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• W2037813174 abstract "The Escherichia coli generluA, coding for the pseudouridine synthase RluA that forms 23 S rRNA pseudouridine 746 and tRNA pseudouridine 32, was deleted in strains MG1655 and BL21/DE3. The rluA deletion mutant failed to form either 23 S RNA pseudouridine 746 or tRNA pseudouridine 32. Replacement of rluA in trans on a rescue plasmid restored both pseudouridines. Therefore, RluA is the sole protein responsible for the in vivo formation of 23 S RNA pseudouridine 746 and tRNA pseudouridine 32. Plasmid rescue of bothrluA − strains using an rluA gene carrying asparagine or threonine replacements for the highly conserved aspartate 64 demonstrated that neither mutant could form 23 S RNA pseudouridine 746 or tRNA pseudouridine 32 in vivo, showing that this conserved aspartate is essential for enzyme-catalyzed formation of both pseudouridines. In vitro assays using overexpressed wild-type and mutant synthases confirmed that only the wild-type protein was active despite the overexpression of wild-type and mutant synthases in approximately equal amounts. There was no difference in exponential growth rate between wild-type and MG1655(rluA −) either in rich or minimal medium at 24, 37, or 42 °C, but when both strains were grown together, a strong selection against the deletion strain was observed. The Escherichia coli generluA, coding for the pseudouridine synthase RluA that forms 23 S rRNA pseudouridine 746 and tRNA pseudouridine 32, was deleted in strains MG1655 and BL21/DE3. The rluA deletion mutant failed to form either 23 S RNA pseudouridine 746 or tRNA pseudouridine 32. Replacement of rluA in trans on a rescue plasmid restored both pseudouridines. Therefore, RluA is the sole protein responsible for the in vivo formation of 23 S RNA pseudouridine 746 and tRNA pseudouridine 32. Plasmid rescue of bothrluA − strains using an rluA gene carrying asparagine or threonine replacements for the highly conserved aspartate 64 demonstrated that neither mutant could form 23 S RNA pseudouridine 746 or tRNA pseudouridine 32 in vivo, showing that this conserved aspartate is essential for enzyme-catalyzed formation of both pseudouridines. In vitro assays using overexpressed wild-type and mutant synthases confirmed that only the wild-type protein was active despite the overexpression of wild-type and mutant synthases in approximately equal amounts. There was no difference in exponential growth rate between wild-type and MG1655(rluA −) either in rich or minimal medium at 24, 37, or 42 °C, but when both strains were grown together, a strong selection against the deletion strain was observed. pseudouridine polymerase chain reaction M9 (Ref. 22Zyskind J.W. Bernstein S.I. Recombinant DNA Laboratory Manual. Academic Press, San Diego, CA1992: 187Crossref Google Scholar) plus 0.4% glucose and 1 mmMgSO4 N-cyclohexyl-N′-β-(4-methylmorpholinium)ethylcarbodiimide Ribosomal RNA, considered to be the functional heart of the ribosome (1Green R. Noller H.F. Annu. Rev. Biochem. 1997; 66: 679-716Crossref PubMed Scopus (424) Google Scholar), contains a variety of modified nucleosides of unknown function (2McClosky J.A. Crain P.F. Nucleic Acids Res. 1998; 26: 196-197Crossref PubMed Scopus (70) Google Scholar). The most common single modification is the conversion of uridine to pseudouridine (Ψ),1 the 5-ribosyl isomer of uridine (3Cohn W. J. Biol. Chem. 1960; 235: 1488-1498Abstract Full Text PDF PubMed Google Scholar). Ψ is formed by isomerization of specific uridines after the RNA chain is formed. The mechanism of the reaction, which involves breaking of the N1-glycosyl bond, rotation of the uracil ring, and formation of a C5-glycosyl bond is unknown but is thought to involve an active site carboxyl group of an aspartate residue (4Huang L. Pookanjanatavip M. Gu X. Santi D.V. Biochemistry. 