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The rluC Gene of Escherichia coli Codes for a Pseudouridine Synthase That Is Solely Responsible for Synthesis of Pseudouridine at Positions 955, 2504, and 2580 in 23 S Ribosomal RNA

1998; Elsevier BV; Volume: 273; Issue: 29 Linguagem: Inglês

10.1074/jbc.273.29.18562

1083-351X

Joel Conrad, Danhui Sun, Nathan Englund, James Ofengand,

Bacterial Genetics and Biotechnology

Escherichia coli ribosomal RNA contains 10 pseudouridines, one in the 16 S RNA and nine in the 23 S RNA. Previously, the gene for the synthase responsible for the 16 S RNA pseudouridine was identified and cloned, as was a gene for a synthase that makes a single pseudouridine in 23 S RNA. The yceCopen reading frame of E. coli is one of a set of genes homologous to these previously identified ribosomal RNA pseudouridine synthases. In this work, the gene was cloned, overexpressed, and shown to code for a pseudouridine synthase able to react with in vitro transcripts of 23 S ribosomal RNA. Deletion of the gene and analysis of the 23 S RNA from the deletion strain for the presence of pseudouridine at its nine known sites revealed that this synthase is solely responsible in vivo for the synthesis of three of the nine pseudouridine residues, at positions 955, 2504, and 2580. Therefore, this gene has been renamed rluC. Despite the absence of one-third of the normal complement of pseudouridines, there was no change in the exponential growth rate in either LB or M-9 medium at temperatures ranging from 24 to 42 °C. From this work and our previous studies, we have now identified three synthases that account for 50% of the pseudouridines in the E. coli ribosome. Escherichia coli ribosomal RNA contains 10 pseudouridines, one in the 16 S RNA and nine in the 23 S RNA. Previously, the gene for the synthase responsible for the 16 S RNA pseudouridine was identified and cloned, as was a gene for a synthase that makes a single pseudouridine in 23 S RNA. The yceCopen reading frame of E. coli is one of a set of genes homologous to these previously identified ribosomal RNA pseudouridine synthases. In this work, the gene was cloned, overexpressed, and shown to code for a pseudouridine synthase able to react with in vitro transcripts of 23 S ribosomal RNA. Deletion of the gene and analysis of the 23 S RNA from the deletion strain for the presence of pseudouridine at its nine known sites revealed that this synthase is solely responsible in vivo for the synthesis of three of the nine pseudouridine residues, at positions 955, 2504, and 2580. Therefore, this gene has been renamed rluC. Despite the absence of one-third of the normal complement of pseudouridines, there was no change in the exponential growth rate in either LB or M-9 medium at temperatures ranging from 24 to 42 °C. From this work and our previous studies, we have now identified three synthases that account for 50% of the pseudouridines in the E. coli ribosome. Pseudouridine (Ψ), 1The abbreviations used are: Ψ, pseudouridine(s); rRNA, ribosomal RNA; SSU, small subunit; LSU, large subunit; ORF, open reading frame; PCR, polymerase chain reaction; kb, kilobase pair(s). the 5-ribosyl isomer of uridine, occurs in rRNA (1Maden B.E.H. Prog. Nucleic Acids Res. Mol. Biol. 1990; 39: 241-300Crossref PubMed Scopus (329) Google Scholar), tRNA (2Sprinzl M. Horn C. Brown M. Ioudovitch A. Steinberg S. Nucleic Acids Res. 1998; 26: 148-153Crossref PubMed Scopus (818) Google Scholar), and small nuclear and nucleolar RNA (3Gu J. Chen Y. Reddy R. Nucleic Acids Res. 1998; 26: 160-162Crossref PubMed Scopus (19) Google Scholar, 4Massenet S. Mougin A. Branlant S. Grosjean H. Benne R. Modification and Editing of RNA. ASM Press, Washington, D. C.1998: 