Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23940
標題: Functional analysis of the regulatory protein LuxZ-R&R/DNA binding from Photobacterium leiognathi
Photobacterium leiognathi 調節基因 luxZ 主導蛋白對調節區域結合之功能與調節機制分析
作者: Hsu, Chien-Ming
徐建明
關鍵字: 海生螢光菌;luxZ
出版社: 生物化學研究所
引用: AbouKhair, N. K., Ziegler, M. M., and Baldwin, T. O. (1985) Bacterial luciferase: demonstration of a catalytically competent altered conformational state following a single turnover. Biochemistry 24, 3942-3947 Alting-Mees, M. A., and Short, J. M. (1989) pBluescript II: gene mapping vectors. Nucleic Acids Res. 17, 9494 Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403-410 Anne Farewell, Alfredo A. Diez, Concetta C. Dirusso, and Thomas Nystrom. (1996) Role of the Escherichia coli FadR regulator in stasis survival and growth phase-dependent expression of the uspA, fad, and fab Genes. J. Bacteriol. 178, 6443-6450 Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (eds.) (1991) Current Protocols in Molecular Biology John Wiley & Sons, New York, NY. Baldwin, T. O., Devine, J. H., Heckel, R. C., Lin, J.-W., and Shadel, G. S. (1989) The complete nucleotide sequence of the lux regulon of Vibrio fischeri and the luxABN region of Photobacterium leiognathi and the mechanism of control of bacterial bioluminescence. J. Biolumin. Chemilumin. 4, 326-341 Baldwin, T. O., Ziegler, M. M., and Powers, D. A. (1979) Covalent structure of subunits of bacterial luciferase: NH2-terminal sequence demonstrates subunits homology. Proc. Natl. Acad. Sci. U.S A. 76, 4887-4889 Baldwin, T. O., Treat, M. L., and Daubner, S. C. (1990) Cloning and expression of the luxY gene from Vibrio fischeri strain Y-1 in Escherichia coli and complete amino acid sequence of the yellow fluorescent protein. Biochemistry 29, 5509-5515 Baumann, P., Baumann, L., Wooklalis, M., and Band, S. (1983) Evolutionary relationships in Vibrio and Photobacterium: a basis for a natural classification. Annu. Rev. Microbiol. 37, 369-398 Bassler, B. L., and Silverman, M. R. (1995) Intercellular communication in marine Vibrio species: density-dependent regulation of the expression of bioluminescence. In: Two-component signal transduction (Eds Hoch, J. A., and Silhavy, T. J.), pp. 431-445, American Society for Microbiology, Washington, D. C. Bassler, B. L., Wright, M., Showalter, R. E., and Silverman, M. R. (1993) Intercellular signalling in Vibrio harveyi: sequence and function of genes regulating expression of luminescence. Mol. Microbiol. 9, 773-786 Bassler, B. L., Wright, M., and Silverman, M. R. (1994a) Sequence and function of LuxO, a negative regulator of luminescence in Vibrio harveyi. Mol. Microbiol. 12, 403-412 Bassler, B. L., Wright, M., and Silverman, M. R. (1994b) Multiple signalling systems controlling expression of luminescence in Vibrio harveyi: sequence and function of genes encoding a second sensory pathway. Mol. Microbiol. 13, 273-286 Bassler, B. L. (1999) How bacteria talk to each other: regulation of gene expression by quorum sensing. Curr. Opin. Microbiol. 2, 582-587 Boylan, M., Graham A. F., and Meighen, E. A. (1985) Functional identification of the fatty acid reductase components encoded in the luminescence operon of Vibrio fischeri. J. Bacteriol. 163, 1186-1190 Chao, Y.