大肠杆菌sRNA编码基因yigP结构与功能研究
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摘要
yigP基因存在于许多微生物中,其序列都相对保守。在大肠杆菌中,yigP (Gene ID:948915)基因位于辅酶Q合成相关基因ubiE和ubiB之间。GeneBank注释yigP基因是一个可能的蛋白质编码基因,然而却没有任何的文献进一步支持该结论。目前,并未发现有针对该基因的研究报道。
我们前期的研究发现,染色体yigP基因缺陷的菌株必需通过外源载体提供一个完整的yigP基因才能够生存,因此认为yigP是大肠杆菌生长必需基因。此外,还发现yigP上游区域具有启动子活性。使用RACE技术还确认了yigP转录一个252nt的RNA。
本论文中,我们发现yigP基因下游297bp的片段足以行使相应功能。利用lacZ作为报告基因,我们精确地鉴定了yigP启动子的边界。同时,结合点突变技术,对启动子的-10区和-35区进行了鉴定,以及发现了启动子上游的一个负转录调控序列。最重要的是,yigP基因对移码突变有免疫能力,这说明yigP并非编码蛋白质,而是一个非编码RNA基因。此外,RT-PCR显示yigP能够与其上下游基因ubiE和ubiB共转录。
总之,本研究显示了yigP是一个生长必需的非编码RNA基因
YigP gene exists in many species, and the sequence is conserved among bacteria. In Escherichia coli, yigP gene(GeneID:948915) locates between ubiquinone biosynthetic genes ubiE and ubiB. GeneBank annotates yigP as a putative protein-coding gene, however, no research focusing on the yigP gene has been reported yet.
Our previous work have confirmed that the strain with yigP inactivation in chromosome must rely on a complementary plasmid containing a wild-type yigP gene, to survive under laboratory growth conditions. Promoter activity was detected in the region of upstream yigP-Using RACE techniques, we confirmed that yigP gene was transcribed to a 252 nt RNA molecule.
In this study, we have shown that a 297bp fragment downstream of yigP was enough to complement the chromosomal defective yigP gene. Using lacZ as a report gene, we have identified the precise region of yigP promoter. Meanwhile, the -10 and -35 region of yigP promoter were identified by site mutations and a negative transcriptional regulatory region was also discovered associating with promoter activity. Notably, the yigP gene is immune to frameshift mutations. This indicates yigP is a non-coding RNA gene, rather than a protein coding gene as predicted in the GeneBank. What's more, our reverse transiption PCR have showed that yigP could cotranscribed with its neighbouring genes ubiE and ubiB.
In conclusion, our studies revealed that yigP is an essential non-coding RNA gene.
我们前期的研究发现,染色体yigP基因缺陷的菌株必需通过外源载体提供一个完整的yigP基因才能够生存,因此认为yigP是大肠杆菌生长必需基因。此外,还发现yigP上游区域具有启动子活性。使用RACE技术还确认了yigP转录一个252nt的RNA。
本论文中,我们发现yigP基因下游297bp的片段足以行使相应功能。利用lacZ作为报告基因,我们精确地鉴定了yigP启动子的边界。同时,结合点突变技术,对启动子的-10区和-35区进行了鉴定,以及发现了启动子上游的一个负转录调控序列。最重要的是,yigP基因对移码突变有免疫能力,这说明yigP并非编码蛋白质,而是一个非编码RNA基因。此外,RT-PCR显示yigP能够与其上下游基因ubiE和ubiB共转录。
总之,本研究显示了yigP是一个生长必需的非编码RNA基因
YigP gene exists in many species, and the sequence is conserved among bacteria. In Escherichia coli, yigP gene(GeneID:948915) locates between ubiquinone biosynthetic genes ubiE and ubiB. GeneBank annotates yigP as a putative protein-coding gene, however, no research focusing on the yigP gene has been reported yet.
Our previous work have confirmed that the strain with yigP inactivation in chromosome must rely on a complementary plasmid containing a wild-type yigP gene, to survive under laboratory growth conditions. Promoter activity was detected in the region of upstream yigP-Using RACE techniques, we confirmed that yigP gene was transcribed to a 252 nt RNA molecule.