201-227Google Scholar) but not in mRNA or viral genomic RNAs. All the RNAs in which Ψ is found share a common characteristic, namely a tertiary structure that must be maintained for proper function. Ψ is made after the polynucleotide chain has been formed by an enzyme-catalyzed but energy-independent isomerization of uridine (reviewed in Ref. 5Ofengand J. Fournier M. Grosjean H. Benne R. Modification and Editing of RNA. ASM Press, Washington, D. C.1998: 229-253Google Scholar). A considerable amount of Ψ is found in ribosomal RNA approaching 8% of the uridines in mammals (6Ofengand J. Bakin A. J. Mol. Biol. 1997; 266: 246-268Crossref PubMed Scopus (179) Google Scholar). The number and distribution of Ψ is different between the two large rRNAs. In small subunit (SSU) RNA, the number varies from 0 or 1 (yeast mitochondria) to 1 (Escherichia coli) to ∼40 (mammals), and Ψ are deployed throughout the molecule, whereas in the large subunit (LSU) RNA, although there is also a wide variation in the number of Ψ from 1 (yeast mitochondria) to 4–9 (prokaryotes) to 55–57 (mammals), the distribution is conserved in all organisms to three defined secondary structural regions at or near the peptidyl transferase center (reviewed in Ref. 5Ofengand J. Fournier M. Grosjean H. Benne R. Modification and Editing of RNA. ASM Press, Washington, D. C.1998: 229-253Google Scholar). In E. coli, the organism studied in this work, there are 10 Ψ residues, one in the SSU RNA at position 516 (7Bakin 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 LSU RNA at positions 746, 955, 1911, 1915, 1917, 2457, 2504, 2580, and 2605 (8Bakin A. Ofengand J. Biochemistry. 1993; 32: 9754-9762Crossref PubMed Scopus (266) Google Scholar, 9Bakin A. Lane B.G. Ofengand J. Biochemistry. 1994; 33: 13475-13483Crossref PubMed Scopus (79) Google Scholar). Ψ1915 is further modified by methylation at N3 (10Kowalak J.A. Bruenger E. Hashizume T. Peltier J.M. Ofengand J. McCloskey J.A. Nucleic Acids Res. 1996; 24: 688-693Crossref PubMed Scopus (59) Google Scholar). Despite the specificity implicit in the conservation of geographic localization in the LSU to the functionally important peptidyl transferase center, there is no known role for Ψ in the ribosome. To address this issue, we have embarked on a program to identify all of the synthases responsible for formation of the 10 Ψ in E. coli rRNA with the aim of deleting specific Ψ residues by inactivating the genes for the corresponding synthases. So far,rsuA, the gene for the synthase responsible for forming Ψ516 in 16 S RNA (11Wrzesinski J. Bakin A. Nurse K. Lane B.G. Ofengand J. Biochemistry. 1995; 34: 8904-8913Crossref PubMed Scopus (85) Google Scholar), and rluA, the gene for the synthase that makes Ψ746 in LSU RNA (12Wrzesinski J. Nurse K. Bakin A. Lane B.G. Ofengand J. RNA. 1995; 1: 437-448PubMed Google Scholar), have been identified. In this work we show that the gene yceC, renamed rluC, makes the synthase responsible for formation of Ψ at positions 955, 2504, and 2580. Deletion of this gene and thus the absence of these three Ψ residues has no detectable effect on the exponential growth rate in either rich or minimal glucose medium at temperatures ranging from 24 to 42 °C. The yceC gene was deleted by the method of Hamilton et al. (13Hamilton 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 KpnI and XbaI sites of pMAK705 was prepared by PCR as described by Nelson and co-workers (see Fig. 2 in Ref. 14Supekova L. Supek F. Nelson N. J. Biol. Chem. 1995; 270: 13726-13732Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). It contained 841 bases 5′ to the AUG start and 870 bases 3′ to the UAA termination codon. 