-F. (1992) Gene expression and regulation of bacterial luciferase: nucleotide sequence and analysis of lux operon regulatory region and neighboring genes from Photobacterium leiognathi. MS Thesis. Institute of Molecular Biology, National Chung Hsing University. Chang, A. C. Y., and Cohen, S. N. (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J. Bacteriol. 134, 1141-1156 Chen, Y.-J. (2001) Cloning and functional analysis of the specific genes that enhanced by the LuxZ from Photobacterium leiognathi. MS Thesis. Institute of Molecular Biology, National Chung Hsing University. Cohn, D. H., Mileham, A. J., Simon, M. I., Nealson, K. H., Rausch, S. K., Bonam, D., and Baldwin, T. O. (1985) Nucleotide sequence of the luxA gene of Vibrio harveyi and the complete amino acid sequence of the a subunit of bacterial luciferase. J. Biol. Chem. 260, 6139-6146 Concetta C. DiRussoS, Tamra L. Heimert, and Amy K. Metzger. (1992) Characterization of FadR, a global transcriptional regulator of fatty acid metabolism in Escherichia coli.J. Biol. Chem. 267, 8685-8691 Concetta C. DiRusso, Vadim Tsvetnitsky, Peter Højrup, and Jens Knudsen. (1998) Fatty acyl-CoA binding domain of the transcription factor fadR. J. Biol. Chem. 273, 33652-33659 Concetta C. DiRusso, Paul N. Black, and James D. Weimar. (1999) Molecular inroads into the regulation and metabolism of fatty acids, lessons from bacteria. Progress in Lipid Research 38, 129-197 Daan M.F.van Aalten, Concetta C.DiRusso, and Jens Knudsem. (2001) The structural basis of acyl coenzyme A-dependent of the transcription factpr FadR. EMBO J. 20, 2041-2050 D. Lane, P. Prentki, M. Chandler. (1992) Use of gel retardation to analyze protien-nucleic acid interaction. Microbiology review 56, 509-528 D.M.F.van Aalten, C.C.DiRusso, J.Knudsen, and R.K.Wierenga. (2000) Crystal structure of FadR, a fatty acid responsive transcription factor with a novel acyl coenzyme A-binding fold. 19, 5167-5177 Doherty, A. J., and Suh, S. W. (2000) Structural and mechanistic conservation in DNA ligases. Nucleic Acids Res. 28, 4051-4058 Dunn, D. K., Michaliszyn, G. A., Bogacki, I. G., and Meighen, E. A. (1973) Conversion of aldehyde to fatty acid in the bacterial bioluminescent reaction. Biochemistry 12, 4911-4918 Engebrecht, J., Nealson, K., Silverman, M. (1983) Bacterial bioluminescence: isolation and genetic analysis of functions from Vibrio fischeri. Cell 32, 773-781 Engebrecht, J., and Silverman, M. (1984) Identification of genes and gene products necessary for bacterial bioluminescence. Proc. Natl. Acad. Sci. U.S.A. 81, 4145-4158 Engebrecht, J., and Silverman, M. (1987) Nucleotide sequence of the regulatory locus controlling expression of bacterial gene for bioluminescence. Nucleic Acids Res. 15, 10455-10467 Farmer, J. J., Jorgensen, J. H., Grimont, P. A. D., Akhurst, R. J., Poinar, G. O., Jr., Ageron, E., Pierce, G. V., Smith, J. A., Carter, G. P., Wilson, K. L., and Jickmanbrenner, F. W. (1989) Xenorhabdus luminescent (DNA hybridization group 5) from human clinical specimen. J. Clinical Microbiol. 27, 1594-1600 Francisco, W. A., Abu-Soud, H. M., DelMonte, A. J., Singleton, D. A., Baldwin, T. O., and Raushel, F. M. (1998) Deuterium kinetic isotope effects and the mechanism of the bacterial luciferase reaction. Biochemistry 37, 2596-2606 Freeman, J. A., and Bsaaler, B. L. (1999a) A genetic analysis of the function of LuxO, a two-component response regulator involved in quorum sensing in Vibrio harveyi. Mol. Microbiol. 