1998; 37: 344-351Crossref PubMed Scopus (132) Google Scholar). Ψ is found in the rRNA of all organisms so far examined (5Ofengand J. Fournier M. Grosjean H. Benne R. Modification and Editing of RNA. ASM Press, Washington, D. C.1998: 229-253Google Scholar), and in Escherichia coli, which has one Ψ in the 16 S RNA (6Bakin A. Kowalak J.A. McCloskey J.A. Ofengand J. Nucleic Acids Res. 1994; 22: 3681-3684Crossref PubMed Scopus (51) Google Scholar) and nine in the 23 S RNA (7Bakin A. Ofengand J. Biochemistry. 1993; 32: 9754-9762Crossref PubMed Scopus (264) Google Scholar, 8Bakin A. Lane B.G. Ofengand J. Biochemistry. 1994; 33: 13475-13483Crossref PubMed Scopus (79) Google Scholar), it is the most prevalent of the modified nucleosides. The single Ψ in the 16 S RNA is found adjacent to the “530” loop, whose sequence has been almost completely conserved in all organisms and is known to be involved in the fidelity of codon recognition (reviewed in Refs. 9Ofengand J. Bakin A. Nurse K. Nierhaus K.H. Subramanian A.R. Erdmann V.A. Franceschi F. Wittman-Liebold B. The Translational Apparatus. Plenum Press, New York1993: 489-500Crossref Google Scholar and 10Santer M. Santer U. Nurse K. Bakin A. Cunningham P. Zain M. O'Connell D. Ofengand J. Biochemistry. 1993; 32: 5539-5547Crossref PubMed Scopus (25) Google Scholar). In 23 S RNA, the nine Ψ residues are distributed among three distinct areas, which, despite their separation in two-dimensional secondary structure representations, are at or near the peptidyl transferase center when in the ribosome (11Ofengand J. Bakin A. J. Mol. Biol. 1997; 266: 246-268Crossref PubMed Scopus (178) Google Scholar). However, despite the congruence of the Ψ residues with the two functional centers of the ribosome, namely decoding and peptide bond formation, there is so far no known role for Ψ in the process of protein synthesis. To approach this problem, we have initiated a program to identify the genes for the specific synthases that make the 10 Ψ in E. coli rRNA on the assumption, subsequently shown to be correct, that distinct synthases are used to form Ψ at the different sites in the rRNA molecule. Once identified, gene inactivation will result in the loss of a specific synthase and should therefore cause the loss of specific Ψ residues for which the effect on cell physiology can then be assessed. Thus far, three Ψ synthase genes have been inactivated in this manner. One, rluC, codes for a synthase solely responsible for formation of Ψ residues 955, 2504, and 2580 in 23 S RNA (12Conrad J. Sun D. Englund N. Ofengand J. J. Biol. Chem. 1998; 273: 18562-18566Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and a second, rluD, codes for the synthase that makes 23 S RNA Ψ1911, 1915, and 1917 (13Raychaudhuri S. Conrad J. Hall B.G. Ofengand J. RNA. 1998; 4: 1407-1417Crossref PubMed Scopus (112) Google Scholar). The third,rsuA, codes for the synthase that forms 16 S RNA Ψ516 (14Conrad J. Niu L. Rudd K. Ofengand J. RNA (N. Y.). 1999; 5: 751-763Crossref PubMed Scopus (65) Google Scholar). Inactivation of rluC and rsuA and the consequent loss of their respective Ψ had no physiological effect. However, disruption of rluD with the loss of its three Ψ severely inhibited cell growth. Another synthase, RluA, was shown to form only Ψ746 when in vitro transcripts of 23 S RNA were the substrate. The synthase also specifically catalyzed the formation of Ψ32 in E. coli tRNAPhe (15Wrzesinski J. Nurse K. Bakin A. Lane B.G. Ofengand J. RNA (N. Y.). 1995; 1: 437-448PubMed Google Scholar). The ability to be highly specific for a single site in more than one class of RNA, a property termed “dual specificity” (15Wrzesinski J. Nurse K. Bakin A. Lane B.G. Ofengand J. RNA (N. Y.). 