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Critical Aspartic Acid Residues in Pseudouridine Synthases

1999; Elsevier BV; Volume: 274; Issue: 32 Linguagem: Inglês

10.1074/jbc.274.32.22225

1083-351X

Vidhyashankar Ramamurthy, Steven L. Swann, Jennifer Paulson, Christopher J. Spedaliere, Eugene G. Mueller,

DNA Repair Mechanisms

The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine at particular positions in certain RNA molecules. Genomic data base searches and sequence alignments using the first four identified pseudouridine synthases led Koonin (Koonin, E. V. (1996) Nucleic Acids Res. 24, 2411–2415) and, independently, Santi and co-workers (Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996)Nucleic Acids Res. 24, 3756–3762) to group this class of enzyme into four families, which display no statistically significant global sequence similarity to each other. Upon further scrutiny (Huang, H. L., Pookanjanatavip, M., Gu, X. G., and Santi, D. V. (1998)Biochemistry37, 344–351), the Santi group discovered that a single aspartic acid residue is the only amino acid present in all of the aligned sequences; they then demonstrated that this aspartic acid residue is catalytically essential in one pseudouridine synthase. To test the functional significance of the sequence alignments in light of the global dissimilarity between the pseudouridine synthase families, we changed the aspartic acid residue in representatives of two additional families to both alanine and cysteine: the mutant enzymes are catalytically inactive but retain the ability to bind tRNA substrate. We have also verified that the mutant enzymes do not release uracil from the substrate at a rate significant relative to turnover by the wild-type pseudouridine synthases. Our results clearly show that the aligned aspartic acid residue is critical for the catalytic activity of pseudouridine synthases from two additional families of these enzymes, supporting the predictive power of the sequence alignments and suggesting that the sequence motif containing the aligned aspartic acid residue might be a prerequisite for pseudouridine synthase function. The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine at particular positions in certain RNA molecules. Genomic data base searches and sequence alignments using the first four identified pseudouridine synthases led Koonin (Koonin, E. V. (1996) Nucleic Acids Res. 24, 2411–2415) and, independently, Santi and co-workers (Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996)Nucleic Acids Res. 24, 3756–3762) to group this class of enzyme into four families, which display no statistically significant global sequence similarity to each other. Upon further scrutiny (Huang, H. L., Pookanjanatavip, M., Gu, X. G., and Santi, D. V. (1998)Biochemistry37, 344–351), the Santi group discovered that a single aspartic acid residue is the only amino acid present in all of the aligned sequences; they then demonstrated that this aspartic acid residue is catalytically essential in one pseudouridine synthase. To test the functional significance of the sequence alignments in light of the global dissimilarity between the pseudouridine synthase families, we changed the aspartic acid residue in representatives of two additional families to both alanine and cysteine: the mutant enzymes are catalytically inactive but retain the ability to bind tRNA substrate. We have also verified that the mutant enzymes do not release uracil from the substrate at a rate significant relative to turnover by the wild-type pseudouridine synthases. Our results clearly show that the aligned aspartic acid residue is critical for the catalytic activity of pseudouridine synthases from two additional families of these enzymes, supporting