31, 665-677 Freeman, J. A., Lilley, B. N., and Bsaaler, B. L. (2000) A genetic analysis of the functions of LuxN: a two-component hybrid sensor kinase that regulates quorum sensing in Vibrio harveyi. Mol. Microbiol. 35, 139-149 Frishman, D., and Argos, P. (1995) Knowledge-based secondary structure assignment. Proteins 23, 566-579 Fuqua, W. C., and Greenberg, E. P. (1998) Self perception in bacteria: quorum sensing with acylated homoserine lactones. Curr. Opin. Microbiol. 1, 183-189 Fuqua, W. C., Winans, S. C., and Greenberg, E. P. (1994) Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators. J. Bacteriol. 176, 269-275 Fuqua, W. C., Winans, S. C., and Greenberg, E. P. (1996) Census and consensus in bacterial ecosystems: the LuxR-LuxI family of quorum-sensing transcriptional regulators. Annu. Rev. Microbiol. 50, 727-751 Gavigan, S. A., Nguyen, T., Nguyen, N., and Senear, D. F. (1999) Role of Multiple CytR binding sites on cooperativity, competition, and induction at the Escherichia coli udp promoter. J. Biol. Chem. 274, 16010-16019 Geourjon, C., and Deleage, G. (1995) SOPMA: Significant improvements in protein secondary structure prediction from multiple alignments. Comput. Applic. Biosci. 11, 681-684 Gray, K. M. (1997) Intercellular communication and group behavior in bacteria. Trends Microbiol. 5, 184-188 Gui, L. Sunnarborg, A., and Laporte, D. C. (1996) Regulated expression of a repressor protein: FadR activates iclR. J. Bacteriol. 178, 4704-4709 Gunsalus-Miguel, A., Meighen, E. A., Nicoli, M. Z., Nealson, K. H., and Hastings, J. W. (1972) Purification and properties of bacterial luciferase. J. Biol. Chem. 247, 398-404 Hanahan, D. (1983) Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 557-580 Hastings, J. W. (1986) Bioluminescence in bacteria and dinoflagellates. In: Light emission in plants and bacteria (Eds. Govindiee, A. J., and Fork, D. C.), pp. 363-398. Acdemic Press, Inc., New York. Hastings, J. W., Baldwin, T. O., and Ziegler-Nicoli, M. M. (1978) Bacterial luciferase: assay, purification, and properties. Methods Enzymol. 57, 135-152 Hastings, J. W., and Nealson, K. H. (1977) Bacterial bioluminescence. Annu. Rev. Microbiol. 31, 549-595 Hastings, J. W., Potrikas, C. J., Gupta, S. C., Kurfurst, M., and Makemson, J. C. (1985) Biochemistry and physiology of bioluminescent bacteria. Adv. Microbiol. Physiol. 26, 235-291 Haygood, M. G. (1990) Relationship of the luminous bacterial symbiont of the Caribbean flashlight fish, Kryptophanaron alfredi (family Anomalopidae), to other luminous bacteria based on bacterial luciferase (luxA) genes. Arch. Microbiol. 154, 496-503 Huang, C.-H. (2002) Cloning and functional analysis of the specific genes that enhance bioluminescence of the lux operon from Photobacterium leiognthi. MS Thesis. Institute of Biochemistry, National Chung Hsing University. Huisman, G. W., and Kolter, R. (1994) Sensing starvation: a homoserine lactone-dependent signaling pathway in Escherichia coli. Science 265, 537-539 Ibarra, J. A., Villalba, M. I., and Puente, J. L. (2003) Identification of the DNA binding sites of PerA, the transcriptional activator of the bfp and per operons in enteropathogenic Escherichia coli. J. Bacteriol. 