1995; 1: 437-448PubMed Google Scholar), has since been reported for another Ψ synthase (16Massenet S. Motorin Y. Lafontaine D.L.J. Hurt E.C. Grosjean H. Branlant C. Mol. Cell. Biol. 1999; 19: 2142-2154Crossref PubMed Scopus (123) Google Scholar) as well as for a ribose methylating enzyme, although in the latter case the dual specificity resides in the guide RNA (17Tycowski K.T. You Z.-H. Graham P.J. Steitz J.A. Mol. Cell. 1998; 2: 629-638Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). A question left open by the work on RluA was whether it is the only synthase in E. coli capable of forming rRNA Ψ746 and tRNA Ψ32 and what the effect of its absence would be on the cell. This issue has now been addressed by deleting rluA and comparing the growth rate with wild type both separately and in a competition experiment. In addition, by mutating aspartate 64, which was predicted to be an essential residue by virtue of its location in a conserved sequence, HRLD (4Huang L. Pookanjanatavip M. Gu X. Santi D.V. Biochemistry. 1998; 37: 344-351Crossref PubMed Scopus (132) Google Scholar), we have shown that RluA, as well as RsuA (14Conrad J. Niu L. Rudd K. Ofengand J. RNA (N. Y.). 1999; 5: 751-763Crossref PubMed Scopus (65) Google Scholar), RluD, 2S. Raychaudhuri and J. Ofengand, unpublished results. and TruA (4Huang L. Pookanjanatavip M. Gu X. Santi D.V. Biochemistry. 1998; 37: 344-351Crossref PubMed Scopus (132) Google Scholar), requires this residue for function. TherluA gene was deleted by the method of Hamilton et al. (18Hamilton C.M. Aldea H. Washburn B.K. Babitzke P. Kushner S.R. J. Bacteriol. 1989; 171: 4617-4622Crossref PubMed Google Scholar). The insert, cloned into the XbaI andKpnI sites of pMAK705, was prepared by PCR as described by Nelson and colleagues (Fig. 2 in Ref. 19Supekova L. Supek F. Nelson N. J. Biol. Chem. 1995; 270: 13726-13732Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). It contained 818 bases 5′ to the AUG start and 785 bases 3′ to the UAA termination codon. Sixteen bases of the N-terminal portion of the gene and 52 bases of the C terminus were retained with the remainder being replaced by the kanamycin resistance gene, obtained by PCR amplification from pUC4K (Amersham Pharmacia Biotech, catalog no. 27-4958-01). The host strain for pMAK705 was the leucine auxotroph MC1061, as described by Hamiltonet al. (18Hamilton C.M. Aldea H. Washburn B.K. Babitzke P. Kushner S.R. J. Bacteriol. 1989; 171: 4617-4622Crossref PubMed Google Scholar). The deleted rluA gene was moved into strains MG1655 (Ref. 20Blattner F.R. Plunkett III, G. Bloch C.A. Perna N.T. Burland V. Riley M. Collado-Vides J. Glasner J.D. Rode C.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick H.A. Goeden M.A. Rose D.J. Mau B. Shao Y. Science. 1997; 277: 1453-1474Crossref PubMed Scopus (6024) Google Scholar; the gift of Dr. Kenneth Rudd, this department) and BL21/DE3 (Novagen, Inc.) by bacteriophage P1 transduction (21Miller J.H. A Short Course in Bacterial Genetics : A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1992: 357-364Google Scholar). Selection was done on either rich (LB, Ref. 22Zyskind J.W. Bernstein S.I. Recombinant DNA Laboratory Manual. Academic Press, San Diego, CA1992: 187Crossref Google Scholar) or minimal (M9+) medium containing 0.05 mg/ml kanamycin. ThemiaA deletion was moved from strain NU426 carrying themiaA::cat insertion (the kind gift of Malcolm Winkler, University of Texas, Houston Medical School) by P1 transduction into the MG1655 and MG1655(rluA −) strains with selection on LB containing 34 mg/ml chloramphenicol in addition to kanamycin. The preparation of wild-type rescue plasmid pET15b/rluA has been described previously (15Wrzesinski J. Nurse K. Bakin A. Lane B.G. Ofengand J. RNA (N. Y.). 