39 bases of the N-terminal portion of the gene and 49 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 number 27-4958-01). The host strain for pMAK705 was MC1061 as described by Hamilton et al. (13Hamilton C.M. Aldea H. Washburn B.K. Babitzke P. Kushner S.R. J. Bacteriol. 1989; 171: 4617-4622Crossref PubMed Google Scholar). The deleted yceC gene was moved into strain SJ134 (Ref. 15Hall B.G. J. Bacteriol. 1998; 180: 2862-2864Crossref PubMed Google Scholar; a gift of Dr. Barry Hall, University of Rochester, Rochester, NY) and from there into strain MG1655 (Ref. 16Blattner 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 (6050) Google Scholar; a gift of Dr. Kenneth Rudd, this department) by bacteriophage P1 transduction (17Miller J.H. A Short Course in Bacterial Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 357-364Google Scholar). This plasmid (pLG338/yceC) was constructed by insertion into the SmaI site of pLG338 (18Stoker N.G. Fairweather N.F. Spratt B.G. Gene ( Amst. ). 1982; 18: 3353-3413Google Scholar) of a PCR-amplified fragment of DNA starting 116 bases 5′ to the AUG initiator of yceC and ending 124 nucleotides 3′ to the termination codon. This insertion site inactivates the KanRgene of the plasmid. The construct includes the 71-base promoter, 45-base spacer, 960-base gene, 77-base spacer, 44-base termination sequence, and 3 bases beyond. pLG338 also carries a tetracycline resistance gene. Putative promoter and terminator sequences were identified by examination of the upstream and downstream regions. For the promoter, a web site was used. 2A. M. Huerta, H. Salgado, and J. Collado-Vides, www.cifn.unam.mx/Computational_Biology/E.coli-predictions/. The terminator sequence was located visually and verified by folding using M-fold version 3.0 (19Jaeger J.A. Turner D.H. Zuker M. Methods Enzymol. 1990; 183: 281-306Crossref PubMed Scopus (374) Google Scholar). 3www.ibc.wustl.edu/∼zuker/rna. Transformants ofyceC-deleted SJ134 and of wild type andyceC-deleted MG1655 with pLG338 and pLG338/yceCwere selected by tetracycline resistance. All growth media contained 10 μg/ml tetracycline to retain the plasmid in the tetracycline-sensitive host cells. Ψ sequencing was performed as described previously (8Bakin A. Ofengand J. Biochemistry. 1993; 32: 9754-9762Crossref PubMed Scopus (266) Google Scholar, 20Bakin A Ofengand J. Methods in Molecular Biology: Protein Synthesis: Methods and Protocols.in: Martin R. Chapter 22. Humana Press, Inc., Totowa, NJ1998Google Scholar). For the 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. From the amino acid sequences of RsuA and RluA, as well as those for two tRNA Ψ synthases, TruB (21Nurse K. Wrzesinski J. Bakin A. Lane B.G. Ofengand J. RNA. 1995; 1: 102-112PubMed Google Scholar) and TruA (22Kammen H.O. Marvel C.C. Hardy L. Penhoet E.E. J. Biol. Chem. 1988; 263: 2255-2263Abstract Full Text PDF PubMed Google Scholar), Koonin (23Koonin E.V. Nucleic Acids Res. 1996; 24: 2411-2415Crossref PubMed Scopus (203) Google Scholar) and Gustafssonet al. (24Gustafsson C. Reid R. Greene P.J. Santi D.V. Nucleic Acids Res. 1996; 24: 3756-3762Crossref PubMed Scopus (120) Google Scholar) were able to identify putative Ψ synthase ORFs by searching for sequence motifs. Five ORFs were identified in E. coli (Table I). A sixth ORF,ymfC, was found subsequently when the entire E. coli genome sequence became available. The ORFs could be divided into four subfamilies based on their sequence motifs (23Koonin E.V. Nucleic Acids Res. 1996; 24: 2411-2415Crossref PubMed Scopus (203) Google Scholar, 24Gustafsson C. Reid R. Greene P.J. Santi D.V. Nucleic Acids Res. 1996; 24: 3756-3762Crossref PubMed Scopus (120) Google Scholar) as indicated in Table I. By chance, the sequences of the four initially identified genes