores R. Nucleic Acids Res. 1992; 20: 6323-6329Crossref PubMed Scopus (42) Google Scholar), in which the extra residue is a C. In the two latter hammerhead structures, the extra C is accompanied by an extra A inserted between the highly conserved C11.1 and the strictly conserved G12 residues. The observation that natural selection has allowed an extra residue between positions A9 and G10.1 in a significant number of natural hammerhead structures is intriguing and raises the question of whether this could provide some adaptive advantage to the corresponding RNAs. A plausible explanation is that the extra residue might be also involved in determining a functional property other than self-cleavage and, on this basis, be preserved. Here we have put this hypothesis to the test using the CChMVd/chrysanthemum system, which is very suitable for this purpose because recombinant plasmids containing dimeric head-to-tail viroid cDNA inserts, or in vitro transcripts thereof, are infectious and incite symptoms in a relatively short time (12–15 days) (13Navarro B. Flores R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11262-11267Crossref PubMed Scopus (135) Google Scholar). Moreover, although an extra residue between positions 9 and 10.1 is compatible with extensive self-cleavage during in vitro transcription and after purification (13Navarro B. Flores R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11262-11267Crossref PubMed Scopus (135) Google Scholar, 25Forster A.C. Symons R.H. Cell. 1987; 49: 211-220Abstract Full Text PDF PubMed Scopus (564) Google Scholar, 34Sheldon C.C. Symons R.H. Nucleic Acids Res. 1989; 17: 5679-5685Crossref PubMed Scopus (87) Google Scholar), a kinetic analysis of the effects of mutations in this particular position on the corresponding rate constants of cleavage is lacking, despite the ample biochemical and biophysical analyses to which the hammerhead ribozyme has been subjected (35Stage-Zimmermann T.K. Uhlenbeck O.C. RNA. 1998; 4: 875-889Crossref PubMed Scopus (184) Google Scholar, 36Murray J.B. Scott W.G. J. Mol. Biol. 2000; 296: 33-41Crossref PubMed Scopus (57) Google Scholar). We have addressed this second issue with atrans-acting hammerhead structure derived from the CChMVd (+) RNA and then by extending the analysis to a well known model hammerhead structure. Our results show that the nature of the extra residue between positions A9 and G10.1 has profound effects on viroid infectivity and on hammerhead-mediated RNA cleavage, leading in some cases to a significant increase in the catalytic efficiency of the ribozyme. Circular forms of the CChMVd, purified by two consecutive PAGE steps, were reverse transcribed and PCR-amplified with primers RF-146 (complementary to nt 133–108) and RF-147 (homologous to nt 134–159 of the CM5 reference sequence of CChMVd (Ref. 37De la Peña M. Navarro B. Flores R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9960-9965Crossref PubMed Scopus (73) Google Scholar; see also Fig. 2)). Reverse transcription, PCR amplification, and cloning were performed as described previously (13Navarro B. Flores R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11262-11267Crossref PubMed Scopus (135) Google Scholar). Inserts were sequenced with an ABI Prism DNA apparatus (PerkinElmer Life Sciences). The protocol reported previously (38Byrappa S. Gavin D.K. Gupta K.C. PCR Methods Appl. 1995; 5: 404-407Crossref Scopus (82) Google Scholar) was followed with minor modifications. The recombinant plasmid pCM5 (5 ng), containing a monomeric insert of the CChMVd reference sequence (13Navarro B. Flores R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11262-11267Crossref PubMed Scopus (135) Google Scholar), was PCR-amplified with Pfu DNA polymerase and 500 ng each of the phosphorylated primers RF-142 (5′-CATGGATCVTCATCAGGACACACCGAC-3′), complementary to nucleotides 11–35 of the CM5 sequence (except the residue in bold that was degenerated to change the A27, corresponding to A10 in the CChMVd plus hammerhead, into C, G, or U), and RF-134 (5′-ACAGGATCGAAACCTCTTCCAGTT-3′), homologous to nucleotides 36–59 (Fig. 2). Plasmid pCM5 was also PCR-amplified with the phosphorylated primers RF-133 (5′-CATGGATCTCATCAGGACACACCGAC-3′), complementary to nucleotides 11–35 of the CM5 sequence (except in the