the predictive power of the sequence alignments and suggesting that the sequence motif containing the aligned aspartic acid residue might be a prerequisite for pseudouridine synthase function. All organisms chemically modify their RNA after transcription, and the isomerization of uridine to its C-glycoside isomer pseudouridine (Ψ) 1The abbreviations used are: Ψ, pseudouridine; [3H]tRNA, E. coli tRNAPhetranscript containing [5-3H]uridine is the most prevalent modification, Fig. 1 (1Grosjean H. Benne R. Modification and Editing of RNA. ASM Press, Washington, D. C.1998Crossref Google Scholar). This isomerization is catalyzed by the pseudouridine synthases, enzymes that display specificity for U residues at particular positions in certain RNA molecules, a specificity that can range from handling a single specific site to mild promiscuity (2Nurse K. Wrzesinski J. Bakin A. Lane B.G. Ofengand J. RNA. 1995; 1: 102-112PubMed Google Scholar, 3Wrzesinski J. Nurse K. Bakin A. Lane B.G. Ofengand J. RNA. 1995; 1: 437-448PubMed Google Scholar, 4Wrzesinski J. Bakin A. Nurse K. Lane B.G. Ofengand J. Biochemistry. 1995; 34: 8904-8913Crossref PubMed Scopus (85) Google Scholar, 5Simos G. Tekotte H. Grosjean H. Segref A. Sharma K. Tollervey D. Hurt E.C. EMBO J. 1996; 15: 2270-2284Crossref PubMed Scopus (155) Google Scholar, 6Raychaudhuri S. Conrad J. Hall B.G. Ofengand J. RNA. 1998; 4: 1407-1417Crossref PubMed Scopus (112) Google Scholar, 7Huang L.X. Ku J. Pookanjanatavip M. Gu X.R. Wang D. Greene P.J. Santi D.V. Biochemistry. 1998; 37: 15951-15957Crossref PubMed Scopus (52) Google Scholar). Physiological ramifications resulting from the lack of Ψ at particular locations have recently become evident, mandating a fuller understanding of Ψ generation in particular and RNA modification generally. In Escherichia coli, severe growth inhibition results from disruption of rluD (formerly denoted sfhB oryfiI), which encodes the Ψ synthase responsible for isomerization of U residues at positions 1911, 1915, and 1917 of 23 S rRNA (6Raychaudhuri S. Conrad J. Hall B.G. Ofengand J. RNA. 1998; 4: 1407-1417Crossref PubMed Scopus (112) Google Scholar, 7Huang L.X. Ku J. Pookanjanatavip M. Gu X.R. Wang D. Greene P.J. Santi D.V. Biochemistry. 1998; 37: 15951-15957Crossref PubMed Scopus (52) Google Scholar). In eukaryotes, Steitz and co-workers (8Yu Y.T. Shu M.D. Steitz J.A. EMBO J. 1998; 17: 5783-5795Crossref PubMed Scopus (209) Google Scholar) have elegantly demonstrated that the presence of Ψ in the U2 small nuclear RNA is required for proper assembly of the spliceosome, work that relied on the inhibition of the responsible Ψ synthase(s) by U2 transcripts containing 5-fluorouridine. Such inhibition of Ψ synthases by RNA containing 5-fluorouracil is well precedented (9Kammen H.O. Marvel C.C. Hardy L. Penhoet E.E. J. Biol. Chem. 1988; 263: 2255-2263Abstract Full Text PDF PubMed Google Scholar, 10Samuelsson T. Nucleic Acids Res. 1991; 19: 6139-6144Crossref PubMed Scopus (49) Google Scholar, 11Patton J.R. Biochem. J. 1993; 290: 595-600Crossref PubMed Scopus (29) Google Scholar), and this inhibition may account for a secondary mode of action of the anticancer drug 5-fluorouracil, which primarily acts by inhibiting thymidylate synthase (12Santi D.V. McHenry C.S. Sommer H. Biochemistry. 