1995; 1: 437-448PubMed Google Scholar). Wild-type rescue plasmid pTrc99A/rluA was constructed by insertion into the NcoI and HindIII sites of pTrc99A (Amersham Pharmacia Biotech, catalog no. 275007-01) of a segment of DNA that was PCR-amplified from pET15b/rluA and consisting of the rluA gene starting from the initiator AUG and ending at the terminator UAA. The N-terminal primer had anNcoI site adjacent to the initiating AUG, whereas in the reverse orientation, the C-terminal primer incorporated aHindIII site after the terminator UAA. These plasmids were prepared by the megaprimer PCR mutagenesis procedure (23Picard V. Ersdal-Badju E. Lu A. Bock S.C. Nucleic Acids Res. 1994; 22: 2587-2591Crossref PubMed Scopus (216) Google Scholar). PCR reactions were performed using the pTrc99A/rluA rescue plasmid as template and three oligonucleotide primers, two outer primers, which were upstream and downstream of the mutation site, and one mutagenic primer. The upstream and downstream primers contained the restriction sites NcoI and HindIII, respectively, so that the product could be ligated directly into pTrc99A. Mutagenesis was carried out in three steps. The initial PCR reaction was performed with either mutagenic primer 5′-CATCGTCTGACTATGGCTACC-3′ for the D64T mutation or 5′-CATCGTCTGAATATGGCTACC-3′ for the D64N mutation (mutation sites shown in bold) and with the downstream primer 5′-GGGAAGCTTTTAAAAATCCGCTGGCGC-3′ having aHindIII site (underlined). A 100-μl reaction contained 50 ng of template plasmid DNA, 15 pmol each of the mutagenic primer and downstream primer, 3 units of Pfu DNA polymerase (Promega), 0.2 mm dNTPs, 20 mm Tris, pH 8.75, 10 mm KCl, 10 mm(NH4)2SO4, 2 mmMgCl2, 0.1% Triton X-100, and 0.1 mg/ml bovine serum albumin. The mixture was denatured at 95 °C for 60 s, and then 10 cycles of amplification (95 °C, 30 s; 47 °C, 60 s; 72 °C, 70 s) were performed, followed by a 5-min extension at 72 °C. Fifty pmol of the upstream primer was added (5′-GGGGCCATGGATGGGGATGGAAAACTAC-3′,NcoI site underlined), and the reaction mixture was subjected to the same amplification program. Finally, 50 pmol of downstream primer was added, and the sample was subjected to the same amplification program again. The amplified product was purified by gel electrophoresis, digested with NcoI and HindIII, and ligated with similarly digested and purified pTrc99A for 16 h at 16 °C. The ligation mixture was transformed into Novablue cells (Novagen, Inc.) by standard methods yielding 4 positive clones of 5 tested for D64T and 3 positive of 4 tested for D64N. DNA sequencing of the isolated plasmids verified that the expected mutation had been produced at the desired site. Transfer of the mutant rluAgenes into pET15b was done by PCR amplification of the mutantrluA genes in pTrc99A. The N-terminal primer extended from −9 to +18, where the A of the initiating AUG of rluA is +1 with changes at −2 to −5, to create an XhoI site adjacent to the initiating AUG. The C-terminal primer, in the reverse orientation, extended from +643 to +669, where the last sense nucleotide is 657, and contained mismatches at +661 to +666 to create aBamHI site. The amplified product was purified, digested with XhoI and BamHI, and subsequently ligated with identically treated pET15b vector for 16 h at 16 °C. DNA sequence analysis verified the constructs (data not shown). pET15b/rluA-D64T and pET15b/rluA-D64N were transformed into Novablue cells (Novagen, Inc.) by standard methods and yielded 7 positive clones of 9 tested (D64T) or 5 of 7 (D64N). They were transferred into BL21/DE3(rluA −) cells by standard methods. Overexpression and affinity purification