position corresponding to A27 that was deleted) and RF-134. The PCR products were electrophoretically separated in 1% agarose gels, and those of plasmid length were eluted and circularized with T4 DNA ligase. After transformation, the inserts of the new plasmids, pCM5-C10, pCM5-G10, pCM5-U10, and pCM5-Δ10, were sequenced to confirm that only the expected mutations had been introduced. From these constructs, plasmids pCM5d, pCM5d-C10, pCM5d-G10, pCM5d-U10, and pCM5d-Δ10, containing the corresponding head-to-tail dimeric inserts, were generated following standard protocols. Chrysanthemum (Dendranthema grandiflora Tzvelez, cv. "Bonnie Jean") was propagated in growth chambers (13Navarro B. Flores R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11262-11267Crossref PubMed Scopus (135) Google Scholar). Plants were mechanically inoculated either with the recombinant plasmids pCM5d, pCM5d-C10, pCM5d-G10, pCM5d-U10, and pCM5d-Δ10 (2 μg of plasmid/plant) or with their monomeric CChMVd RNAs (0.1 μg of RNA/plant) resulting from self-cleavage during in vitrotranscription. CChMVd replication in the inoculated plants was analyzed by dot-blot hybridization following extraction of leaves with buffer-saturated phenol and chromatography on nonionic cellulose (CF11, Whatman) (37De la Peña M. Navarro B. Flores R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9960-9965Crossref PubMed Scopus (73) Google Scholar). The recombinant plasmids pCM5, pCM5-C10, pCM5-G10, pCM5-U10, and pCM5-Δ10 were linearized with BamHI and in vitro transcribed with T3 RNA polymerase (39Forster A.C. Davies C. Hutchins C.J. Symons R.H. Methods Enzymol. 1990; 181: 583-607Crossref PubMed Scopus (21) Google Scholar). The primary transcripts and their self-cleavage products were separated by PAGE in 5% gels containing 8 m urea and 40% formamide that were quantitatively scanned with a bioimage analyzer (Fuji BAS1500). Ribozymes with the sequence of the CChMVd (+) hammerhead from positions 14 to 53 of the CM5 reference sequence (Fig. 2) and mutants thereof at position 27, which corresponds to position A10 of the hammerhead (Fig. 4), were synthesized by in vitro transcription ofXbaI-linearized plasmids containing these sequences, immediately preceded and followed by the T7 promotor and theXbaI site, respectively. Transcription reactions (50 μl) contained 40 mm Tris-HCl, pH 8, 6 mmMgCl2, 2 mm spermine, 10 mmdithiothreitol, 2 mm each of ATP, CTP, GTP and UTP, 2 units/μl human placental ribonuclease inhibitor, 20 ng/μl plasmid DNA, and 4 units/μl T7 RNA polymerase. After incubation at 37 °C for 1 h, transcription products were separated by PAGE in 15% denaturing gels and those with the expected length were eluted, recovered by ethanol precipitation, and resuspended in 50 mm Tris-HCl, pH 7.5. The model hammerhead ribozyme HH8 (Fig. 6) and its mutants at position 10 of the hammerhead were also prepared by in vitro transcription following the same protocol. Substrate RNAs (5′-AAGAGGUCGGCACC-3′) and (5′-GAAUGUCGGUCG-3′) for the CChMVd (+) and the HH8 hammerheads, respectively (Figs. 4 and 6), were obtained by chemical synthesis using 2′-orthoester protection (Dharmacon Research, Boulder, CO) and sequentially deprotected with 0.2 m acetic acid and Tris-HCl, pH 8.7. After purification by PAGE in 20% denaturing gels, the substrate RNAs were eluted and labeled at their 5′ termini using [α-32P]ATP (Amersham Pharmacia Biotech, 3000 Ci/mmol) and T4 polynucleotide kinase (40Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar).Figure 6Ribozyme and substrate complexes derived from the HH8 hammerhead structure. Ribozymes and substrates are inblack and gray letters, respectively. Residues conserved in most natural hammerhead structures are on ablack background, and the self-cleavage sites are denoted by arrows. HH8 refers to the consensus hammerhead structure (without a residue at position 10), andHH8-A10, HH8-C10, HH8-G10, andHH8-U10 refer to the mutant forms with an extra A10, C10, G10, and U10 residues (outlined fonts), respectively. The extra A10, C10, and U10 residues could potentially interact with G12, distort the catalytic core, and reduce the corresponding rate constants. In the case of HH8-G10, an alternative catalytically inactive complex is also presented. Other details are as described in the legend to Fig. 