happened to define each of the four subclasses. Note, however, that the six putative synthase genes subsequently identified are all in class A or B. The predicted protein properties and the site(s) of Ψ formation recognized by the synthases are also indicated in Table I when known.Table IKnown and potential pseudouridine synthases of E. coliNameMotif subclassGene locusPredicted massNumber of amino acidsSWISS-PROT accession numberRNA substratePosition modifiedminkDaRsuAaWrzesinski et al. (11).A49.1225.9231P33918SSU RNA516RluAbWrzesinski et al. (12).B1.3024.9219P39219LSU RNA746tRNA32TruBcNurse et al. (21).C71.3635.1314P09171tRNA55TruAdKammen et al. (22).D52.4630.4270P07649tRNA38–40SfhBeN-terminal sequence obtained on a purified Ψ synthase and ORF identified (J. Wrzesinski, D. Sun, and J. Ofengand, unpublished results)., fKoonin (23). In this report, SfhB was YfiI, and YqcB was ECU29581_5., gGustafsson et al. (24). In this report, SfhB was YfiI, and YqcB was f260.B58.9337.1326P33643YceCfKoonin (23). In this report, SfhB was YfiI, and YqcB was ECU29581_5.,gGustafsson et al. (24). In this report, SfhB was YfiI, and YqcB was f260.B24.6636.0319P23851YciLfKoonin (23). In this report, SfhB was YfiI, and YqcB was ECU29581_5.,gGustafsson et al. (24). In this report, SfhB was YfiI, and YqcB was f260.A28.5632.7291P37765YjbCfKoonin (23). In this report, SfhB was YfiI, and YqcB was ECU29581_5.,gGustafsson et al. (24). In this report, SfhB was YfiI, and YqcB was f260.A91.1432.5290P32684YqcBfKoonin (23). In this report, SfhB was YfiI, and YqcB was ECU29581_5.,gGustafsson et al. (24). In this report, SfhB was YfiI, and YqcB was f260.B62.9829.7260Q46918YmfChValues in parentheses were obtained using the next downstream initiator AUG. There is uncertainty regarding the true start site (K. Rudd, personal communication).A25.7424.9217P75966(23.7)(207)a Wrzesinski et al. (11Wrzesinski J. Bakin A. Nurse K. Lane B.G. Ofengand J. Biochemistry. 1995; 34: 8904-8913Crossref PubMed Scopus (85) Google Scholar).b Wrzesinski et al. (12Wrzesinski J. Nurse K. Bakin A. Lane B.G. Ofengand J. RNA. 1995; 1: 437-448PubMed Google Scholar).c Nurse et al. (21Nurse K. Wrzesinski J. Bakin A. Lane B.G. Ofengand J. RNA. 1995; 1: 102-112PubMed Google Scholar).d Kammen et al. (22Kammen H.O. Marvel C.C. Hardy L. Penhoet E.E. J. Biol. Chem. 1988; 263: 2255-2263Abstract Full Text PDF PubMed Google Scholar).e N-terminal sequence obtained on a purified Ψ synthase and ORF identified (J. Wrzesinski, D. Sun, and J. Ofengand, unpublished results).f Koonin (23Koonin E.V. Nucleic Acids Res. 1996; 24: 2411-2415Crossref PubMed Scopus (203) Google Scholar). In this report, SfhB was YfiI, and YqcB was ECU29581_5.g Gustafsson et al. (24Gustafsson C. Reid R. Greene P.J. Santi D.V. Nucleic Acids Res. 1996; 24: 3756-3762Crossref PubMed Scopus (120) Google Scholar). In this report, SfhB was YfiI, and YqcB was f260.h Values in parentheses were obtained using the next downstream initiator AUG. There is uncertainty regarding the true start site (K. Rudd, personal communication). Open table in a new tab Because homologs to yceC are common in other species (23Koonin E.V. Nucleic Acids Res. 1996; 24: 2411-2415Crossref PubMed Scopus (203) Google Scholar, 24Gustafsson C. Reid R. Greene P.J. Santi D.V. Nucleic Acids Res. 1996; 24: 3756-3762Crossref PubMed Scopus (120) Google Scholar), we selected this ORF for our initial study. The gene was cloned, the protein was overexpressed, and Ψ formation activity was detected using 5-[3H]uridine-labeled 23 S RNA as anin vitro substrate, confirming that this gene product was indeed a Ψ synthase. To determine the specificity of the synthase under in vivo conditions, the gene was deleted by insertion of the kanamycin resistance gene (13Hamilton C.M. Aldea H. Washburn B.K. Babitzke P. Kushner S.R. J. Bacteriol. 