178, 4704-4709 Ish-Horowitzs, D., and Burke, J. F. (1981) Rapid and efficient cosmid cloning. Nucleic. Acids Res. 9, 2989-2998 Johansson, J., Mandin, P., Renzoni, A., Chiaruttini, C., Springer, M., and Cossart, P. (2002) An RNA thermosensor controls expression of virulence genes in Listeria monocytogenes. Cell 110, 551-561 Johnston, T. C., Thompson, R. B., and Baldwin, T. O. (1986) Nucleotide sequence of the luxB gene of Vibrio harveyi and the complete amino acid sequence of the b subunit of bacterial luciferase. J. Biol. Chem. 261, 4805-4811 Kita, A., Kasai, S., and Miki, K. (1995) Crystal structure determination of a flavoprotein FP390 from a luminescent bacterium, Photobacterium phosphoreum. J. Biochem. 117, 575-578 Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 Lee, J. (1985) The mechanism of bacterial bioluminescence. In: Chemiluminescence and Bioluminescence. (Ed. Burr, J. G.), pp. 401-437, Marcel Dekker, Inc., New York. Lee, J., O¢Kane, D. J., and Visser, A. J. W. G. (1985) Spectral properties and function of two lumazine proteins from Photobacterium. Biochemistry 24, 1476-1483 Lin, J.-W. (1989) Active center studies of bacterial luciferase by nucleotide sequence analysis and site-directed mutagenesis. Ph. D. Dissertation of Texas A&M University, College Station, Texas, USA. Lin, J.-W., Chao, Y.-F., and Weng, S.-F. (1995a) Nucleotide sequence and functional analysis of regulatory region of the lumP and the lux operon from Photobacterium leiognathi. Biochem. Biophys. Res. Commun. 210, 938-947 Lin, J.-W., Chao, Y.-F., and Weng, S.-F. (1996b) Nucleotide sequence and functional analysis of the luxE gene encoding acyl-protein sythetase of the lux operon from Photobacterium leiognthi. Biochem. Biophys. Res. Commum. 228, 764-773 Lin, J.-W., Chao, Y.-F., and Weng, S.-F. (1998a) Characteristic analysis of the luxG gene encoding the probable flavin reductase that resides in the lux operon of Photobacterium leiognathi. Biochem. Biophys. Res. Commun. 246, 446-452 Lin, J.-W., Chao, Y.-F., and Weng, S.-F. (2001) Riboflavin synthesis genes ribE, ribA, ribH, ribA reside in the the lux operon of Photobacterium leiognathi. Biochem. Biophys. Res. Commun. 284, 587-595 Lin, J.-W., Lin, B.-J., and Weng, S.-F. (1998b) Characteristic analysis of the luzA gene encoding chaperon from Photobacterium leiognthi related to bioluminescence. Biochem. Biophys. Res. Commum. 244, 838-842 Lin, J.-W., Lu, H.-C., Chen, H.-Y., and Weng, S.-F. (1997) The pkI gene encoding pyruvate kinase I links to the luxZ gene which enhances bioluminescence of the lux operon from Photobacterium leiognthi. Biochem. Biophys. Res. Commum. 239, 228-234 Lin, J.-W., Yu, K.-Y., Chao, Y.-F., and Weng, S.-F. (1995b). The lumQ gene is linked to the lumP gene and the lux operon in Photobacterium leiognathi. Biochem. Biophys. Res. Commun. 210, 938-947 Lin, J.-W., Yu, K.-Y., Chen, H.-Y., and Weng, S.-F. (1996a). Regulatory region with putA gene of proline dehydrogenase that links to the lum and lux operons in Photobacterium leiognathi. Biochem. Biophys. Res. Commun. 219, 868-875 Lu, H.-C. (1996) Identification and functional analysis of the luxZ gene that enhances bioluminescence of the lux operon from Photobacterium leiognthi. MS Thesis. Institute of Agricultural Biotechnology, National Chung Hsing University. Meighen, E. A. (1988) Enzymes and genes from the lux operons of bioluminescent bacteria. Annu. Rev. Microbiol. 