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Single-turnover experiments with excess ribozyme (covering a range from 100 to 1000 nm in different experiments in order to assure saturating conditions) and trace32P-labeled substrate (less than 1 nm) were used to determine the rate constant of cleavage (41Hertel K.J. Herschlag D. Uhlenbeck O.K. Biochemistry. 1994; 33: 3374-3385Crossref PubMed Scopus (253) Google Scholar). Cleavage reactions were carried out in 50 mm Tris-HCl, pH 7.5, 10 mm MgCl2 at 25 °C as described previously (42Fedor M.J. Uhlenbeck O.C. Biochemistry. 1992; 31: 12042-12054Crossref PubMed Scopus (211) Google Scholar). The ribozyme and substrate were first annealed in 50 mm Tris-HCl, pH 7.5, by heating at 95 °C for 1 min and slowly cooling down to 25 °C for 15 min. Reactions were initiated by adding MgCl2 to a final concentration of 10 mm. Aliquots were removed at appropriate time intervals and quenched with a 5-fold excess of stop solution (8 m urea, 50% formamide, 50 mm EDTA, 0.1% xylene cyanol, and bromphenol blue dyes) at 0 °C. Substrate and product from each time point were separated by PAGE in 20% denaturing gels. The fraction of product at different times F was determined by radioactivity quantitation of the corresponding gel bands with a bioimage analyzer and fitted to the equation F = F∞ (1 −e −kt), whereF∞ is the fraction of product at the end point of the reaction and k the first order rate constant of cleavage (kcat). In the case of the HH8 hammerhead, cleavage rates were also measured under multiple-turnover conditions (43Fedor M.J. Uhlenbeck O.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1668-1672Crossref PubMed Scopus (241) Google Scholar) using at least six different substrate concentrations, ranging from 50 to 1000 nm, in excess over those of the ribozyme (from 10 to 40 nm depending on the catalytic activity of the ribozyme). Reactions were initiated by mixing at 25 °C the ribozyme (15 μl) and the substrate (15 μl) previously heated at 95 °C for 1 min in 50 mm Tris-HCl, pH 7.5, containing 10 mm MgCl2. Aliquots were removed as before, and data were fitted to Eadie-Hofstee plots to obtain the values for kcat and Km. The errors reported for kinetic parameters were obtained from triplicate experiments with different preparations of RNA. Hereafter, we will refer to the position occupied by the extra A in the CChMVd (+) hammerhead structure, and by any other residue in this or in other hammerhead structures, as position number 10 considering that it is located between the strictly conserved A9 and the highly conserved G10.1 in the consensus hammerhead structure (Figs.1 and 2). The extra A10 of the CChMVd (+) hammerhead structure also holds a special place in the branched secondary structure of lowest free energy predicted for the (+) strand of this viroid (position A27 in the genomic reference sequence of CChMVd), connecting two helices of a cruciform domain (Fig. 2). It is worth noting that the in vivo significance of this proposed branched conformation, which is inactive for self-cleavage, is strongly supported by the analysis of the sequence heterogeneity found in more than 100 natural CChMVd variants, because the observed changes are located in loops or when affecting a base pair the substitutions are compensatory, and also because no variability has been observed at this particular position A27 (13Navarro B. Flores R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11262-11267Crossref PubMed Scopus (135) Google Scholar, 37De la Peña M. Navarro B. Flores R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9960-9965Crossref PubMed Scopus (73) Google Scholar). 