1989; 171: 4617-4622Crossref PubMed Google Scholar). Verification of gene deletion was done by PCR amplification from the N and C termini of theyceC gene in the chromosomal DNA of the deletion mutant. The wild type control gave the expected 1.0-kb band, whereas the mutant supposed to contain the KanR insert was, as expected, 1.4 kb in size. Further evidence was obtained by amplification from the N and C termini of the KanR gene. The mutant gave the expected 1.3-kb band, but nothing was obtained from the wild type. Preliminary Ψ sequence analysis showed the absence of Ψ 955, 2504, and 2580 as a result of this single gene deletion. For further experiments, the deleted gene was transferred by P1 transduction (17Miller J.H. A Short Course in Bacterial Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 357-364Google Scholar) into strain SJ134. Mutant cells were then transformed with a low copy number plasmid, pLG338 (18Stoker N.G. Fairweather N.F. Spratt B.G. Gene ( Amst. ). 1982; 18: 3353-3413Google Scholar), or a rescue plasmid, pLG338 containing the yceC gene inserted at itsSmaI site, creating strainsyceC−(pLG338) and yceC−(pLG338/yceC). The yceC gene insert included the putative yceC gene promoter and terminator elements, and there were no other apparent ORFs in this operon. Because pLG338 carries a tetracycline resistance gene, the plasmid-containing cultures were grown in the presence of tetracycline to retain the plasmid in its tetracycline-sensitive host strain. Ribosomal RNA from the wild type and yceC-disrupted strain SJ134 and from the two transformed strains,yceC−(pLG338) andyceC−(pLG338/yceC), were isolated and sequenced for the presence of Ψ at the nine known sites in 23 S RNA (Fig. 1). This figure shows clearly that of the five Ψ shown, the deletion mutant is lacking Ψ at 955, 2504, and 2580 but not at 2457 or 2605. Because the bands for 2457 and 2605 are found in the same lanes as 2504 and 2580, respectively, they serve as effective internal controls for the absence of bands at 2504 and 2580. The other four Ψ, at positions 746, 1911, 1915, and 1917, were present in the mutant as well as the wild type (data not shown). When the deletion strain was supplemented with the rescue plasmid pLG338/yceC, but not with pLG338 alone, all three Ψ reappear. We conclude that the yceC gene makes a Ψ synthase that can recognize these three sites in 23 S RNA and that no other synthase in the cell can take over this function. In view of this result, we have renamed the gene rluC, ribosomallarge subunit pseudouridine synthaseC. The location of the nine Ψ sites in 23 S RNA and the five sites monitored in Fig. 1 are shown in Fig. 2 A on the secondary structure of 23 S RNA, and in Fig. 2 B, the immediate vicinity of the three sites recognized by RluC are shown in expanded form. Clearly, the three sites are separated from each other and do not share any obvious common structural elements that would be suitable for recognition purposes. Deletion of these three Ψ residues from the ribosome was not lethal. To assess any more subtle metabolic defects, growth rates were measured at different temperatures in both rich and minimal glucose media (TableII). For this purpose, therluC deletion was moved by P1 transduction into strain MG1655, the same strain whose genome was sequenced by Blattner et al. (16Blattner 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 (6050) Google Scholar), to provide a well defined background. The MG1655 deletion strain was selected by its kanamycin resistance, and PCR amplification from the yceC termini confirmed the deletion (the expected 1.4-kb band was found