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. 1999 Jun;5(6):751–763. doi: 10.1017/s1355838299990167

16S ribosomal RNA pseudouridine synthase RsuA of Escherichia coli: deletion, mutation of the conserved Asp102 residue, and sequence comparison among all other pseudouridine synthases.

J Conrad 1, L Niu 1, K Rudd 1, B G Lane 1, J Ofengand 1
PMCID: PMC1369802  PMID: 10376875

Abstract

The gene for RsuA, the pseudouridine synthase that converts U516 to pseudouridine in 16S ribosomal RNA of Escherichia coli, has been deleted in strains MG1655 and BL21/DE3. Deletion of this gene resulted in the specific loss of pseudouridine516 in both cell lines, and replacement of the gene in trans on a plasmid restored the pseudouridine. Therefore, rsuA is the only gene in E. coli with the ability to produce a protein capable of forming pseudouridine516. There was no effect on the growth rate of rsuA- MG1655 either in rich or minimal medium at either 24, 37, or 42 degrees C. Plasmid rescue of the BL21/DE3 rsuA- strain using pET15b containing an rsuA gene with aspartate102 replaced by asparagine or threonine demonstrated that neither mutant was active in vivo. This result supports a role for this aspartate, located in a unique GRLD sequence in this gene, at the catalytic center of the synthase. Induction of wild-type and the two mutant synthases in strain BL21/DE3 from genes in pET15b yielded a strong overexpression of all three proteins in approximately equal amounts showing that the mutations did not affect production of the protein in vivo and thus that the lack of activity was not due to a failure to produce a gene product. Aspartate102 is found in a conserved motif present in many pseudouridine synthases. The conservation and distribution of this motif in nature was assessed.