42, 151-176 Meighen, E. A. (1991) Molecular biology of bacterial bioluminescence. Microbiol. Rev. 55, 123-142 Meighen, E. A., and Dunlap, P. V. (1993) Physiological, biochemical and genetic control of bacterial bioluminescence. Adv. Mibrob. Physiol. 34, 1-67 Meighen, E. A., and MacKenzie, R. E. (1973) Flavin specificity of enzyme-substrate intermediates in the bacterial bioluminescent reaction. Biochemistry 12, 1482-1491 Michael P. Spector, Concetta C. DiRusso, Mark J. Pallen, Francisco Garcia del Portillo, Gordon Dougan and B. Brett Finlay. (1999) The medium-/long-chain fatty acyl-CoA dehydrogenase (fadF) gene of Salmonella typhimurium is a phase 1 starvation-stress response (SSR) locus. Microbiology. 145, 15-31 Moore, S. A., and James, M. N. G. (1995) Structural refinement of the non-fluorescent flavoprotein from Photobacterium leiognathi at 1.60 Å resolution. J. Mol. Biol. 249, 195-214 Moore, S. A., James, M. N. G., O¢Kane, D. J., and Lee, J. (1993) Crystal structure of a flavoprotein related to the subunits of bacterial luciferase. EMBO J. 12, 1767-1774 Narayan Raman, and Concetta C. DiRusso. (1995) Analysis of Acyl Coenzyme A Binding to the Transcription Factor FandR and Identification of Amino Acid Residues in the Carboxyl Terminus Required for Ligand Binding. J. Biol. Chem. 270, 1092-1097 Narayan Raman, Paul N. Black, and Concetta C. DiRusso. (1997) Characterization of the Fatty Acid-responsive Transcription Factor FadR. J. Biol. Chem. 272, 30645-30650 Nealson, K. H., and Hastings, J. W. (1979) Bacterial bioluminescence: its control and ecological significance. Microbiol. Rev. 43, 496-518 Petushkov, V. N., Ketelaars, M., Gibson, B. G., and Lee, J. (1996) Interaction of Photobacterium leiognathi and Vibrio fischeri Y1 luciferases with fluorescent (antenna) proteins: bioluminescence effects of the aliphatic additive. Biochemistry 35, 12086-12093 Riendeau, D., Rodriguez, A., and Meighen, E. A. (1982) Resolution of the fatty acid reductase from Photobacterium phosphoreum into acyl-protein synthetase and acyl-CoA reductase activities. Evidence for an enzyme complex. J. Biol. Chem. 257, 6908-6915 Riendeau, D., and Meighen, E. A. (1979) Evidence for a fatty acid reductase catalyzing the synthesis of aldehydes for the bacterial bioluminescent reaction. J. Biol. Chem. 154, 7488-7490 Rigali, S., Derouaux, A., Giannotta, F., and Dusart, J. (2002) Subdivision of the helix-turn-helix GntR family of bacterial regulators in the FadR, HutC, MocR, and YtrA subfamilies. J. Biol. Chem. 277, 12507-12515 Rodriguez, A., Riendeau, D., and Meighen, E. A. (1983a) Purification of the acyl-CoA reductase component from a complex responsible for the reduction of fatty acids in bioluminescent bacteria. Properties and acyl-transferase activity. J. Biol. Chem. 258, 5233-5238 Rodriguez, A., Wall, L., Riendeau, D., and Meighen, E. A. (1983b) Fatty acid acylation of proteins in bioluminescent bacteria. Biochemistry 22, 5604-5611 Sambrook, F., Fritsch, E. F., and Maniatis, T. (1989) Molecular cloning: A Laboratory Manual, 2nd edit., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Sancar, A., Hack, A. M., and Rupp, W. D. (1979) Simple method for identification of plasmid-coded proteins. J. Bacteriol. 