1974; 13: 471-481Crossref PubMed Scopus (582) Google Scholar). Consistent with 5-fluorouracil cytotoxicity resulting from Ψ synthase inhibition is a long string of observations concerning cell lines treated with both 5-fluorouracil and thymidine (eliminating the need for thymidylate synthase). This treatment affects many RNA-mediated events, including disruption of rRNA maturation (13Wilkinson D.S. Pitot H.C. J. Biol. Chem. 1973; 248: 63-68Abstract Full Text PDF PubMed Google Scholar, 14Ghoshal K. Jacob S.T. Cancer Res. 1994; 54: 632-636PubMed Google Scholar), disruption of pre-mRNA splicing (15Doong S.L. Dolnick B.J. J. Biol. Chem. 1988; 263: 4467-4473Abstract Full Text PDF PubMed Google Scholar, 16Sierakowska H. Shukla R.R. Dominski Z. Kole R. J. Biol. Chem. 1989; 264: 19185-19191Abstract Full Text PDF PubMed Google Scholar, 17Lenz H.J. Manno D.J. Danenberg K.D. Danenberg P.V. J. Biol. Chem. 1994; 269: 31962-31968Abstract Full Text PDF PubMed Google Scholar), and, perhaps, reducing translational accuracy (18Dolnick B.J. Pink J.J. J. Biol. Chem. 1985; 260: 3006-3014Abstract Full Text PDF PubMed Google Scholar). The link is thus established between Ψ synthases and critical RNA-mediated cellular processes, the disruption of which can lead to dire consequences. One such consequence is likely the X-linked human disease dyskeratosis congenita. Young men suffering from this disease have blotchy skin, poor dental health, sparse hair (including a lack of eyebrows), and evanescent nails; these men also tend to develop gastrointestinal tumors and suffer bone marrow failure (19Luzzatto L. Karadimitris A. Nat. Genet. 1998; 19: 6-7Crossref PubMed Scopus (56) Google Scholar). The gene responsible for dyskeratosis congenita has recently been identified and encodes a protein dubbed dyskerin, which contains a nuclear localization sequence and has two stretches of amino acids highly similar to the E. coli Ψ synthase TruB (20Heiss N.S. Knight S.W. Vulliamy T.J. Klauck S.M. Wiemann S. Mason P.J. Poustka A. Dokal I. Nat. Genet. 1998; 19: 32-38Crossref PubMed Scopus (751) Google Scholar). Although the detected similarity to TruB is in itself rather weak evidence for concluding that dyskerin is a Ψ synthase, the sequence alignments of the known Ψ synthases support that assessment (see below), and probing the functional significance of these alignments was the purpose of the experiments described in this communication. A brief presentation of the alignments is, therefore, imperative before recounting and discussing the experiments. After Penhoet and co-workers (9Kammen H.O. Marvel C.C. Hardy L. Penhoet E.E. J. Biol. Chem. 1988; 263: 2255-2263Abstract Full Text PDF PubMed Google Scholar) and Ofengand and co-workers (2Nurse K. Wrzesinski J. Bakin A. Lane B.G. Ofengand J. RNA. 1995; 1: 102-112PubMed Google Scholar, 3Wrzesinski J. Nurse K. Bakin A. Lane B.G. Ofengand J. RNA. 1995; 1: 437-448PubMed Google Scholar, 4Wrzesinski J. Bakin A. Nurse K. Lane B.G. Ofengand J. Biochemistry. 1995; 34: 8904-8913Crossref PubMed Scopus (85) Google Scholar) cloned the first four Ψ synthase genes, both Koonin (21Koonin E.V. Nucleic Acids Res. 1996; 24: 2411-2415Crossref PubMed Scopus (203) Google Scholar) and Santi and co-workers (22Gustafsson C. Reid R. Greene P.J. Santi D.V. Nucleic Acids Res. 1996; 24: 3756-3762Crossref PubMed Scopus (120) Google Scholar) undertook alignments and data base searches with these Ψ synthases. Both studies found statistically insignificant identity between the four proteins over their entire length, but both studies also determined that three short stretches of amino acids (5–13 residues) displayed significant similarity. Interestingly, each of the four Ψ synthases (RluA, RsuA, TruB, and TruA, which was formerly named either HisT or Ψ synthase I) had clear homologs in the data base, which led Koonin (21Koonin E.V. Nucleic Acids Res. 1996; 24: 2411-2415Crossref PubMed Scopus (203) Google Scholar) to conclude that each of the first four cloned Ψ synthases represented a different family of these enzymes. Indeed, similarity to the known Ψ synthases has been used to target unidentified open reading frames as Ψ synthases, and this strategy has led to the cloning of eight more Ψ synthases fromE. coli, yeast, mouse, and human (as a partial clone) (6Raychaudhuri S. Conrad J. Hall B.G. Ofengand J. RNA. 1998; 4: 1407-1417Crossref PubMed Scopus (112) Google Scholar, 7Huang L.X. Ku J. Pookanjanatavip M. Gu X.R. Wang D. Greene P.J. Santi D.V. Biochemistry. 