instead of a 1.0-kb band for the wild type). Wild type and mutant MG1655 were transformed with both the rescue plasmid and its control. Exponential growth rates for all four strains are shown in Table II. Even though both rich and minimal media were tested over a range of temperature from 24 to 42 °C, no significant change in growth rate was observed. Moreover, no instance was found where the maximum cell density at stationary phase was limited by the deletion mutation. We conclude that at least under the described conditions, there is no effect of the loss of three of the nine Ψ present in the large subunit of the ribosome.Table IIGrowth rate of rluC deletion and rescue strainsStrainMediumDoubling timeaValues in parentheses are the number of exponential phase doublings over which the doubling time was measured.24 °C37 °C42 °CminMG1655/pLG338RichbLB broth (27).121 (2)25.3 (5)24.7 (3)MG1655/pLG338(rluC)RichbLB broth (27).123 (2)25.5 (5)24.5 (3)MG1655(rluC−)/pLG338RichbLB broth (27).122 (2)25.0 (5)25.0 (3)MG1655(rluC−)/pLG338(rluC)RichbLB broth (27).119 (2)25.3 (5)25.0 (3)MG1655/pLG338MinimalcM-9 (27) plus 0.4% glucose, 1 mm MgSO4, and 0.01 mg/ml tetracycline.282 (2)67.5 (3)57.9 (4)MG1655/pLG338(rluC)MinimalcM-9 (27) plus 0.4% glucose, 1 mm MgSO4, and 0.01 mg/ml tetracycline.287 (2)67.0 (3)59.4 (4)MG1655(rluC−)/pLG338MinimalcM-9 (27) plus 0.4% glucose, 1 mm MgSO4, and 0.01 mg/ml tetracycline.306 (2)71.5 (3)59.3 (4)MG1655(rluC−)/pLG338(rluC)MinimalcM-9 (27) plus 0.4% glucose, 1 mm MgSO4, and 0.01 mg/ml tetracycline.291 (2)70.7 (3)60.0 (4)a Values in parentheses are the number of exponential phase doublings over which the doubling time was measured.b LB broth (27Zyskind J.W. Bernstein S.I. Recombinant DNA Laboratory Manual. Academic Press, San Diego, CA1992: 187Crossref Google Scholar).c M-9 (27Zyskind J.W. Bernstein S.I. Recombinant DNA Laboratory Manual. Academic Press, San Diego, CA1992: 187Crossref Google Scholar) plus 0.4% glucose, 1 mm MgSO4, and 0.01 mg/ml tetracycline. Open table in a new tab In this work, we have shown that yceCis indeed a gene for a Ψ synthase and that its gene product is the only synthase which in vivo is capable of forming Ψ955, Ψ2504, and Ψ2580 in E. coli 23 S RNA. In view of its specificity, the gene has been renamed rluC. Because this work was done in vivo in the presence of all of the other Ψ synthases of the cell, it is clear that none of the other synthases share the specificity for positions 955, 2504, and 2580 with RluC. However, the reverse is not necessarily the case. RluC might share the ability with another synthase for recognition of one or more of the remaining Ψ sites. This can only be determined with certainty when all of the other rRNA Ψ synthases are identified and deleted. RluC does not share the ability to form Ψ516 in 16 S RNA with RsuA (TableI), because deletion of the rsuA gene blocks formation of Ψ516 in vivo. 4L. Niu and J. Ofengand, unpublished results. A possible dual specificity of recognition with one of the sites in tRNA, like that found for RluA (12Wrzesinski J. Nurse K. Bakin A. Lane B.G. Ofengand J. RNA. 1995; 1: 437-448PubMed Google Scholar), has not yet been tested. Although all three sites for Ψ formation are well separated in the primary structure of 23 S RNA, 2504 and 2580, but not 955, approach each other when the RNA is folded into its secondary structure (Fig. 2 A). However, in the tertiary structure existing in the ribosome, Ψ955 is probably not far away either, because cross-linking results place A960 next to C2475 (28Dokudovskaya S. Dontsova O. Shpanchenko O. Bogdanov A. Brimacombe R. RNA. 1996; 2: 146-152PubMed Google Scholar). These results suggest a possible mode of recognition of these three sites, which