were carried out as described previously (13Raychaudhuri S. Conrad J. Hall B.G. Ofengand J. RNA. 1998; 4: 1407-1417Crossref PubMed Scopus (112) Google Scholar). pET15b/rluA was processed in the same way. 5-[3H]Uridine-labeled transcripts of full-length 23 S RNA (specific activity 168 dpm/pmol uridine residues) were prepared as described previously (13Raychaudhuri S. Conrad J. Hall B.G. Ofengand J. RNA. 1998; 4: 1407-1417Crossref PubMed Scopus (112) Google Scholar). The 5-[3H]uridine-labeled transcripts of tRNAPhe(199 dpm/pmol), tRNACys (1269 dpm/pmol), and tRNA4Leu (454 dpm/pmol) were prepared as described for tRNAPhe (15Wrzesinski J. Nurse K. Bakin A. Lane B.G. Ofengand J. RNA (N. Y.). 1995; 1: 437-448PubMed Google Scholar) but using pTFMa-ECys (the gift of Ya-Ming Hou, Thomas Jefferson University, Philadelphia, PA) as template for tRNACys and pUC19/tRNA4Leu(UAA) for tRNA4Leu. Ribosomal RNA for Ψ sequencing was prepared according to King and Schlessinger (24King T.C. Schlessinger D. J. Biol. Chem. 1983; 258: 12034-12042Abstract Full Text PDF PubMed Google Scholar) with omission of the LiCl precipitation step. Ψ sequencing of rRNA was performed as described previously (7Bakin A. Ofengand J. Biochemistry. 1993; 32: 9754-9762Crossref PubMed Scopus (264) Google Scholar, 25Bakin A. Ofengand J. Martin R. Methods in Molecular Biology: Protein Synthesis: Methods and Protocols. 77. Humana Press, Inc., Totowa, NJ1998: 297-309Google Scholar). tRNA for Ψ sequencing was isolated as described (26Deutscher M.P. Hilderman R.H. J. Bacteriol. 1974; 118: 621-627Crossref PubMed Google Scholar) from cells grown to anA 550 of 1.0 in LB medium. Ψ sequencing of tRNACys was done exactly as for rRNA using a primer complementary to residues 61–76. For the individual exponential phase growth experiments, overnight cultures at 37 °C in the medium to be tested were diluted 50-fold (minimal medium) or 100-fold (rich medium) and placed at the testing temperature. Cell density was monitored at 600 nm. Viable cells in the mixed competition experiment were determined by plating on LB, in which both wild-type and mutant grow, and on LB plus kanamycin, in which only rluA −grows. For the 1:103 dilution series (Fig. 8), the first four cycles were analyzed by first plating aliquots on LB and then direct transfer of individual colonies to LB plus kanamycin (patch analysis). 100 colonies originating from each of four individual flasks (400 colonies total) were analyzed per time point. For the last two cycles, direct plating of three aliquots per each of four flasks to LB with and without kanamycin was used for a total of 12 platings per medium per time point. For the 1:1.6 × 106 dilution series, direct plating of two aliquots per each of three flasks (six platings per time point) to LB with and without kanamycin was used for the rluA − cells. Patch analysis using 100 colonies from each of two flasks (200 colonies total) was used for thersuA − and rluC − cells. The fraction of rluA − in the mixture is the number of colonies on LB plus kanamycin divided by the number on LB alone. The number of cell doublings (G) is calculated from the dilution factor (DF) at each cycle using the relation DF =N/N o = 2 G. Thus, to reach the same cell density after a 1:103 dilution requires 9.97 cell doublings, and the 1.6 × 106 dilution corresponds to 20.6 cell doublings. Transformants of wild-type, MG1655(rluA −), and MG1655(miaA − rluA −) strains with pTrc99A and pTrc99A/rluA as well as wild-type and BL21/DE3(rluA −) with pET15b and pET15b/rluA were selected on LB plates containing 0.1 mg/ml carbenicillin. All subsequent growth media for the transformants also contained 0.1 mg/ml carbenicillin to retain the plasmid in the carbenicillin-sensitive host cells. In vitro assays of Ψ synthase activity were done as described previously (15Wrzesinski J. Nurse K. Bakin A. Lane B.G. Ofengand J. RNA (N. Y.). 