1998; 37: 15951-15957Crossref PubMed Scopus (52) Google Scholar,23Chen J. Patton J.R. RNA. 1999; 5: 409-419Crossref PubMed Scopus (45) Google Scholar, 24Conrad J. Sun D.H. Englund N. Ofengand J. J. Biol. Chem. 1998; 273: 18562-18566Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 25Lecointe F. Simos G. Sauer A. Hurt E.C. Motorin Y. Grosjean H. J. Biol. Chem. 1998; 273: 1316-1323Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 26Becker H.F. Motorin Y. Planta R.J. Grosjean H. Nucleic Acids Res. 1997; 25: 4493-4499Crossref PubMed Google Scholar). The degree of relatedness among the four families, however, was rather ambiguous. In fact, as a result of the relatively low levels of similarity, each report of these alignments omitted at least one of the genes from the figure showing the alignments: Koonin (21Koonin E.V. Nucleic Acids Res. 1996; 24: 2411-2415Crossref PubMed Scopus (203) Google Scholar) excluded TruA, and Santi and co-workers (22Gustafsson C. Reid R. Greene P.J. Santi D.V. Nucleic Acids Res. 1996; 24: 3756-3762Crossref PubMed Scopus (120) Google Scholar) excluded both TruA and TruB. Reexamination of the sequence data by Santi and co-workers (27Huang L.X. Pookanjanatavip M. Gu X.G. Santi D.V. Biochemistry. 1998; 37: 344-351Crossref PubMed Scopus (132) Google Scholar) led to the insight that among all of the Ψ synthasesand their identified homologs, a single residue, an aspartic acid, is found aligned in all sequences. This aspartic acid residue in TruA (Asp-60) was mutated to alanine, asparagine, glutamate, serine, and lysine, and all of the mutant TruA proteins were catalytically inactive although still able to bind tRNA with near wild-type affinity (27Huang L.X. Pookanjanatavip M. Gu X.G. Santi D.V. Biochemistry. 1998; 37: 344-351Crossref PubMed Scopus (132) Google Scholar). The critical catalytic participation of the aligned aspartic acid residue strongly supports the functional significance of the aligned sequence motifs. Fig. 2 shows the region, which Koonin (21Koonin E.V. Nucleic Acids Res. 1996; 24: 2411-2415Crossref PubMed Scopus (203) Google Scholar) named motif II, containing the aligned aspartic acid residue, including the first four cloned Ψ synthases and dyskerin, which shares strong similarity with TruB in this region (20Heiss N.S. Knight S.W. Vulliamy T.J. Klauck S.M. Wiemann S. Mason P.J. Poustka A. Dokal I. Nat. Genet. 1998; 19: 32-38Crossref PubMed Scopus (751) Google Scholar). Interestingly, this motif was also identified in both deoxycytidine triphosphate deaminase (catalyzes dCTP → dUTP) and deoxyuridine triphosphatase (catalyzes dUTP → dUMP). Based on this observation, Koonin hypothesized that this stretch of amino acids was involved in uridine binding (21Koonin E.V. Nucleic Acids Res. 1996; 24: 2411-2415Crossref PubMed Scopus (203) Google Scholar), which appears to conflict with the ability of the TruA mutants to bind tRNA (27Huang L.X. Pookanjanatavip M. Gu X.G. Santi D.V. Biochemistry. 