otherwise appear dissimilar in both sequence and secondary structure. Indeed, the only common structural element is that all three Us destined to be isomerized to Ψ are followed by a G residue. We postulate that any UG sequence within a given short distance of the catalytic center will be recognized. Thus if there is a tertiary structure of the 23 S RNA at some stage of ribosome biogenesis in which U955 is sufficiently close to the recognition site of the synthase, which must also be near to U2504 and U2580, it might be possible for the synthase to catalyze Ψ formation at all three sites. In this regard it should be noted that the closest other UG sequence to any of the three sites is 11 residues away at U2493. An alternative way to account for this unusual specificity would be for the synthase to possess separate polypeptide binding domains specific for each site that would each have the property that upon binding, the desired U residue would be brought into the catalytic center. No effect on exponential phase growth rate was found when Ψ955, 2504, and 2580 were absent, even when both medium and temperature were varied. Other growth parameters such as survival in stationary phase and the length of the lag phase were not examined in detail, but preliminary observations suggest that there is little or no effect (data not shown). In the absence of any such clues as to Ψ function, the ribosomes from Ψ-deficient cells will need to be examined for their ability to support the partial reactions of protein synthesis in vitro. This has been done previously to study the effects of rRNA mutations on ribosome function (29Cunningham P.R. Nurse K. Bakin A. Weitzmann C.J. Pflumm M. Ofengand J. Biochemistry. 1992; 31: 12012-12022Crossref PubMed Scopus (33) Google Scholar, 30Santer 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, 31Cunningham P.R. Nurse K. Weitzmann C. Ofengand J. Biochemistry. 1993; 32: 7172-7180Crossref PubMed Scopus (29) Google Scholar). Failure to find a physiological effect upon Ψ deletion is disappointing but not surprising. Neither the rsuA deletion strain mentioned above nor a strain with the rluA gene deleted showed any major metabolic defects,4 and deletion of various Ψ residues from yeast SSU and LSU RNAs by deletion of their guide RNAs also had no effect (reviewed in Ref. 5Ofengand J. Fournier M. Grosjean H. Benne R. Modification and Editing of RNA. ASM Press, Washington, D. C.1998: 229-253Google Scholar). Nevertheless, the geographic juxtaposition of at least two of these Ψ residues, namely 2504 and 2580, to sites known to be functionally important in peptide bond formation (reviewed in Ref. 6Ofengand J. Bakin A. J. Mol. Biol. 1997; 266: 246-268Crossref PubMed Scopus (179) Google Scholar) is a strong indication that these Ψ residues play an important, if still undiscovered, role. This conclusion is reinforced by the recent finding that of the four 23 S RNA residues whose chemical modification interferes with binding of tRNA to the ribosomal P site, U2506 and U2585 are within 2 and 5 bases, respectively, of Ψ2504 and Ψ2580 (35Bochetta M. Xiong L. Mankin A.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3525-3530Crossref PubMed Scopus (56) Google Scholar). TableIII summarizes the properties of all known cloned Ψ synthases and their genes. Table III includes synthases for E. coli and Bacillus subtilis rRNA and for E. coli and yeast tRNA. It is clear that the specificity of each synthase varies from being limited to one kind of RNA at one site to multiple sites in one class of RNA or even sites in different kinds of RNA. To help in categorizing these synthases and those still to be discovered, four specificity classes have been defined. Class I specificity