Characterization of the Adenylation Site in the RNA 3′-Terminal Phosphate Cyclase from Escherichia coli

RNA 3′-terminal phosphate cyclases are a family of evolutionarily conserved enzymes that catalyze ATP-dependent conversion of the 3′-phosphate to the 2′,3′-cyclic phosphodiester at the end of RNA. The precise function of cyclases is not known, but they may be responsible for generating or regenerating cyclic phosphate RNA ends required by eukaryotic and prokaryotic RNA ligases. Previous work carried out with human andEscherichia coli enzymes demonstrated that the initial step of the cyclization reaction involves adenylation of the protein. The AMP group is then transferred to the 3′-phosphate in RNA, yielding an RNA-N3′pp5′A (N is any nucleoside) intermediate, which finally undergoes cyclization. In this work, by using different protease digestions and mass spectrometry, we assign the site of adenylation in the E. coli cyclase to His-309. This histidine is conserved in all members of the class I subfamily of cyclases identified by phylogenetic analysis. Replacement of His-309 with asparagine or alanine abrogates both enzyme-adenylate formation and cyclization of the 3′-terminal phosphate in a model RNA substrate. The cyclase is the only known protein undergoing adenylation on a histidine residue. Sequences flanking the adenylated histidine in cyclases do not resemble those found in other proteins modified by nucleotidylation. RNA 3′-terminal phosphate cyclases are a family of evolutionarily conserved enzymes that catalyze ATP-dependent conversion of the 3′-phosphate to the 2′,3′-cyclic phosphodiester at the end of RNA. The precise function of cyclases is not known, but they may be responsible for generating or regenerating cyclic phosphate RNA ends required by eukaryotic and prokaryotic RNA ligases. Previous work carried out with human andEscherichia coli enzymes demonstrated that the initial step of the cyclization reaction involves adenylation of the protein. The AMP group is then transferred to the 3′-phosphate in RNA, yielding an RNA-N3′pp5′A (N is any nucleoside) intermediate, which finally undergoes cyclization. In this work, by using different protease digestions and mass spectrometry, we assign the site of adenylation in the E. coli cyclase to His-309. This histidine is conserved in all members of the class I subfamily of cyclases identified by phylogenetic analysis. Replacement of His-309 with asparagine or alanine abrogates both enzyme-adenylate formation and cyclization of the 3′-terminal phosphate in a model RNA substrate. The cyclase is the only known protein undergoing adenylation on a histidine residue. Sequences flanking the adenylated histidine in cyclases do not resemble those found in other proteins modified by nucleotidylation. nucleoside dithiothreitol LC-ESIMS, high performance liquid chromatography interfaced with ESIMS tandem mass spectrometry nanoelectrospray ionization polyacrylamide gel electrophoresis carboxyamidomethyl The RNA 3′-terminal phosphate cyclase, an enzyme originally identified in extracts from human HeLa cells and Xenopusoocyte nuclei, catalyzes the ATP-dependent conversion of the 3′-terminal phosphate group into a 2′,3′-cyclic phosphodiester at the 3′-end of RNA (Refs. 1Filipowicz W. Konarska M. Gross H.J. Shatkin A.J. Nucleic Acids Res. 1983; 11: 1405-1418Crossref PubMed Scopus (69) Google Scholar, 2Filipowicz W. Strugala K. Konarska M. Shatkin A.J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1316-1320Crossref PubMed Scopus (38) Google Scholar, 3Reinberg D. Arenas J. Hurwitz J. J. Biol. Chem. 1985; 260: 6088-6097Abstract Full Text PDF PubMed Google Scholar; reviewed in Ref. 4Filipowicz W. Vicente O. Methods Enzymol. 1990; 181: 499-510Crossref PubMed Scopus (16) Google Scholar). The exact biological role of the cyclase in RNA metabolism remains unknown, but the demonstration that several eukaryotic and prokaryotic RNA ligases require 2′,3′-cylic phosphate RNA ends (Refs. 1Filipowicz W. Konarska M. Gross H.J. Shatkin A.J. Nucleic Acids Res. 1983; 11: 1405-1418Crossref PubMed Scopus (69) Google Scholar and 5Konarska M. Filipowicz W. Domdey H. Gross H.J. Nature. 