1998; 37: 344-351Crossref PubMed Scopus (132) Google Scholar). This seeming contradiction between prediction and experimental results and the weak similarity between the four families of Ψ synthases dictated further testing of the alignment generally and the "conserved" aspartic acid residue specifically. To this end, we undertook the mutation of the aligned aspartic acid residues in two other Ψ synthases of different families, RluA and TruB. Hen egg white lysozyme was purchased from Sigma. Activated charcoal (Norit SA-3) was purchased from Aldrich. Nucleoside triphosphates and competent JM109(DE3) E. coli cells were purchased from Promega Corp. (Madison, WI). The restriction enzyme BstNI was purchased from New England Biolabs (Beverly, MA). Isopropyl-β-d-thiogalactopyranoside, HEPES, and Tris were purchased from Roche Molecular Biochemicals. Oligonucleotides were purchased from the Great American Gene Company (Ramona, CA). QuikChange™ site-directed mutagenesis kits were purchased from Stratagene (La Jolla, CA). [5-3H]UTP was purchased from Amersham Pharmacia Biotech. Prime RNase inhibitor was purchased from 5 Prime → 3 Prime, Inc. (Boulder, CO). Ni-NTA superflow resin and RNA/DNA purification kits were purchased from Qiagen (Chatsworth, CA). Competent BLR(DE3) pLysS E. coli cells were purchased from Novagen (Madison, WI). Spectra/Por dialysis tubing (12,000–14,000 NMWCO) and all other chemicals were purchased from Fisher or its Acros Organics division (Pittsburgh, PA). A Robocylcer Gradient 96 thermal cycler (Stratagene) was used for the polymerase chain reaction component of the site-directed mutagenesis. DNA sequencing was performed in the University of Delaware Cell Biology Core Facility using a Long Readir 4200 DNA sequencer (Li-Cor, Inc., Lincoln, NE). The plasmid p67CF23, which contains the gene forE. coli tRNAPhe behind a T7 promoter, was a generous gift from O. Uhlenbeck (28Peterson E.T. Uhlenbeck O.C. Biochemistry. 1992; 31: 10380-10389Crossref PubMed Scopus (87) Google Scholar). Plasmids containing the genestruB (2Nurse K. Wrzesinski J. Bakin A. Lane B.G. Ofengand J. RNA. 1995; 1: 102-112PubMed Google Scholar) and rluA (3Wrzesinski J. Nurse K. Bakin A. Lane B.G. Ofengand J. RNA. 1995; 1: 437-448PubMed Google Scholar) in pET15b vectors were generously provided by J. Ofengand. Because the reports of these plasmids do not assign them names, they will be referred to as pΨ55 (containing truB) and pΨ746 (containing rluA). The plasmid pT7–911Q for expression of bacteriophage T7 RNA polymerase gene was a gift from T. Shrader, and the overexpressed T7 RNA polymerase was isolated as described (29Ichetovkin I.E. Abramochkin G. Shrader T.E. J. Biol. Chem. 1997; 272: 33009-33014Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). All of these overexpressed proteins have a His6 tag fused to their N terminus to simplify purification. Site-directed mutagenesis was performed using the QuikChangeTM protocol (Stratagene) according to the manufacturer's instructions except for the thermal cycling program. Because the Robocycler moves sample tubes between heating blocks maintained at different temperatures, additional time is required for the sample to achieve thermal equilibrium than with a single block thermal cycler (in which the sample temperature equilibrates as the block is adjusted to the new temperature). The final program for the mutagenesis polymerase chain reaction was as follows: 95 °C for 1 min; 16 cycles of 95 °C for 1 min, 55 °C for 1.5 min, and 68 °C for 15 min; and holding at 4 °C. The QuikChangeTM protocol uses complementary primers containing a mismatch to alter a codon by polymerase chain reaction amplification of the entire plasmid. For TruB mutants, pΨ55 was used; for RluA mutants, the plasmid template was pΨ746. The primers used for each mutation are presented here with the upper primer broken into codons and the altered codon (and its complement on the other primer) denoted in boldface type. TruB D48A, CC GGT GCG CTG GCC CCG CTG GCG ACC GGC and GCC GGT CGC CAG CGG GGC CAG CGC ACC GG; TruB D48C, CAT ACC GGT GCG CTG TGC CCG CTG GCG ACC GGC and GCC GGT CGC CAG CGG GCA CAG CGC ACC GGT ATG; RluA D64A, GAA TCG GTG CAT CGT CTG GCT ATG GCT ACC AGC GGC GTG and CAC GCC GCT GGT AGC CAT AGC CAG ACG