1995; 1: 437-448PubMed Google Scholar).Pfu DNA polymerase was from Promega. All other enzymes and primers were obtained and polyacrylamide gel electrophoresis was performed as described previously (13Raychaudhuri S. Conrad J. Hall B.G. Ofengand J. RNA. 1998; 4: 1407-1417Crossref PubMed Scopus (112) Google Scholar). Overexpressed and purified RluA converts U746 inE. coli 23 S rRNA transcripts to Ψ746 and U-32 in tRNAPhe transcripts to Ψ32 (15Wrzesinski J. Nurse K. Bakin A. Lane B.G. Ofengand J. RNA (N. Y.). 1995; 1: 437-448PubMed Google Scholar). In vitro, the enzyme was highly specific for these two sites. Comparison of the sequence surrounding the sites of the two U residues selected for conversion to Ψ revealed that both possessed the same sequence immediately 3′ to the U in question, thus providing a rationale for the dual specificity exhibited by this enzyme (15Wrzesinski J. Nurse K. Bakin A. Lane B.G. Ofengand J. RNA (N. Y.). 1995; 1: 437-448PubMed Google Scholar). These experiments did not, however, show whether additional enzymes existed in the cell that were also capable of Ψ formation at these sites, nor did they show the effect of deletion of these Ψ residues. Therefore, the gene was deleted by insertion of the kanamycin resistance gene (18Hamilton C.M. Aldea H. Washburn B.K. Babitzke P. Kushner S.R. J. Bacteriol. 1989; 171: 4617-4622Crossref PubMed Google Scholar) in strain MC1061. The deletion was confirmed by PCR amplification from the N and C terminii of the rluA gene in the chromosomal DNA of the deletion mutant. The wild-type control had the expected 670-base pair band, whereas the mutant having a kan insert was 1.4 kilobase pairs in size. The presence of the kan gene was further confirmed by amplification from the N and C termini of thekan gene. The mutant produced the expected 1.3-kilobase pair band, whereas no band was obtained from the wild type. To assess the physiological effects of this gene deletion uncomplicated by the other mutant genetic loci present in MC1061 (18Hamilton C.M. Aldea H. Washburn B.K. Babitzke P. Kushner S.R. J. Bacteriol. 1989; 171: 4617-4622Crossref PubMed Google Scholar), the deletion was transferred by bacteriophage P1 transduction into MG1655 in which the sequenced genome (20Blattner F.R. Plunkett III, G. Bloch C.A. Perna N.T. Burland V. Riley M. Collado-Vides J. Glasner J.D. Rode C.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick H.A. Goeden M.A. Rose D.J. Mau B. Shao Y. Science. 1997; 277: 1453-1474Crossref PubMed Scopus (6024) Google Scholar) provided a well defined background. Transductants were selected by resistance to kanamycin. PCR amplification confirmed the presence of the kanamycin insert, and Ψ sequencing analysis of the ribosomal RNA from the mutant strain showed unequivocally that Ψ746 was absent (data not shown). To prove that the loss of Ψ attendant on the deletion ofrluA was a direct consequence of the deletion and not because of some downstream polarity or other indirect effect, the gene was replaced in trans by transformation of the deletion strain with a rescue plasmid that contained only the rluAgene inserted into pTrc99A. Wild type and MG1655(rluA −) were transformed with both the rescue plasmid and the control vector pTrc99A and selected on carbenicillin plates. Ribosomal RNA was isolated and sequenced for the presence of Ψ (Fig. 1). Comparing therluA + lanes with therluA − lanes, it is clear that the stop at residue 746 in the + CMC lane of therluA + set is absent in the + CMC laneof the rluA − pair. Recall that in this method of sequencing, reverse transcriptase halts one residue 3′ to the CMC-Ψ (7Bakin A. Ofengand J. Biochemistry. 