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Selected References

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  1. Altschul S. F., Koonin E. V. Iterated profile searches with PSI-BLAST--a tool for discovery in protein databases. Trends Biochem Sci. 1998 Nov;23(11):444–447. doi: 10.1016/s0968-0004(98)01298-5. [DOI] [PubMed] [Google Scholar]
  2. Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997 Sep 1;25(17):3389–3402. doi: 10.1093/nar/25.17.3389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bairoch A., Apweiler R. The SWISS-PROT protein sequence data bank and its supplement TrEMBL in 1999. Nucleic Acids Res. 1999 Jan 1;27(1):49–54. doi: 10.1093/nar/27.1.49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bakin A. V., Ofengand J. Mapping of pseudouridine residues in RNA to nucleotide resolution. Methods Mol Biol. 1998;77:297–309. doi: 10.1385/0-89603-397-X:297. [DOI] [PubMed] [Google Scholar]
  5. Bakin A., Kowalak J. A., McCloskey J. A., Ofengand J. The single pseudouridine residue in Escherichia coli 16S RNA is located at position 516. Nucleic Acids Res. 1994 Sep 11;22(18):3681–3684. doi: 10.1093/nar/22.18.3681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bakin A., Lane B. G., Ofengand J. Clustering of pseudouridine residues around the peptidyltransferase center of yeast cytoplasmic and mitochondrial ribosomes. Biochemistry. 1994 Nov 15;33(45):13475–13483. doi: 10.1021/bi00249a036. [DOI] [PubMed] [Google Scholar]
  7. Bakin A., Ofengand J. Four newly located pseudouridylate residues in Escherichia coli 23S ribosomal RNA are all at the peptidyltransferase center: analysis by the application of a new sequencing technique. Biochemistry. 1993 Sep 21;32(37):9754–9762. doi: 10.1021/bi00088a030. [DOI] [PubMed] [Google Scholar]
  8. Bakin A., Ofengand J. Mapping of the 13 pseudouridine residues in Saccharomyces cerevisiae small subunit ribosomal RNA to nucleotide resolution. Nucleic Acids Res. 1995 Aug 25;23(16):3290–3294. doi: 10.1093/nar/23.16.3290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Becker H. F., Motorin Y., Planta R. J., Grosjean H. The yeast gene YNL292w encodes a pseudouridine synthase (Pus4) catalyzing the formation of psi55 in both mitochondrial and cytoplasmic tRNAs. Nucleic Acids Res. 1997 Nov 15;25(22):4493–4499. doi: 10.1093/nar/25.22.4493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Benson J. R., Querci della Rovere G. Sentinel lymph node in breast cancer. Lancet. 1999 Dec 4;354(9194):1998–1999. doi: 10.1016/s0140-6736(05)76770-2. [DOI] [PubMed] [Google Scholar]
  11. Blattner F. R., Plunkett G., 3rd, Bloch C. A., Perna N. T., Burland V., Riley M., Collado-Vides J., Glasner J. D., Rode C. K., Mayhew G. F. The complete genome sequence of Escherichia coli K-12. Science. 1997 Sep 5;277(5331):1453–1462. doi: 10.1126/science.277.5331.1453. [DOI] [PubMed] [Google Scholar]
  12. Conrad J., Sun D., Englund N., Ofengand J. 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. J Biol Chem. 1998 Jul 17;273(29):18562–18566. doi: 10.1074/jbc.273.29.18562. [DOI] [PubMed] [Google Scholar]
  13. Grosjean H., Szweykowska-Kulinska Z., Motorin Y., Fasiolo F., Simos G. Intron-dependent enzymatic formation of modified nucleosides in eukaryotic tRNAs: a review. Biochimie. 1997 May;79(5):293–302. doi: 10.1016/s0300-9084(97)83517-1. [DOI] [PubMed] [Google Scholar]
  14. Hamilton C. M., Aldea M., Washburn B. K., Babitzke P., Kushner S. R. New method for generating deletions and gene replacements in Escherichia coli. J Bacteriol. 1989 Sep;171(9):4617–4622. doi: 10.1128/jb.171.9.4617-4622.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Huang L., Pookanjanatavip M., Gu X., Santi D. V. A conserved aspartate of tRNA pseudouridine synthase is essential for activity and a probable nucleophilic catalyst. Biochemistry. 1998 Jan 6;37(1):344–351. doi: 10.1021/bi971874+. [DOI] [PubMed] [Google Scholar]
  16. Jensen K. F. The Escherichia coli K-12 "wild types" W3110 and MG1655 have an rph frameshift mutation that leads to pyrimidine starvation due to low pyrE expression levels. J Bacteriol. 1993 Jun;175(11):3401–3407. doi: 10.1128/jb.175.11.3401-3407.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. King T. C., Schlessinger D. S1 nuclease mapping analysis of ribosomal RNA processing in wild type and processing deficient Escherichia coli. J Biol Chem. 1983 Oct 10;258(19):12034–12042. [PubMed] [Google Scholar]
  18. Koonin E. V. Pseudouridine synthases: four families of enzymes containing a putative uridine-binding motif also conserved in dUTPases and dCTP deaminases. Nucleic Acids Res. 1996 Jun 15;24(12):2411–2415. doi: 10.1093/nar/24.12.2411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Korber P., Zander T., Herschlag D., Bardwell J. C. A new heat shock protein that binds nucleic acids. J Biol Chem. 1999 Jan 1;274(1):249–256. doi: 10.1074/jbc.274.1.249. [DOI] [PubMed] [Google Scholar]
  20. Kowalak J. A., Bruenger E., Hashizume T., Peltier J. M., Ofengand J., McCloskey J. A. Structural characterization of U*-1915 in domain IV from Escherichia coli 23S ribosomal RNA as 3-methylpseudouridine. Nucleic Acids Res. 1996 Feb 15;24(4):688–693. doi: 10.1093/nar/24.4.688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lafontaine D. L., Bousquet-Antonelli C., Henry Y., Caizergues-Ferrer M., Tollervey D. The box H + ACA snoRNAs carry Cbf5p, the putative rRNA pseudouridine synthase. Genes Dev. 1998 Feb 15;12(4):527–537. doi: 10.1101/gad.12.4.527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lecointe F., Simos G., Sauer A., Hurt E. C., Motorin Y., Grosjean H. Characterization of yeast protein Deg1 as pseudouridine synthase (Pus3) catalyzing the formation of psi 38 and psi 39 in tRNA anticodon loop. J Biol Chem. 1998 Jan 16;273(3):1316–1323. doi: 10.1074/jbc.273.3.1316. [DOI] [PubMed] [Google Scholar]