ATG CAC CGA TTC; and RluA D64C, GAA TCG GTG CAT CGT CTG TGT ATG GCT ACC AGC GGC GTG and CAC GCC GCT GGT AGC CAT ACA CAG ACG ATG CAC CGA TTC. The success of the mutagenesis protocol was confirmed by sequencing the entire gene. Typically, two of three sequenced plasmids had the desired mutation and no other mutations. The mutant Ψ synthases and the names of the plasmids encoding them are as follows: TruB D48A, pBH305; TruB D48C, pBH301; RluA D64A, pBH206; and RluA D64C, pBH207. For expression of wild-type TruB and RluA, JM109(DE3) cells were transformed with pΨ55 or pΨ746, respectively, transformed cells were used to inoculate LB medium (500 ml), and the culture in a baffled flask was shaken vigorously at 37 °C. When the culture reachedA 600 nm = 0.6, isopropyl-β-d-thiogalactopyranoside (100 mm) was added (final concentration, 1 mm). After 3 h, the cells were harvested at 6000 × g for 20 min at 4 °C, quick frozen, and stored at −80 °C. For purification of the enzyme, the cell pellet was thawed and resuspended in 50 mm sodium phosphate buffer (10 ml), pH 8.0, containing NaCl (300 mm) and imidazole (3 mm). All subsequent steps were performed at 4 °C. Lysozyme (100 mg/ml; final concentration, 0.1 mg/ml) was added, and the mixture was stirred for 30 min to effect cell lysis. After sonication to reduce viscosity, the lysate was centrifuged (18,000 ×g for 30 min) to pellet cell debris. Ni-NTA superflow resin (2.5 ml) was added to the supernatant, and the mixture was gently stirred for 1.5 h to bind the His-tagged enzyme to the resin. After centrifugation (1100 × g for 3 min), the pelleted resin was resuspended in the same volume of buffer initially used for cell resuspension and packed into a column. The column was washed (three times, 7 ml each) with 50 mm sodium phosphate buffer, pH 8.0, containing NaCl (300 mm) and imidazole (20 mm). Bound enzyme was then eluted with a step (10 ml) of 50 mm sodium phosphate buffer, pH 8.0, containing NaCl (300 mm) and imidazole (500 mm). Eluted TruB was dialyzed overnight against 50 mm HEPES buffer, pH 7.5, containing ammonium chloride (100 mm) and EDTA (0.1 mm) and stored at 4 °C. Eluted RluA was dialyzed overnight against 20 mm triethanolamine·HCl buffer, pH 7.9, containing NaCl (0.35 m). This concentrated solution was either used within one day or was diluted to 0.24 mg/ml with buffer and then an equal volume of glycerol and stored at −20 °C. The overexpression and purification of the mutant Ψ synthases were identical to that of the wild-type enzyme except that the expression host was BLR(DE3) pLysS, which produces small amounts of T4 lysozyme so that a separate treatment with lysozyme was not required. The isolated enzymes were judged to be >98% pure by SDS-polyacrylamide gel electrophoresis with Coomassie Blue staining, and 15–40 mg of purified protein was obtained from 500 ml of culture. Substrate tRNAPhe was synthesized by in vitrotranscription catalyzed by T7 RNA polymerase withBstNI-linearized p67CF23 as template. The transcription reaction (1 ml) was carried out in 40 mm Tris·HCl buffer, pH 8.1, containing NTPs (4 mm each), MgCl2 (24 mm), spermidine (1 mm), dithiothreitol (5 mm), Triton X-100 (0.01%), Prime RNase Inhibitor (120 units), GMP (16 mm), and T7 RNA polymerase (0.1 mg/ml). After 9 h at 37 °C, protein was extracted into phenol:chloroform:isoamyl alcohol (25:24:1), and the aqueous phase was removed and washed with chloroform:isoamyl alcohol (24:1). RNA was precipitated from the removed aqueous phase by addition of 0.3m sodium acetate buffer, pH 5.2 (0.1 volume) and then ethanol (95%, 3 volumes). After incubation at −20 °C for at least 2 h, the precipitate was pelleted by centrifugation, and the supernatant was decanted. The pellet was dissolved in 10 mmTris·HCl buffer, pH 7.5, containing EDTA (1 mm), and the tRNA transcript was purified using the Qiagen RNA/DNA system according to the manufacturer's instructions. Preparation of tRNAPhecontaining [5-3H]uridine ([5-3H]tRNA) was accomplished by substituting [5-3H]UTP (0.1 mm, 1.0 Ci/mmol) for UTP (27Huang L.X. Pookanjanatavip M. Gu X.G. Santi D.V. Biochemistry. 