1993; 32: 9754-9762Crossref PubMed Scopus (264) Google Scholar, 25Bakin A. Ofengand J. Martin R. Methods in Molecular Biology: Protein Synthesis: Methods and Protocols. 77. Humana Press, Inc., Totowa, NJ1998: 297-309Google Scholar). However, when the rescue plasmid was introduced into the rluA − strain, Ψ746 was again found. The stop seen in all lanes results from m1G745, because all of the RNAs were isolated from cells. We conclude that the loss of Ψ746 is a direct result of deletion of rluA and that RluA is the sole gene product capable of synthesis of Ψ746. Additional sequencing analyses verified that only Ψ746 was absent from therluA − strain (data not shown). Recently, it has been shown that the replacement of Asp-60 in a conserved (G/H)(R/a)(L/t)(D) motif (lowercase identifies a rare event), by Ala, Asn, Glu, Lys, or Ser in the pseudouridine synthase TruA resulted in the loss of catalytic activity while retaining binding ability (4Huang L. Pookanjanatavip M. Gu X. Santi D.V. Biochemistry. 1998; 37: 344-351Crossref PubMed Scopus (132) Google Scholar). There is an equivalent residue, Asp-64, in a similarly conserved motif, HRLD, in the RluA synthase, and it is the only Asp residue in such a motif in the molecule. To test the possibility that Asp-64 could be an essential residue of this enzyme, we mutated it to Thr and Asn. This was done by megaprimer mutagenesis (23Picard V. Ersdal-Badju E. Lu A. Bock S.C. Nucleic Acids Res. 1994; 22: 2587-2591Crossref PubMed Scopus (216) Google Scholar). The two mutated rluA were cloned in pTrc99A and transformed into MG1655(rluA −) cells to assess the function of these mutant enzymes in vivo. The wild-type rescue plasmid served as a control. Ribosomal RNA was isolated and sequenced for the presence of Ψ (Fig.2). It is clear that the only strain able to make Ψ746 is the strain carrying the wild-type rescue plasmid. Neither plasmid carrying a mutant rluA was any more effective than the vector alone. Thus, in vivo, the single mutation D64T or D64N is sufficient to block synthesis of an active Ψ746 synthase. One might conclude from this experiment that Asp-64 is an amino acid that actively participates in catalysis, but it could also be that its role is in the maintenance of the correct conformation of the enzyme. In the latter event, the replacement of Asp-64 by another amino acid might make the protein susceptible to protease degradation. To address this question, we turned to the BL21/DE3 strain and pET15b to obtain stable overexpression of the mutant proteins. TherluA − gene was transferred into BL21/DE3 by P1 transduction from MC1061 with selection by kanamycin resistance. PCR amplification confirmed the presence of the kanamycin insert, and Ψ sequencing analysis of the ribosomal RNA from the mutant strain showed the absence of Ψ746 (data not shown, but see Fig. 4 for an equivalent result). Both the wild-type and mutant rluA constructs were subcloned into pET15b. DNA sequencing analysis (data not shown) confirmed that the desired mutants had been produced in pET15b. The BL21/DE3(rluA −) cells were then transformed with vector alone or with the rluA constructs in pET15b. Transformants were selected on carbenicillin plates. The BL21/DE3 cells carrying either the vector or the various rluA constructs were then induced. After a 3-h induction with isopropyl-1-thio-β-d-galactopyranoside, samples from each cultures were taken out for protein analysis on SDS-polyacrylamide gels as well as for ribosomal RNA isolation and Ψ sequencing analysis. Fig. 3 shows that a strongly overexpressed protein band at about 27 kDa, the expected size, was found in the cells carrying both wild-type and mutant rluAconstructs, whereas