  23. Marvel C. C., Arps P. J., Rubin B. C., Kammen H. O., Penhoet E. E., Winkler M. E. hisT is part of a multigene operon in Escherichia coli K-12. J Bacteriol. 1985 Jan;161(1):60–71. doi: 10.1128/jb.161.1.60-71.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Massenet S., Motorin Y., Lafontaine D. L., Hurt E. C., Grosjean H., Branlant C. Pseudouridine mapping in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (snRNAs) reveals that pseudouridine synthase pus1p exhibits a dual substrate specificity for U2 snRNA and tRNA. Mol Cell Biol. 1999 Mar;19(3):2142–2154. doi: 10.1128/mcb.19.3.2142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. McGeoch D. J. Protein sequence comparisons show that the 'pseudoproteases' encoded by poxviruses and certain retroviruses belong to the deoxyuridine triphosphatase family. Nucleic Acids Res. 1990 Jul 25;18(14):4105–4110. doi: 10.1093/nar/18.14.4105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Niu L., Lane B. G., Ofengand J. Cloning and characterization of the 23S RNA pseudouridine 2633 synthase from Bacillus subtilis. Biochemistry. 1999 Jan 12;38(2):629–635. doi: 10.1021/bi9821869. [DOI] [PubMed] [Google Scholar]
  27. Nurse K., Wrzesinski J., Bakin A., Lane B. G., Ofengand J. Purification, cloning, and properties of the tRNA psi 55 synthase from Escherichia coli. RNA. 1995 Mar;1(1):102–112. [PMC free article] [PubMed] [Google Scholar]
  28. Ofengand J., Bakin A. Mapping to nucleotide resolution of pseudouridine residues in large subunit ribosomal RNAs from representative eukaryotes, prokaryotes, archaebacteria, mitochondria and chloroplasts. J Mol Biol. 1997 Feb 21;266(2):246–268. doi: 10.1006/jmbi.1996.0737. [DOI] [PubMed] [Google Scholar]
  29. Perrière G., Gouy M. WWW-query: an on-line retrieval system for biological sequence banks. Biochimie. 1996;78(5):364–369. doi: 10.1016/0300-9084(96)84768-7. [DOI] [PubMed] [Google Scholar]
  30. Picard V., Ersdal-Badju E., Lu A., Bock S. C. A rapid and efficient one-tube PCR-based mutagenesis technique using Pfu DNA polymerase. Nucleic Acids Res. 1994 Jul 11;22(13):2587–2591. doi: 10.1093/nar/22.13.2587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Prangishvili D., Klenk H. P., Jakobs G., Schmiechen A., Hanselmann C., Holz I., Zillig W. Biochemical and phylogenetic characterization of the dUTPase from the archaeal virus SIRV. J Biol Chem. 1998 Mar 13;273(11):6024–6029. doi: 10.1074/jbc.273.11.6024. [DOI] [PubMed] [Google Scholar]
  32. Raychaudhuri S., Conrad J., Hall B. G., Ofengand J. A pseudouridine synthase required for the formation of two universally conserved pseudouridines in ribosomal RNA is essential for normal growth of Escherichia coli. RNA. 1998 Nov;4(11):1407–1417. doi: 10.1017/s1355838298981146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Santer M., Santer U., Nurse K., Bakin A., Cunningham P., Zain M., O'Connell D., Ofengand J. Functional effects of a G to U base change at position 530 in a highly conserved loop of Escherichia coli 16S RNA. Biochemistry. 1993 Jun 1;32(21):5539–5547. doi: 10.1021/bi00072a007. [DOI] [PubMed] [Google Scholar]
  34. Simos G., Tekotte H., Grosjean H., Segref A., Sharma K., Tollervey D., Hurt E. C. Nuclear pore proteins are involved in the biogenesis of functional tRNA. EMBO J. 1996 May 1;15(9):2270–2284. [PMC free article] [PubMed] [Google Scholar]
  35. Supeková L., Supek F., Nelson N. The Saccharomyces cerevisiae VMA10 is an intron-containing gene encoding a novel 13-kDa subunit of vacuolar H(+)-ATPase. J Biol Chem. 1995 Jun 9;270(23):13726–13732. doi: 10.1074/jbc.270.23.13726. [DOI] [PubMed] [Google Scholar]
  36. Tatusov R. L., Altschul S. F., Koonin E. V. Detection of conserved segments in proteins: iterative scanning of sequence databases with alignment blocks. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):12091–12095. doi: 10.1073/pnas.91.25.12091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Thompson J. D., Gibson T. J., Plewniak F., Jeanmougin F., Higgins D. G. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 1997 Dec 15;25(24):4876–4882. doi: 10.1093/nar/25.24.4876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tsui H. C., Arps P. J., Connolly D. M., Winkler M. E. Absence of hisT-mediated tRNA pseudouridylation results in a uracil requirement that interferes with Escherichia coli K-12 cell division. J Bacteriol. 1991 Nov;173(22):7395–7400. doi: 10.1128/jb.173.22.7395-7400.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Watkins N. J., Gottschalk A., Neubauer G., Kastner B., Fabrizio P., Mann M., Lührmann R. Cbf5p, a potential pseudouridine synthase, and Nhp2p, a putative RNA-binding protein, are present together with Gar1p in all H BOX/ACA-motif snoRNPs and constitute a common bipartite structure. RNA. 1998 Dec;4(12):1549–1568. doi: 10.1017/s1355838298980761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wrzesinski J., Bakin A., Nurse K., Lane B. G., Ofengand J. Purification, cloning, and properties of the 16S RNA pseudouridine 516 synthase from Escherichia coli. Biochemistry. 1995 Jul 11;34(27):8904–8913. doi: 10.1021/bi00027a043. [DOI] [PubMed] [Google Scholar]
  41. Wrzesinski J., Nurse K., Bakin A., Lane B. G., Ofengand J. A dual-specificity pseudouridine synthase: an Escherichia coli synthase purified and cloned on the basis of its specificity for psi 746 in 23S RNA is also specific for psi 32 in tRNA(phe). RNA. 1995 Jun;1(4):437–448. [PMC free article] [PubMed] [Google Scholar]

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