1998; 37: 344-351Crossref PubMed Scopus (132) Google Scholar). This [5-3H]tRNA was typically diluted with unlabeled tRNA transcript to afford [5-3H]tRNA of suitable specific activity for the Ψ synthase assays. The assay for Ψ synthase activity was a slight modification of the tritium release assay reported by Nurse et al. (2Nurse K. Wrzesinski J. Bakin A. Lane B.G. Ofengand J. RNA. 1995; 1: 102-112PubMed Google Scholar), which measures the liberation of tritium from C5 when labeled U in RNA is isomerized to Ψ. A typical assay mixture (500 μl) was 50 mm HEPES buffer, pH 7.5, containing ammonium chloride (100 mm), dithiothreitol (5 mm), EDTA (1 mm), Prime RNase inhibitor (30 units), and [5-3H]tRNA (0.1–4.2 μm, 1.216 μCi/nmol tRNA). After incubation for 5 min at 37 °C, reaction was initiated by addition of a small volume (<5 μl) of a concentrated solution of a Ψ synthase (final concentration, 20 nm to 2 μm). Aliquots (95 μl) were removed periodically (30 s to 30 min) and quenched by dilution into 0.1 m HCl (1 ml) containing Norit-SA3 (12% w/v). Mixtures were centrifuged for 5 min at maximum speed in a microcentrifuge, and the supernatants were filtered through a plug of glass wool. The pellet was washed twice by resuspension in 0.1 m HCl (1 ml), followed by centrifugation. The supernatants from these washes were separately passed through the glass wool plug and combined with the original filtrate. An aliquot (1ml) of the combined filtrate was mixed with Scintisafe Econo 2 scintillation fluid (10 ml) and subjected to scintillation counting. A solution of tRNAPhe transcript containing [5-3H]uridine (0.54 μl, 5.55 μm tRNA, 16 Ci/mmol) was added to a solution of Ψ synthase (0–50 μm) in 50 mm HEPES buffer, pH 7.5, containing 100 mm NH4Cl (final volume, 300 μl; final [tRNA] = 10 nm). After 10 min at room temperature, an aliquot (95 μl) of each mixture was very slowly filtered through a 25 mm cellulose nitrate membrane filter (0.45 μm; Whatman, Maidstone, United Kingdom), which were prewetted with the HEPES buffer. The filter was rinsed rapidly with 25 mmpotassium phosphate buffer, pH 7.4 (two times, 1 ml each). After air-drying, the filter was put into a scintillation vial along with scintillation fluid (5 ml), shaken vigorously, and then counted in a liquid scintillation counter. Each protein concentration provided three filter binding data points, and the computer program GraphPad InPlot was used to plot the data and fit them to the binding curvee·tRNA = (e Σ)(e·tRNAmax)/(e Σ+ K d), where e Σ is the total enzyme concentration, e·tRNA is the concentration of tRNA bound to enzyme,e·tRNAmax is the maximum concentration of tRNA bound to enzyme, and K d is the dissociation constant for the enzyme-tRNA complex. The use of this simplified binding curve is allowed because the concentration of tRNA (10 nm) is 1% of the lowest enzyme concentration (1 μm), so that the free enzyme concentration essentially equals the total enzyme concentration. An assay mixture was prepared as described above except that the volume was doubled. A mutant Ψ synthase was added (final concentration, 100 nm), and after 30 min, half of the reaction mixture was quenched and processed as described above. To the other half of the reaction mixture, wild-type Ψ synthase was added (final concentration, 100 nm); after an additional 30 min of incubation, the reaction mixture was worked up as described above. The site-directed mutagenesis proceeded smoothly, affording the D48A and D48C mutant TruB and D64A and D64C mutant RluA. By virtue of an N-terminal His6 tag, all four mutant enzymes were purified to very near homogeneity by chromatography over a column of Ni-NTA resin. No major differences were noted in the yield, isolation, or storage of the mutant enzymes versus the wild-type enzymes. Nitrocellulose binding assays (30Arluison V. Hountondji C. Robert B. Grosjean H. Biochemistry. 