1981; 293: 112-116Crossref PubMed Scopus (99) Google Scholar, 6Konarska M. Filipowicz W. Gross H.J. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1474-1478Crossref PubMed Scopus (74) Google Scholar, 7Filipowicz W. Shatkin A.J. Cell. 1983; 32: 547-557Abstract Full Text PDF PubMed Scopus (111) Google Scholar, 8Furneaux H. Pick L. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3933-3937Crossref PubMed Scopus (16) Google Scholar, 9Greer C.L. Peebles C.L. Gegenheimer P. Abelson J. Cell. 1983; 32: 537-546Abstract Full Text PDF PubMed Scopus (178) Google Scholar, 10Greer C.L. Javor B. Abelson J. Cell. 1983; 33: 899-906Abstract Full Text PDF PubMed Scopus (42) Google Scholar, 11Schwartz R.C. Greer C.L. Gegenheimer P. Abelson J. J. Biol. Chem. 1983; 258: 8374-8383Abstract Full Text PDF PubMed Google Scholar, 12Perkins K.K. Furneaux H. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 684-688Crossref PubMed Scopus (29) Google Scholar, 13Phizicky E.M. Schwartz R.C. Abelson J. J. Biol. Chem. 1986; 261: 2978-2986Abstract Full Text PDF PubMed Google Scholar, 14Pick L. Furneaux H. Hurwitz J. J. Biol. Chem. 1986; 261: 6694-6704Abstract Full Text PDF PubMed Google Scholar, 15Zillmann M. Gorovsky M.A. Phizicky E.M. Mol. Cell. Biol. 1991; 11: 5410-5416Crossref PubMed Scopus (79) Google Scholar, 16Arn E.A. Abelson J.N. J. Biol. Chem. 1996; 271: 31145-31153Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar; reviewed in Refs. 17Filipowicz W. Gross H.J. Trends Biochem. Sci. 1984; 9: 68-71Abstract Full Text PDF Scopus (12) Google Scholar, 18Phizicky E.M. Greer C. Trends Biochem. Sci. 1993; 18: 31-34Abstract Full Text PDF PubMed Scopus (6) Google Scholar, 19Arn E.A. Abelson J.N. Simons R.W. Grunberg-Manago M. RNA Structure and Function. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1998: 695-726Google Scholar) suggests that the enzyme may be involved in generation or maintenance of cyclic termini in RNA ligation substrates (4Filipowicz W. Vicente O. Methods Enzymol. 1990; 181: 499-510Crossref PubMed Scopus (16) Google Scholar, 20Genschik P. Billy E. Swianiewicz M. Filipowicz W. EMBO J. 1997; 16: 2955-2967Crossref PubMed Scopus (61) Google Scholar). Alternatively, the cyclase could be responsible for producing cyclic phosphate 3′-ends identified in the spliceosomal U6 small nuclear RNA (21Lund E. Dahlberg J.E. Science. 1992; 255: 327-330Crossref PubMed Scopus (110) Google Scholar) and some other small RNAs (Ref. 22Gu J. Shumyatsky G. Makan N. Reddy R. J. Biol. Chem. 1997; 272: 21989-21993Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar; for discussion of additional possible functions, see Ref. 20Genschik P. Billy E. Swianiewicz M. Filipowicz W. EMBO J. 1997; 16: 2955-2967Crossref PubMed Scopus (61) Google Scholar) The cyclase has been purified from HeLa cell extracts, and its cDNA had been cloned (20Genschik P. Billy E. Swianiewicz M. Filipowicz W. EMBO J. 1997; 16: 2955-2967Crossref PubMed Scopus (61) Google Scholar, 23Vicente O. Filipowicz W. Eur. J. Biochem. 1988; 176: 431-439Crossref PubMed Scopus (13) Google Scholar). The enzyme is expressed in all mammalian tissues and cell lines investigated, and has a nucleoplasmic localization, consistent with its postulated role in RNA processing (20Genschik P. Billy E. Swianiewicz M. Filipowicz W. EMBO J. 1997; 16: 2955-2967Crossref PubMed Scopus (61) Google Scholar). The cyclase has no apparent motifs in common with any proteins of known function. However, data base searches indicated that genes encoding proteins with a significant similarity to the human cyclase are conserved among eucarya, bacteria, and archaea. When the protein encoded in the Escherichia coli genome was overexpressed, it showed RNA 3′-phosphate cyclase activity. The