跳到主要內容

臺灣博碩士論文加值系統

(3.236.68.118) 您好!臺灣時間:2021/07/31 21:20
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:張志強
研究生(外文):Chih-Chiang Chang
論文名稱:以X-射線結晶學探討汞離子誘發雙重功能轉錄調節因子MerR構型變化之結構機轉
論文名稱(外文):Structural Basis of the Hg2+-Mediated Conformational Switching of the Dual-Function Transcriptional Regulator MerR
指導教授:詹迺立
口試委員:蕭傳鐙徐駿森黃介辰曾秀如
口試日期:2015-05-29
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:生物化學暨分子生物學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:77
中文關鍵詞:汞抗性操作組轉錄調控區塊蛋白質結構轉錄抑制態轉錄活化態MerR 家族汞結合位點
外文關鍵詞:mer operonoperator/promoter regioncrystal structurerepressoractivatorMerR familyHg2+ binding site
相關次數:
  • 被引用被引用:0
  • 點閱點閱:143
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
汞抗性操作組 (mer operon) 上帶有許多功能性蛋白質基因,可對環境中有毒性的汞離子 (Hg2+) 以及有機汞複合物 (organomercurial compounds) 產生抗性,這些蛋白質具有感應 (sensing protein) 、運輸 (transport protein) 和去毒性 (detoxification protein) 等功能。雙重功能之轉錄調控因子MerR 能對汞抗性操作組之表現與否進行嚴緊的調控。當細菌中沒有汞離子時,MerR 會與汞抗性操作組的轉錄調控區塊 (operator/promoter (O/P)) 結合,抑制汞抗性操作組之轉錄作用,而當汞離子存在時,MerR可與汞離子結合並轉換為轉錄活化因子,啟始汞抗性操作組之轉錄作用,以生成與汞抗性相關之功能性蛋白質。大多數啟動子(promoter)之RNA聚合酶 (RNA polymerase) 結合位-35和-10區塊之間隔為17±1 bps,DNA序列分析發現汞抗性操作組轉錄調控區塊 (mer operon O/P) RNA聚合酶結合位之間隔為19~20 bps,且中間部分帶有一段偽迴文序列 (pseudo-palindromic sequence),使汞抗性操作組之轉錄調控不同於一般基因之轉錄調控機制。汞抗性操作組轉錄調控區塊與MerR結合後,可經由汞離子引發之MerR二聚體發生構型變化,導致汞抗性操作組轉錄調控區 -10和-35區塊之距離縮短,並旋轉至相同位面上,因而活化轉錄作用。為了瞭解汞離子如何與MerR二聚體結合並活化汞抗性操作組之轉錄,在本實驗中,我們解出了Bacillus megaterium strain MB1之汞抗性操作組轉錄調控因子MerR之原態(apo)與汞離子結合態(Hg2+-bound)兩種狀態下之蛋白質結構(簡稱為apo-MerR和 Hg2+-MerR),分別代表MerR抑制態 (repressor) 和活化態 (activator)的蛋白質構型。其中apo-MerR結構更是目前已知結構之MerR 家族 (MerR family) 蛋白質中,唯一沒有配體 (ligand) 結合的全長蛋白質構型。比較兩者之蛋白構型差異,不但可以得知MerR二聚體如何組成一對以平面三角配位與汞離子結合的作用位點,並且可了解汞離子與apo-MerR結合之後所引發的二級、三級和四級結構變化。藉由汞離子所引起MerR二聚體之DNA結合位構型變化,可合理解釋MerR與汞離子之交互作用,如何影響轉錄調控區塊之構型變化並調控汞抗性操作組之轉錄作用。

The mer operon confers bacterial resistance to environmental inorganic mercury (Hg2+) and organomercurial compounds by encoding proteins involved in the sensing, transport, and detoxification of these cytotoxic agents. Expression of the mer operon is tightly regulated by the dual-function transcriptional regulatory protein MerR. Whereas in the absence of Hg2+, MerR binds to the operator/promoter region (O/P) of mer operon to supress transcription, MerR is converted into a transcriptional activator upon Hg2+-binding to induce mer operon expression. Sequence analysis suggests that the O/P of mer operon is pseudopalindromic with the -35 and -10 boxes being spaced by 19~20 bps, deviate from the optimal spacing of 17 bp. Therefore, a Hg2+-dependent DNA distortion by the MerR dimer, which brings closer and reorients the two polymerase binding sites, is required to activate transcription. To understand the structural basis by which Hg2+-binding modulates MerR function, we have determined the crystal structures of apo- and Hg2+-bound MerR dimer form Bacillus megaterium MB1, which correspond to the suppressor and activator conformation of MerR, respectively. To our knowledge, the apo-MerR structure represents the first visualization of an inducer-free form of a MerR family protein. Structural comparison not only illustrated how a buried trigonal planar Hg2+-binding pocket is assembled, but also revealed functionally relevant tertiary and quaternary changes between the apo- and Hg2+-bound MerR dimer. The pronounced Hg2+-dependent reposition of the DNA-binding domains suggests a plausible mechanism of transcription regulation by MerR.