1998; 37: 7268-7276Crossref PubMed Scopus (33) Google Scholar) were used to probe the ability of the wild-type and mutant Ψ synthases to bind substrate. A small concentration of [3H]tRNA was incubated with an increasing concentration of enzyme, and protein was adsorbed to nitrocellulose filters, which were subjected to liquid scintillation counting to quantitate bound [3H]tRNA. A representative binding curve is shown in Fig. 3. Binding of [3H]tRNA by enzyme plateaus at 70% of the tritium present, which corresponds well with the release of only 70% of the theoretical maximum when this batch of substrate was incubated with wild-type TruB. Although the presence of non-substrate tritiated RNA might initially seem disadvantageous, this contamination allowed the determination of the counting efficiency for tritium bound to the filter by quantitation of the tritium in the filtrate for a sample on the binding plateau. Substitution of bovine serum albumin for Ψ synthase resulted in no retention of [3H]tRNA on the filter (data not shown). As shown in Table I, the mutant Ψ synthases all bind tRNA substrate with measured K d< 2 μm. The higher K d of wild-type TruB versus mutants (Table I) likely reflects that the former is interacting with product (Ψ in place), whereas the latter interacts with substrate (U in place). The measured values are probably greater than the true K d values due to expected dissociation from the filter-bound enzyme at this binding affinity. Even with K d = 2 μm, under the standard assay conditions (500 nm [3H]tRNA, 50 nm enzyme), a significant part (20%) of the mutant Ψ synthases would be bound by substrate.Table IValues of Kd for [ 3H]tRNA determined by nitrocellulose binding assay (30Arluison V. Hountondji C. Robert B. Grosjean H. Biochemistry. 1998; 37: 7268-7276Crossref PubMed Scopus (33) Google Scholar)EnzymeK d for [3H]tRNAμmTruB wild-type8.8 ± 2.0TruB D48A1.6 ± 0.4TruB D48C1.3 ± 0.2RluA wild-type4.0 ± 1.1RluA D64A1.8 ± 0.9RluA D64C1.2 ± 0.5 Open table in a new tab The Ψ synthase activity of each mutant was assessed using the well established tritium release assay (2Nurse K. Wrzesinski J. Bakin A. Lane B.G. Ofengand J. RNA. 1995; 1: 102-112PubMed Google Scholar). As shown in Table IIand Fig. 4, the mutants did not catalyze the release of tritium from [3H]tRNA significantly above the background levels of the method. To reduce the lower limit of detectable catalytic activity, the amounts of mutant Ψ synthase included in the incubation mixture was increased past that of the wild-type enzyme in the comparator assay: the tritium released still did not vary significantly from the background of the method, even in one case in which the [3H]tRNA (500 nm) was saturated with 20 μm mutant enzyme (D48A TruB).Table IIActivity assays for wild-type and mutant TruB and RluAEnzymeActivityIdentityConcentration3H releasedPercentage of wild-typenmcpm%TruBBackground040 ± 4Wild-type201098 ± 20100D48A20,00038 ± 4<0.1D48C20,00052 ± 4<0.1RluABackground040 ± 4Wild-type20592 ± 6100D64A20038 ± 4<0.5D64C20036 ± 4<0.5Release of tritium from [3H]tRNA (500 nM, 76 Ci/mol U) was used to monitor the reaction. After a 5-min (TruB) or 10-min (RluA) incubation at 37 °C, the reaction was quenched by addition of charcoal in dilute acid.