E. colicyclase gene forms part of a previously uncharacterized operon, expression of which is controlled by an alternative sigma factor, ς54 (20Genschik P. Billy E. Swianiewicz M. Filipowicz W. EMBO J. 1997; 16: 2955-2967Crossref PubMed Scopus (61) Google Scholar, 24Genschik P. Drabikowski K. Filipowicz W. J. Biol. Chem. 1998; 273: 25516-25526Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The properties of the human and bacterial cyclases are very similar. Both enzymes catalyze conversion of the 3′-terminal phosphate to a 2′,3′-cyclic phosphodiester in a reaction dependent on ATP, other nucleoside triphosphates being much less active co-factors. With both enzymes, the cyclization of the 3′-phosphate at the 3′-end of RNA occurs by a three-step mechanism (2Filipowicz W. Strugala K. Konarska M. Shatkin A.J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1316-1320Crossref PubMed Scopus (38) Google Scholar, 3Reinberg D. Arenas J. Hurwitz J. J. Biol. Chem. 1985; 260: 6088-6097Abstract Full Text PDF PubMed Google Scholar, 4Filipowicz W. Vicente O. Methods Enzymol. 1990; 181: 499-510Crossref PubMed Scopus (16) Google Scholar, 20Genschik P. Billy E. Swianiewicz M. Filipowicz W. EMBO J. 1997; 16: 2955-2967Crossref PubMed Scopus (61) Google Scholar, 23Vicente O. Filipowicz W. Eur. J. Biochem. 1988; 176: 431-439Crossref PubMed Scopus (13) Google Scholar, 24Genschik P. Drabikowski K. Filipowicz W. J. Biol. Chem. 1998; 273: 25516-25526Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) as follows. (i) Enzyme + ATP → enzyme-AMP + PPi. (ii) RNA-N3′p + enzyme-AMP → RNA-N3′pp5′A + enzyme, where N1 is any nucleoside, and p is a phosphate group. (iii) RNA-N3′pp5′A → RNA-N>p + AMP, where N>p is nucleoside 2′,3′-cyclic phosphate. Evidence for step (i) comes from identification by either SDS-polyacrylamide gel electrophoresis (SDS-PAGE) or gel filtration of the covalent cyclase-AMP complex. Step (ii) is supported by the ability of 3′-phosphorylated RNA but not 3′-OH-terminated RNA to release AMP from the preformed adenylated cyclase complexes and by accumulation of the RNA-N3′pp5′A molecules when the ribose at the RNA 3′ terminus is replaced with the 2′-deoxy- or 2′-O-methylribose (2Filipowicz W. Strugala K. Konarska M. Shatkin A.J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1316-1320Crossref PubMed Scopus (38) Google Scholar, 3Reinberg D. Arenas J. Hurwitz J. J. Biol. Chem. 1985; 260: 6088-6097Abstract Full Text PDF PubMed Google Scholar, 20Genschik P. Billy E. Swianiewicz M. Filipowicz W. EMBO J. 1997; 16: 2955-2967Crossref PubMed Scopus (61) Google Scholar, 23Vicente O. Filipowicz W. Eur. J. Biochem. 1988; 176: 431-439Crossref PubMed Scopus (13) Google Scholar, 24Genschik P. Drabikowski K. Filipowicz W. J. Biol. Chem. 1998; 273: 25516-25526Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Step (iii) probably takes place nonenzymatically as the result of nucleophilic attack by the adjacent 2′-OH on the phosphorus in the phosphodiester linkage. Mechanistically, with respect to formation of the covalent protein-nucleoside monophosphate intermediate and transfer of nucleoside monophosphate to the terminal phosphate (or pyrophosphate) in nucleic acid, the cyclase resembles RNA and DNA ligases and capping enzymes (reviewed in Ref. 25Shuman S. Schwer B. Mol. Microbiol. 1995; 17: 405-410Crossref PubMed Scopus (190) Google Scholar). In all the later cases, nucleotidyl transfer occurs through a covalent lysyl-nucleoside monophosphate phosphoamide intermediate; the active-site lysine is present in a conserved short sequence motif, KXDG. RNA ligases, ATP-dependent DNA ligases, and capping enzymes also contain several additional conserved motifs (25Shuman S. Schwer B. Mol. Microbiol. 