137, 692-693 Sanger, F., Nicklen, S., and Coulson, A. R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74, 5463-5467 Schagger, H., and von Jagow, G. (1987) Tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 368-379 Short, J. M., Fernandez, J. M., Sorge, J. A., and Huse, W. D. (1988) Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties. Nucleic Acids Res. 16, 7583-7600 Showalter, R. E., Martin, M. O., and Silverman, M. R. (1990) Cloning and nucleotide sequence of luxR, a regulatory gene controlling bioluminescence in Vibrio harveyi. J. Bacteriol. 172, 2946-2954 Shyu, J.-H. (1998) Identification and characterization of the specific gene that enhances bioluminescence of the lux operon from Photobacterium leiognthi. MS Thesis. Institute of Agricultural Biotechnology, National Chung Hsing University. Silverman, M., Martin, M., and Engebrecht, J. (1988) Regulation of luminescence in marine bacteria. In: Genetics of bacterial diversity (Eds. Hopwood, D., and Chater, K. F.), pp. 71-85, Academic Press, Inc., New York. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185, 60-89 Swartzman, E., Kapoor, S., Graham, A. F., and Meighen, E. A. (1990a) A new Vibrio fischeri lux gene precedes a bidirectional termination site for the lux operon. J. Bacteriol. 172, 6796-6802 Swartzman, E., Miyamoto, C., Graham, A., and Meighen, E. A. (1990b) Delineation of the transcriptional boundaries of the lux operon of Vibrio harveyi demonstrates the presence of two new lux genes. J. Biol. Chem. 265, 3513-3517 Swift, S., Stewart, G. S. A. B., and Williams, P. (1996b) The inner workings of a quorum sensing signal generator. Trends. Microbiol. 4, 463-465 Swift, S., Throup, J. P., Williams, P., Salmond, G. P. C., and Stewart, G. S. A. B. (1996a) Quorum sensing: a population-density component in the determinants of bacterial phenotype. Trends. Biochem. Sci. 21, 214-219 Szallas, E., Koch, C., Fodor, A., Burghardt, J., Buss, O., Szentirmai, A., Nealson, K. H., and Stackebrandt, E. (1997) Phylogenetic evidence for the taxonomic heterogeneity of Photorhabdus luminescent. Int. J. Syst. Bacteriol. 47, 402-407 Szittner, R., and Meighen, E. (1990) Nucleotide sequence, expression and properties of luciferase coded by lux genes from a terrestrial bacterium. J. Biol. Chem. 265, 16581-16587 Tabor, S., and Richardson, C. C. (1985) A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc. Natl. Acad. Sci. U.S.A. 82, 1074-1078 Tabor, S., and Richardson, C. C. (1987) DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc. Natl. Acad. Sci. U.S A. 84, 4767-4771 Tu, S.-C., and Mager, H. I. X. (1995) Biochemistry of bacterial bioluminescence. Photochem. Photobiol. 62, 615-624 Ulitzur, S., and Hastings, J. W. (1979) Evidence for tetradecanal as the natural aldehyde in bacterial bioluminescence. Proc. Natl. Acad. Sci. U.S A. 76, 265-267 Watanabe, T., Inoue, R., Kimura, N., and Furukawa, K. (2000) Versatile transcription of biphenyl catabolic bph operon in Pseudomonas pseudoalcaligenes KF707. J. Biol. Chem. 275, 31016-31023 Yanisch-Perron, C., Vieira, J., and Messing, J. (1985) Improved M13 phage cloning vectors and host strains nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103-119 Ziegler, M. M., and Baldwin, T. O. (1981) Biochemistry of bacterial biolumine- scence. Curr. Top. Bioenerg. 12, 65-113