there was no such overexpressed protein band in the cells carrying the vector only. Furthermore, induction was required to produce the band. The intensity of the 27-kDa band appeared the same in both wild-type and mutant constructs. Ψ sequencing analysis of the rRNA showed that, as with the results obtained in Fig. 2, the mutant rescue plasmids were unable to form Ψ746 (Fig.4). We conclude that the two mutantrluA constructs produced stable proteins that had, nevertheless, lost the capability to isomerize U746 to Ψ as a result of the replacement of Asp-64 by Thr-64 or Asn-64.Figure 3Overexpression of the wild type and mutantrluA gene products in BL21/DE3(rluA −) cells. Cells grown at 37 °C were transformed with the various pET15b/rluAconstructs, harvested either before (−) or after (+) induction with 1 mm isopropyl-1-thio-β-d-galactopyranoside, lysed by boiling in SDS, and analyzed on SDS-polyacrylamide gels.Lanes: A, pET15b; B, pET15b/rluA-D64T; C, pET15b/rluA-D64N;D, pET15b/rluA-D64D; S, molecular mass standards of the indicated sizes.View Large Image Figure ViewerDownload (PPT) Affinity purification of the overexpressed proteins shown in Fig. 3 and assay of in vitro activity using 23 S [3H]RNA transcripts as substrate gave the same result, namely that whereas unit stoichiometry of Ψ formation could be obtained for the wild-type construct, as reported previously (15Wrzesinski J. Nurse K. Bakin A. Lane B.G. Ofengand J. RNA (N. Y.). 1995; 1: 437-448PubMed Google Scholar), both the D64T and D64N mutants were totally inactive (Fig. 5). Ψ32 is found in four tRNAs of E. coli, tRNAPhe, tRNACys, tRNA4Leu(UAA), and tRNA5Leu(CAA) (28Sprinzl M. Horn C. Brown M. Ioudovitch A. Steinberg S. Nucleic Acids Res. 1998; 26: 148-153Crossref PubMed Scopus (815) Google Scholar, 29Horie N. Yamaizumi Z. Kuchino Y. Takai K. Goldman E. Miyazawa T. Nishimura S. Yokoyama S. Biochemistry. 1999; 38: 207-217Crossref PubMed Scopus (19) Google Scholar). We previously showed that RluA formed Ψ32 on a transcript of tRNAPhe in an in vitro reaction (15Wrzesinski J. Nurse K. Bakin A. Lane B.G. Ofengand J. RNA (N. Y.). 1995; 1: 437-448PubMed Google Scholar), a result that led to the concept of dual specificity for this enzyme. All five of these RNAs, the 23 S RNA and the four tRNAs, share a common sequence surrounding the Ψ residue, namely (A/G)ΨUN(A/C)AAA. Therefore, it seemed reasonable that these other tRNAs could also serve as a substrate for RluA. To test this hypothesis, tRNACys and tRNA4Leu transcripts were assayed for their ability to react with RluA (Fig.6). Both transcripts were active. The rate and yield with tRNACys was virtually identical to that with tRNAPhe, whereas tRNA4Leu was somewhat less reactive for unknown reasons. To determine whether RluA is the only protein in E. colicapable of tRNA Ψ32 formation, tRNA from therluA − strain was analyzed. However, before the reverse transcription assay could be used, obstacles created by the presence of other modified nucleosides in the tRNAs that block reverse transcriptase had to be overcome. ms2i6A37, present in all three tRNAs, is a strong inhibitor of reverse transcription. Moreover, tRNAPhe also has acp3U47, another strong blocker, and tRNA4Leu has cmnm5Um34 (29Horie N. Yamaizumi Z. Kuchino Y. Takai K. Goldman E. Miyazawa T. Nishimura S. Yokoyama S. Biochemistry. 1999; 38: 207-217Crossref PubMed Scopus (19) Google Scholar) only two residues away from Ψ32. To replace ms2i6A37 by A37, a deletion of miaA, the gene" @default.
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• W2037813174 title "Functional Effect of Deletion and Mutation of theEscherichia coli Ribosomal RNA and tRNA Pseudouridine Synthase RluA" @default.
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