1995; 17: 405-410Crossref PubMed Scopus (190) Google Scholar). Neither KXDG nor these additional sequence motifs are identifiable in cyclases. In this work, we determined the adenylation site of the E. coli cyclase. The adenylated amino acid His-309 is conserved in a large subfamily of cyclases encompassing all bacterial and archaeal proteins and also some metazoan proteins. Mutations of His-309 in the E. coli cyclase abrogate formation of the AMP-cyclase intermediate and cyclization of the 3′-phosphate in RNA. The pET11d vector-based plasmid for overexpression of the wild-type E. coli cyclase containing a 6 × His tag at the C terminus has been previously described (20Genschik P. Billy E. Swianiewicz M. Filipowicz W. EMBO J. 1997; 16: 2955-2967Crossref PubMed Scopus (61) Google Scholar, 24Genschik P. Drabikowski K. Filipowicz W. J. Biol. Chem. 1998; 273: 25516-25526Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Plasmids for overexpression of mutant proteins were generated by a polymerase chain reaction approach as described previously (26Brøns-Poulsen J. Petersen N.E. Hørder M. Kristiansen K. Mol. Cell. Probes. 1998; 12: 345-348Crossref PubMed Scopus (61) Google Scholar). The identity of mutants was checked by DNA sequencing. Overexpression was performed in the E. coli strain BL21(DE3)pLysS as described before (20Genschik P. Billy E. Swianiewicz M. Filipowicz W. EMBO J. 1997; 16: 2955-2967Crossref PubMed Scopus (61) Google Scholar). For purification, the bacterial pellet was resuspended in buffer A (50 mmTris-HCl, pH 8.0, 0.3 m NaCl, 10 mm imidazole, 1 mm DTT) supplemented with 0.5% Triton X-100 and protease inhibitors (complete protease inhibitors-EDTA mixture, Roche Molecular Biochemicals). The pellet was lysed by sonication, and a lysate, cleared by centrifugation, was applied to a Ni+-silica gel column (Qiagen) pre-equilibrated with buffer A. The column was washed with buffer A containing 40 mm imidazole, and the cyclase protein was eluted with buffer A containing 0.4 mimidazole. Samples were desalted into 50 mm HEPES-NaOH, pH 7.8, 0.1 m NaCl, 0.5 mm DTT, 5% glycerol, concentrated using the UltraFree Biomax system (Millipore), and stored at −20 °C. The protein was more than 95% pure as judged by SDS-PAGE (20Genschik P. Billy E. Swianiewicz M. Filipowicz W. EMBO J. 1997; 16: 2955-2967Crossref PubMed Scopus (61) Google Scholar). Protein concentration was determined using the Bradford procedure with bovine serum albumin as a standard (27Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215589) Google Scholar). Cyclase activity was assayed by the Norit method as described elsewhere (4Filipowicz W. Vicente O. Methods Enzymol. 1990; 181: 499-510Crossref PubMed Scopus (16) Google Scholar). Unless otherwise indicated, the 10-μl assays contained 40 fmol of the substrate, and incubations were for 20 min at 25 °C. Other details are indicated in the figure legends. Reactions (15 μl) containing 20 ng of wild-type or mutant cyclase and 2.5 μm[α-32P]ATP (specific activity, 300 Ci/mmol) were incubated at 25 °C for 3 h in 50 mm HEPES-NaOH buffer, pH 8.0, containing 0.2 m NaCl, 10 mmMgCl2, 1 mm DTT, and 10% glycerol. The reactions were analyzed by SDS-PAGE and autoradiography. Immediately before loading onto the gel, samples were supplemented with unlabeled ATP (final concentration 10 mm) to decrease the background. Twenty-five μg (0.7 nmol) of cyclase was adenylated in 60 μl of buffer T (50 mm Tris-HCl, pH 8.0, 0.2m NaCl, 2 mm MgCl2, 1 mm CaCl2, 1 mm DTT) containing 20 μm [α-32P]ATP. After incubation for 3 h at 25 °C, the sample was divided into three equal aliquots, which were submitted to digestion with either trypsin, from or protease at °C or °C and A 10% of digestion reaction was analyzed by SDS-PAGE using a system described by and H. G. Anal. Biochem. PubMed Scopus Google Scholar) the samples in the of and were by and autoradiography. The of was in the electrophoresis and the were onto a After on the were and to sequence by using an model and the μg nmol) of cyclase was incubated in buffer T with 60 μm After incubation for 2 h at 25 °C, proteins were with mm for min in the adenylation The protein was then at °C for 1 h with μg of by a with μg of adenylated was with 0.2 μg of for min at 25 °C in 50 were on a 1 × column in A mm pH and mm pH and a was from to in 60 min at a of 50 The system was as described previously J. S. A. W. D. J. Mol. Biol. Cell. 1998; 9: PubMed Scopus Google Scholar). was performed to the method of and M. M. Anal. Chem. 1996; PubMed Scopus Google Scholar). The mass were on an 300 mass with a A model of the human cyclase was using the of the E. E. W. and A. submitted for The enzymes were using the The identity with was by the (Ref. A. J. Mol. Biol. 1993; PubMed Scopus Google Scholar; to and 6 human cyclase cyclase is than the E. coli was to model this part of the The on the of the functions, was using the (Ref. R. D. Nature. 1992; PubMed Scopus Google Scholar; was than that for a chain of this the of the A of the E. coli and human cyclases and proteins encoded in different indicated that they be into of class I all prokaryotic the and of the proteins expressed and The previously E. coli and human cyclases to this class of the class proteins encoded in of and and also forms of proteins expressed in and of the members of the cyclase class I subfamily is in 1 B. proteins and also class not no apparent motifs in common with other proteins in data The and of class I cyclases are conserved at the sequence with of proteins being The of similarity are to and of the E. the sequence of the human protein is more to the D. protein than to protein is possible that human and proteins are not and that additional cyclase genes are expressed in Previous demonstration that the covalent human cyclase-AMP is when in 0.1 or when with at pH but to in has that AMP is to the protein a phosphoamide the lysine group D. Arenas J. Hurwitz J. J. Biol. Chem. 1985; 260: 6088-6097Abstract Full Text PDF PubMed Google O. Filipowicz W. Eur. J. Biochem. 1988; 176: 431-439Crossref PubMed Scopus (13) Google Scholar). The E. coli cyclase-AMP is to with 1 min at but is in 0.1 min at consistent with the phosphoamide not from the in 1 no conserved lysine is present in class I cyclases. the protein containing the adenylation protease of the adenylated E. colicyclase was The protein was adenylated in the of and with either trypsin, or In this and other it was to to adenylation reaction to the adenylated by gel filtration in of as previously for the human enzyme D. Arenas J. Hurwitz J. J. Biol. Chem. 1985; 260: 6088-6097Abstract Full Text PDF PubMed Google Scholar, 23Vicente O. Filipowicz W. Eur. J. Biochem. 1988; 176: 431-439Crossref PubMed Scopus (13) Google the E. coli cyclase-AMP may in the of The from protease digestion of the cyclase-AMP were by by and analyzed by This demonstrated that the adenylated amino acid is and not see 1 the adenylated amino acid the enzyme adenylated in the of ATP and enzyme were with by The were by of of the adenylated and proteins indicated the in the of additional with a mass of not that it to + AMP The of this was by and of the cyclase before protease The were by LC-ESIMS, and a with a mass at the as the was mass be consistent with the + + AMP The identity of this was by This not the adenylated amino acid, the AMP was the and only were of the adenylated of 1 was performed in the liquid chromatography The of from the is and identified from the in the were in the The adenylated and was with yielding a of short that were analyzed by The mass and are in A. The generated were by which the amino acid sequence of the + + AMP and The with a mass of showed a of a to an residue. a only were the the originally the was The of the AMP was by of this in the were could be to AMP and AMP The active-site could in be adenylated on or and could also place on either S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). the AMP is present at be the in for the and in the tandem mass of the it be that is the that His-309 is not This was by of this which in not His-309 as the only possible site of This is by (i) The is the of the cyclase-AMP in and its to This is consistent with a than a A.J. The of B. Scholar). The acid of the also than a modified was in of the not (ii) the histidine is conserved in all class I the is only found in the E. coli enzyme 1 the of His-309 for activity of the E. coli cyclase, acid mutant enzymes were and of the of the that of His-309 by either or not the of the protein. proteins were for activity as in the adenylation reaction and for ability to catalyze cyclization of the 3′-phosphate in the model substrate, a Both of the His-309 adenylation of the protein and its activity to catalyze cyclization of the 3′-phosphate The mutant protein was also overexpressed, and less that the wild-type this mutant adenylation and cyclization of the 3′-phosphate not for His-309 as an In this work we the site of adenylation in the E. coli cyclase to His-309 by using protease digestions and mass with this histidine as an AMP its with asparagine or alanine both formation of the enzyme-AMP and the cyclization of the 3′-terminal phosphate in a model RNA substrate. on the of the E. coli the His-309 not with the of the this is to it into a or the of it is that these on enzyme activity by of the protein than its The human class I cyclase has identity and similarity with the E. coli of the human sequence using the of the E. coli enzyme showed an that is very not the human enzyme a histidine at which to His-309 of the E. coli as for a its in the model also to be conserved In the of and is these been found to with the histidine in the E. coli of proteins to the class subfamily not a conserved histidine in the or in this group of proteins. D. J. and W. remains to be class proteins cyclase activity. the of the E. coli cyclase is the only of a protein adenylated on a histidine and using ATP as a However, three other proteins are known to of a histidine with nucleotidyl other then an enzyme involved in the for is at the of the of a histidine is a group in this reaction Methods Enzymol. 1982; PubMed Scopus (32) Google Scholar, J.E. 1996; PubMed Scopus Google Scholar). A is the protein of the S. RNA of this protein a covalent with is a nucleophilic attack by the on the phosphate of the in The of by the protein in of A. N. Mol. Cell. Biol. PubMed Scopus Google Scholar). and Biochem. PubMed Scopus (12) Google Scholar) that the the enzyme responsible for of forms a reaction intermediate of a histidine residue. only proteins are known to be on a transfer reactions a are more common and found in proteins that are members of in both and H. M. 1997; PubMed Scopus Google Scholar). acid flanking the adenylated His-309 in the E. coli cyclase and in other members of the class I family of cyclases do not resemble found in other proteins that on histidine or other amino acid For the RNA other RNA and DNA ligases, forms a covalent intermediate and AMP to the phosphate in nucleic acid to the ligation intermediate (reviewed in Ref. 19Arn E.A. Abelson J.N. Simons R.W. Grunberg-Manago M. RNA Structure and Function. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1998: 695-726Google Scholar). in the of the substrate, RNA transfer AMP to 3′-terminal in formation and 3′-phosphate a mechanism probably very to that of the RNA cyclase R.W. Nucleic Acids Res. Scholar, Nucleic Acids Res. 1982; PubMed Scopus Google Scholar). However, in RNA and DNA ligases, the nucleotidyl transfer occurs to the lysine in a conserved sequence motif, this is not present in cyclase class I or class previously in the of the 3′-phosphorylated the human cyclase has a to the phosphate in RNA. of formation was when a large of the enzyme was In no of or ligation of either or 3′-phosphorylated was (20Genschik P. Billy E. Swianiewicz M. Filipowicz W. EMBO J. 1997; 16: 2955-2967Crossref PubMed Scopus (61) Google Scholar). these data that RNA ligases and RNA 3′-phosphate cyclases are very for and for the cyclase to and for The for and The for sequence data to