摘要: 
P. leiognathi ATCC 25521 genome library was constructed in E. coli to select the specific regulatory gene(s) that enhances the lux operon by in trans Lux-bioassays. The luxZ gene was cloned. The Mr of the encoded LuxZ protein is Mr 25,807 Da and pI = 5.85. Sequence alignment of the LuxZ and the related proteins revealed that the N-terminal has a helix-turn-helix (H-T-H) DNA-binding motif and the C-terminal domain has a dimerization domain which, was subclassified into FadR sub-family of GntR family. Gel filtration chromatography assays demonstrated that the LuxZ could form homodimer in EMSA reaction buffer. Electrophoretic mobility shift assays (EMSA) elicited that the LuxZ-R&R/DNA binding affinity increased with the LuxZ concentration, which make the retarded bands as ladder-shape pattern; it showed that the LuxZ kept RNAP-R&R/DNA binding affinity. DNase I footprinting assays revealed that the consensus LuxZ-binding sequence was 5'-CTnnTTATTTAnTTnAnnT-3' in R&R[ufo], which located downstream and upstream of ufo promoter and partial overlapped; the consensus LuxZ-binding sequence was 5'-TtnAnnTAnATTnnTnnT-3' in R&R[orf2], which located partial and downstream of P(II)-promoter, and downstream intern-promoter; the LuxZ-binding sequences was 5'-CGTCGAAGCTAATTATACCTT-3' in R&R[luxZ], which located P*-promoter -10 sequence; the consensus LuxZ-binding sequence was 5'-nGnnTA nGTnnnnTnTT-3' in PL741 R&R[lux], which located partial P(I)-promoter and SRR; the consensus LuxZ-binding sequence was 5'-ATAATGGATnnATTATTTTT-3' in ATCC 25521 R&R[lux], which located P(II)-promoter and upstream of it. The Lux-bioassays showed that there were only two-fold increased when luxZ in trans with the above regulatory regions. The overall consensus LuxZ-binding sequences are 5'-TnnAnnTnnATTnTTnnT-3'.

前之研究構築 P. leiognathi ATCC 25521 genome library 選殖出可增強螢光表現之基因 luxZ,其主導調節蛋白 LuxZ 生成,計算分子量為 Mr = 25,807 Da;pI = 5.85。胺基酸序列比對顯示 LuxZ 之 N-terminal 含高度保留之 helix-turn-helix (H-T-H) motif,具 DNA 結合能力,其 C-terminal 含十個 helix 之 dimerization domain,歸類為 GntR family 之 FadR sub-family。Gel filtration chromatography 分析顯示 LuxZ 可結合為 homodimer 形式。Electrophoretic mobility shift assays (EMSA) 分析顯示 LuxZ 對所調控之調節區域的結合,會隨蛋白濃度增加而有一個以上之 dimer 結合,使 retarded band 呈現梯狀 pattern。於 LuxZ 與 RNA polymerase (RNAP) 同時存在下,LuxZ 於特定濃度下可強化 RNAP 對調節區域之結合。DNase I footprinting 分析顯示 LuxZ 於 R&R[ufo] 之 consensus binding sequence 為 5’-CTnnTTATTTAnTTnA nnT-3’,位於 promoter 上、下游並部分涵蓋 -10 序列;於 R&R[orf2] 之 LuxZ-binding sequence 為 5’-TTnAnnTAnATTnnTnnT-3’,涵蓋部份 P(II)- promoter 與其下游處以及基因內 intern-promoter 下游 SD sequence 附近;於 luxZ 調節區域 LuxZ-binding sequence 為 5’-CGTCGAAGCTAATTATACC TT-3’,位於 P*-promoter 之 -10 序列;於 PL741 R&R[lux] 之 LuxZ-binding sequence 為 5’-nGnnTAnGTnnnnTnTT-3’,涵蓋部份 P(I)-promoter 及上游 SRR;於 ATCC 25521 R&R[lux] 之 LuxZ-binding sequence 為 5’-ATAATGGATnnATTATTTTT-3’,位於 P(II)-promoter 及其上游處。前之 Lux-bioassays 顯示 luxZ 可增強上述調節區域之基因表現約二倍。綜觀上述 LuxZ binding sequences,推測 LuxZ 可能以 5’-TnnAnnTnnATTnTTnnT-3’ 之序列為其結合特徵。LuxZ 之所以可調節此等基因,可能因其結合之位置若可協助 RNAP binding 即可強化該基因之 transcription。LuxZ 之 binding locus 決定其功能。
URI: http://hdl.handle.net/11455/23940
其他識別: U0005-0806200616380900
Appears in Collections:生物化學研究所

Show full item record
 

Google ScholarTM

Check


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.