In this study, we have shown that a 297bp fragment downstream of yigP was enough to complement the chromosomal defective yigP gene. Using lacZ as a report gene, we have identified the precise region of yigP promoter. Meanwhile, the -10 and -35 region of yigP promoter were identified by site mutations and a negative transcriptional regulatory region was also discovered associating with promoter activity. Notably, the yigP gene is immune to frameshift mutations. This indicates yigP is a non-coding RNA gene, rather than a protein coding gene as predicted in the GeneBank. What's more, our reverse transiption PCR have showed that yigP could cotranscribed with its neighbouring genes ubiE and ubiB.
In conclusion, our studies revealed that yigP is an essential non-coding RNA gene.
引文
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[2]Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants:the Keio collection. Mol Syst Biol. 2006;2:2006 0008.
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[4]Kim JN, Youm GW, Kwon YM. Essential genes in Salmonella enteritidis as identified by TnAraOut mutagenesis. Curr Microbiol.2008;57:391-4.
[5]Hamilton CM, Aldea M, Washburn BK, Babitzke P, Kushner SR. New method for generating deletions and gene replacements in Escherichia coli. J Bacteriol.1989;171:4617-22.
[6]Jennie E. Mitchell DZ, Stephen J. W. Busby and Stephen D. Minchin*. Identification and analysis of'extended ±10' promoters in Escherichia coli. Nucleic Acids Research. 2003;31:4689±95.
[7]Lim K, Chae CB. A simple assay for DNA transfection by incubation of the cells in culture dishes with substrates for beta-galactosidase. Biotechniques.1989;7:576-9.
[8]James L. Lissemore, Bayes, J, Calvey, M,, LR,, AC,, MT, et al. Green fluorescent protein is superior to blue fluorescent protein as a quantitative reporter of promoter activity in E. coli. Mol Biol Rep.2009; 36:1107-12.
[9]Ellis J, Bagshaw CR, Shaw WV. Kinetic mechanism of chloramphenicol acetyltransferase: the role of ternary complex interconversion in rate determination. Biochemistry. 1995;34:16852-9.
[10]Leclerc GM, Boockfor FR, Faught WJ, Frawley LS. Development of a destabilized firefly luciferase enzyme for measurement of gene expression. Biotechniques.2000;29:590-1,4-6, 8 passim.
[11]Miller WG, Leveau JH, Lindow SE. Improved gfp and inaZ broad-host-range promoter-probe vectors. Mol Plant Microbe Interact.2000;13:1243-50.
[12]Beckwith JR. Regulation of the lac operon. Recent studies on the regulation of lactose metabolism in Escherichia coli support the operon model. Science.1967;156:597-604.
[13]Kuo JT, Chang YJ, Tseng CP. Growth rate regulation of lac operon expression in Escherichia coli is cyclic AMP dependent. FEBS Lett.2003;553:397-402.
[14]Morse DE, Mosteller RD, Yanofsky C. Dynamics of synthesis, translation, and degradation of trp operon messenger RNA in E. coli. Cold Spring Harb Symp Quant Biol. 1969;34:725-40.
[15]Schleif R. AraC protein, regulation of the 1-arabinose operon in Escherichia coli, and the light switch mechanism of AraC action. FEMS Microbiol Rev.2010;34:779-96.
[16]Salgado H, Moreno-Hagelsieb G, Smith TF, Collado-Vides J. Operons in Escherichia coli: genomic analyses and predictions. Proc Natl Acad Sci U S A.2000;97:6652-7.
[17]Ermolaeva MD, White O, Salzberg SL. Prediction of operons in microbial genomes. Nucleic Acids Res.2001;29:1216-21.
[18]Keis S, Kaim G, Dimroth P, Cook GM. Cloning and molecular characterization of the atp operon encoding for the F1F0-ATP synthase from a thermoalkaliphilic Bacillus sp. strain TA2.A1. Biochim Biophys Acta.2004; 1676:112-7.
[19]ten Broeke-Smits NJ, Pronk TE, Jongerius I, Bruning O, Wittink FR, Breit TM, et al. Operon structure of Staphylococcus aureus. Nucleic Acids Res.2010;38:3263-74.
[20]Birney E, Stamatoyannopoulos JA, Dutta A, Guigo R, Gingeras TR, Margulies EH, et al. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature.2007;447:799-816.