is most stringent, in that only a single specific site in one kind of RNA is recognized. RsuA, RluB, TruB, and its homolog in yeast, Pus4p, are examples of this class. Class II defines those synthases that modify any U residue within a span of 5–6 nucleotides, but only in one kind of RNA. TruA and its yeast homolog, Pus3p, are examples. In class III, the specificity is relaxed even more, and distant sites become recognizable, although again only in the same class of RNA. Pus1p in yeast is an example of this type because it can recognize up to eight different sites in tRNA, and RluC is another example. Although both synthases recognize sites spread some distance apart, the maximum distance in the case of Pus1p is 41 residues, whereas for RluC it is 1625 nucleotides. Class IV specificity is reserved for those synthases that recognize specific single sites in more than one class of RNA, a property we have termed "dual specificity" (12Wrzesinski J. Nurse K. Bakin A. Lane B.G. Ofengand J. RNA. 1995; 1: 437-448PubMed Google Scholar). So far, RluA is the only member of this class, but this may be at least in part because of the fact that testing for dual specificity is not straightforward because it requires a predetermination of which site in which RNA to test. The dual specificity of RluA was only discovered serendipitously.Table IIICloned rRNA and tRNA pseudouridine synthasesNameGene locationRNA substratePosition modifiedSpecificity classMonomer mass of ORFNumber of amino acidsSWISS-PROT Accession NumberReferencekDaRsuA (E.c.)49.12 min16 S rRNA516I25.9231P3391811Wrzesinski J. Bakin A. Nurse K. Lane B.G. Ofengand J. Biochemistry. 1995; 34: 8904-8913Crossref PubMed Scopus (85) Google ScholarRluA (E.c.)1.30 min23 S rRNA746IV24.9219P3921912Wrzesinski J. Nurse K. Bakin A. Lane B.G. Ofengand J. RNA. 1995; 1: 437-448PubMed Google ScholartRNA32Grosjean H. Szweykowska-Kulinska Z. Motorin Y. Fasiolo F. Simos G. Biochimie ( Paris ). 1997; 79: 293-302Crossref PubMed Scopus (61) Google ScholarRluC (E.c.)24.66 min23 S rRNA955III36.0319P23851this work (YceC)25042580RluB (B.s.)206.80 °23 S rRNA2605I26.0229P35159L. Niu and J. Ofengand, unpublished (YpuL)TruB (E.c.)71.36 mintRNA55I35.1314P0917121Nurse K. Wrzesinski J. Bakin A. Lane B.G. Ofengand J. RNA. 1995; 1: 102-112PubMed Google ScholarTruA (E.c.)52.46 mintRNA38–40II30.4270P0764922Kammen H.O. Marvel C.C. Hardy L. Penhoet E.E. J. Biol. Chem. 1988; 263: 2255-2263Abstract Full Text PDF PubMed Google ScholarPus1p (S.c.)tRNA26–28III62.1544Q1221132; H. Grosjean, personal communication34–366567Pus3p (S.c.)tRNA38, 39II50.9442P3111533Lecointe 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 ScholarPus4p (S.c.)tRNA55I45.3403P4856734Becker H.F. Motorin Y. Planta R.J. Grosjean H. Nucleic Acids Res. 1997; 25: 4493-4499Crossref PubMed Google ScholarE.c., E. coli; B.s., B. subtilis;S.c., Saccharomyces cerevisiae. Open table in a new tab E.c., E. coli; B.s., B. subtilis;S.c., Saccharomyces cerevisiae. The intriguing RNA recognition properties of the RluC synthase coupled with the ease of overexpression of the recombinant enzyme with demonstrable Ψ formation activity of appropriate specificity make this protein a logical candidate for x-ray crystallographic analysis. Major questions are how RNA recognition at three such disparate sites is accomplished and what is the mechanism of U isomerization to Ψ. Such studies, in collaboration with R. Fenna of this department, are underway. We thank Ferez Nallaseth for participation in the preliminary sequence analysis of the rluC-disrupted strain, Daanish Kazi for assistance in constructing the rescue plasmid, and K. Rudd for assistance with sequence analysis and for enthusiastic moral support of this project.