2M. De la Peña and R. Flores, unpublished data. In the interest of simplicity and to avoid any confusion that would result from referring to the same residue with two numbers, 10 in the CChMVd (+) hammerhead and 27 in the genomic reference sequence, we will only use the first number. To determine whether the extra A10 plays any role in infectivity, chrysanthemum plants were inoculated with recombinant plasmids containing dimeric tandem inserts of CChMVd cDNA with all possible mutations at this position introduced by site-directed mutagenesis. Ten days later, only those control plants inoculated with the plasmid containing the wild-type CChMVd cDNA (pCM5d-A10) developed the characteristic symptoms of the chlorotic mottle disease. All plants inoculated with plasmids containing the substitutions A10→C and A10→U (pCM5d-C10 and pCM5d-U10, respectively) showed symptoms 15–20 days after inoculation, but only two of the four plants inoculated with pCM5d-G10, containing the substitution A10→G, displayed the typical symptoms 25 days after inoculation. Interestingly, none of the plants inoculated with the plasmid not containing A10 (pCM5d-Δ10) developed symptoms during the observation period (up to 3 months). Analysis by dot-blot hybridization confirmed that the inoculated plants showing symptoms were indeed infected, whereas no signal was observed in those remaining symptomless (data not shown). These experiments were repeated twice with similar results. When the inoculations were performed with the monomeric CChMVd RNAs resulting from self-cleavage during in vitro transcription of the dimeric cDNA inserts, symptoms induced by RNAs with the substitutions A10→C, A10→U, and A10→G, appeared with only a short delay (1–2 days) with respect to those induced by the wild-type RNA, but again none of the plants inoculated with the RNA without A10 developed symptoms, and dot-blot hybridization confirmed that they had not been infected. Reverse transcription-PCR amplifications of viroid progenies from the infected chrysanthemum plants and sequencing of the resulting full-length clones (from 5 to 9 for each construct) revealed that the three substitutions at position 10 had reverted to the original A10 in all cases. Altogether, these results demonstrate that the A10 residue is indispensable for infectivity. CChMVd sequences with substitutions at this position are less infectious, most likely because they have to revert to the wild type, whereas the reversion does not occur when this residue is deleted. The higher infectivity of CChMVd RNAs when compared with their cDNAs is not surprising considering that the latter must be recognized and transcribed by a host RNA polymerase before entering into the standard RNA-RNA replication cycle. To initially assess the effect of the extra A10 residue on the catalytic activity of the CChMVd (+) hammerhead structure, we determined the extent of self-cleavage of CChMVd (+) RNAs transcribed from five recombinant plasmids containing monomeric inserts with the wild-type CChMVd sequence (pCM5-A10), with the three possible substitutions at this position (pCM5-C10, pCM5-G10 and pCM5-U10) and with the A10 deleted (pCM5-Δ10). The extent to which the five RNAs self-cleaved during transcription ranged from ∼55% (the wild-type RNA and those with the changes A10→U and A10→G) to 65% (the A10→C and A10→Δ RNAs) (Fig.3). When the uncleaved monomeric transcripts were purified and incubated under standard self-cleavage conditions (39Forster A.C. Davies C. Hutchins C.J. Symons R.H. Methods Enzymol. 1990; 181: 583-607Crossref PubMed Scopus (21) Google Scholar), the differences were even smaller with the extent of self-cleavage varying between 65 and 70% (data not shown). Although these experiments seemed to suggest that the extra A10 does not play a major role in the catalytic efficiency of the CChMVd (+) hammerhead structure, the extent of self-cleavage measured using full-length CChMVd (+) transcripts and only one reaction time was probably too rough an estimate for this aim. Therefore, we decided to re-examine this question using a more accurate approach. Since the observed self-cleavage could be influenced by either vector or viroid sequences external to the hammerhead structure, or even by the cloning site of the viroid cDNA and by some of the components present in the in vitro transcription reaction, a kinetic analysis under protein-free conditions of the minimal CChMVd (+) hammerhead structure in the well known I/III trans format (35Stage-Zimmermann T.K. Uhlenbeck O.C. RNA. 1998; 4: 875-889Crossref PubMed Scopus (184) Google Scholar) (Fig.4), was performed. The substrate was the same in all cases, whereas the ribozyme contained the wild-type sequence of CChMVd (+) hammerhead structure and the mutants A10→Δ, A10→C, A10→G, and A10→U. The cleavage rate constants for these ham
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