[21]Washietl S, Pedersen JS, Korbel JO, Stocsits C, Gruber AR, Hackermuller J, et al. Structured RNAs in the ENCODE selected regions of the human genome. Genome Res. 2007;17:852-64.
[22]Huttenhofer A, Schattner P, Polacek N. Non-coding RNAs:hope or hype? Trends Genet. 2005;21:289-97.
[23]Poole AM, Jeffares DC, Penny D. The path from the RNA world. J Mol Evol.1998;46:1-17.
[24]Waters LS, Storz G. Regulatory RNAs in bacteria. Cell.2009;136:615-28.
[25]Altuvia S. Identification of bacterial small non-coding RNAs:experimental approaches. Curr Opin Microbiol.2007; 10:257-61.
[26]Urbanowski ML, Stauffer LT, Stauffer GV. The gcvB gene encodes a small untranslated RNA involved in expression of the dipeptide and oligopeptide transport systems in Escherichia coli. Mol Microbiol.2000;37:856-68.
[27]Altuvia S, Zhang A, Argaman L, Tiwari A, Storz G. The Escherichia coli OxyS regulatory RNA represses fhlA translation by blocking ribosome binding. EMBO J.1998; 17:6069-75.
[28]Majdalani N, Chen S, Murrow J, St John K, Gottesman S. Regulation of RpoS by a novel small RNA:the characterization of RprA. Mol Microbiol.2001;39:1382-94.
[29]Pulvermacher SC, Stauffer LT, Stauffer GV. The small RNA GcvB regulates sstT mRNA expression in Escherichia coli. J Bacteriol.2009;191:238-48.
[30]Walker SC, Engelke DR. Ribonuclease P:the evolution of an ancient RNA enzyme. Crit Rev Biochem Mol Biol.2006;41:77-102.
[31]Keenan RJ, Freymann DM, Stroud RM, Walter P. The signal recognition particle. Annu Rev Biochem.2001;70:755-75.
[32]Keiler KC, Waller PR, Sauer RT. Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA. Science.1996;271:990-3.
[33]Gillet R, Felden B. Emerging views on tmRNA-mediated protein tagging and ribosome rescue. Mol Microbiol.2001;42:879-85.
[34]Klein RJ, Misulovin Z, Eddy SR. Noncoding RNA genes identified in AT-rich hyperthermophiles. Proc Natl Acad Sci U S A.2002;99:7542-7.
[35]Kawano M, Reynolds AA, Miranda-Rios J, Storz G. Detection of 5'- and 3'-UTR-derived small RNAs and cis-encoded antisense RNAs in Escherichia coli. Nucleic Acids Res. 2005;33:1040-50.
[36]Liu JM, Livny J, Lawrence MS, Kimball MD, Waldor MK, Camilli A. Experimental discovery of sRNAs in Vibrio cholerae by direct cloning,5S/tRNA depletion and parallel sequencing. Nucleic Acids Res.2009;37:e46.
[37]Beckmann BM, Grunweller A, Weber MH, Hartmann RK. Northern blot detection of endogenous small RNAs (approximately 14 nt) in bacterial total RNA extracts. Nucleic Acids Res.2010;38:e147.
[38]Livny J, Fogel MA, Davis BM, Waldor MK. sRNAPredict:an integrative computational approach to identify sRNAs in bacterial genomes. Nucleic Acids Res.2005;33:4096-105.
[39]Bouvier M, Sharma CM, Mika F, Nierhaus KH, Vogel J. Small RNA binding to 5'mRNA coding region inhibits translational initiation. Mol Cell.2008;32:827-37.
[40]Heidrich N, Moll I, Brantl S. In vitro analysis of the interaction between the small RNA SRI and its primary target ahrC mRNA. Nucleic Acids Res.2007;35:4331-46.
[41]Lustig Y, Wachtel C, Safro M, Liu L, Michaeli S.'RNA walk' a novel approach to study RNA-RNA interactions between a small RNA and its target. Nucleic Acids Res.2010;38:e5.
[42]Urban JH, Vogel J. Translational control and target recognition by Escherichia coli small RNAs in vivo. Nucleic Acids Res.2007;35:1018-37.
[43]Lutz R, Bujard H. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res. 1997;25:1203-10.
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