Contents
口試委員會審定書 ........................................................................................................ I
謝誌............................................................................................................................... II
中文摘要 .................................................................................................................... III
Abstract ........................................................................................................................ V
Contents .................................................................................................................... VII
List of Figures ............................................................................................................. IX
List of Tables .............................................................................................................. XI
1. Introduction .......................................................................................................... 1
1.1. Heavy metal resistance system ........................................................................... 2
1.2. The regulatory proteins of the MerR family ..................................................... 3
1.3. The mer operon ................................................................................................. 4
1.4. The TnMERI1 transposon from Bacillus megaterium MB1 strain .................... 6
1.5. Specific aims of this study ................................................................................. 7
2. Methods and Materials ........................................................................................ 9
2.1. Cloning of the Bacillus megaterium MB1 strain MerR ................................... 10
2.2. MerR expression and purification .................................................................... 10
2.3. DNA sequence derived from the mer operon for crystallography ................... 11
2.4. Protein crystallization ....................................................................................... 11
2.5. Structure determination .................................................................................... 13
2.6. Structural modeling .......................................................................................... 14
VIII
2.7 Analyzing the MerR-DNA crystals by gel electrophoresis ............................... 15
3. Results and Discussion ....................................................................................... 16
3.1. Structure determination of apo-MerR and Hg2+-bound MerR .......................... 17
3.2. Overall structure of apo-MerR and Hg2+-bound MerR .................................... 18
3.3. MerR undergoes extensive secondary, tertiary and quaternary structural changes
upon Hg2+ binding ............................................................................................ 20
3.4. Structure of the Hg2+-binding site and structural basis of metal selectivity ...... 23
3.5. Functional implications for the MerR from Gram-negative bacteria and the
prospective transcriptional regulator MerR2 of TnMERI1 .............................. 26
3.6. Structural basis of MerR-mediated regulation of the mer operon .................... 28
4. Conclusion ......................................................................................................... 32
5. Figures ................................................................................................................ 34
6. Tables ................................................................................................................. 64
7. References .......................................................................................................... 71

1.Tai, H.C. and Lim, C. (2006) Computational studies of the coordination stereochemistry, bonding, and metal selectivity of mercury. J. Phys. Chem.. A, 110, 452-462.
2.Barkay, T., Miller, S.M. and Summers, A.O. (2003) Bacterial mercury resistance from atoms to ecosystems. FEMS Microbiol. Rev., 27, 355-384.
3.Giedroc, D.P. and Arunkumar, A.I. (2007) Metal sensor proteins: nature''s metalloregulated allosteric switches. Dalton Trans., 3107-3120.
4.O''Halloran, T.V. (1993) Transition metals in control of gene expression. Science, 261, 715-725.
5.Silver, S. and Phung le, T. (2005) A bacterial view of the periodic table: genes and proteins for toxic inorganic ions. J. Ind. Microbiol. Biotechnol., 32, 587-605.
6.Osborn, A.M., Bruce, K.D., Strike, P. and Ritchie, D.A. (1997) Distribution, diversity and evolution of the bacterial mercury resistance (mer) operon. FEMS Microbiol. Rev., 19, 239-262.
7.O''Halloran, B.A.G.a.T.V. (2013) Mechanisms Controlling the Cellular Metal Economy, Encyclopedia of Inorganic and Bioinorganic Chemistry.
8.Reyes-Caballero, H., Campanello, G.C. and Giedroc, D.P. (2011) Metalloregulatory proteins: metal selectivity and allosteric switching. Biophys. Chem., 156, 103-114.
9.Hobman, J.L. (2007) MerR family transcription activators: similar designs, different specificities. Mol. Microbiol., 63, 1275-1278.
10.Hobman, J.L., Wilkie, J. and Brown, N.L. (2005) A design for life: prokaryotic metal-binding MerR family regulators. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine, 18, 429-436.
11.Brown, N.L., Stoyanov, J.V., Kidd, S.P. and Hobman, J.L. (2003) The MerR family of transcriptional regulators. FEMS Microbiol. Rev., 27, 145-163.
12.Helmann, J.D., Wang, Y., Mahler, I. and Walsh, C.T. (1989) Homologous metalloregulatory proteins from both gram-positive and gram-negative bacteria control transcription of mercury resistance operons. J. Bacteriol., 171, 222-229.
13.Watanabe, S., Kita, A., Kobayashi, K. and Miki, K. (2008) Crystal structure of the [2Fe-2S] oxidative-stress sensor SoxR bound to DNA. Proc. Natl. Acad. Sci. U.S.A., 105, 4121-4126.
14.Changela, A., Chen, K., Xue, Y., Holschen, J., Outten, C.E., O''Halloran, T.V. and Mondragon, A. (2003) Molecular basis of metal-ion selectivity and zeptomolar sensitivity by CueR. Science, 301, 1383-1387.
15.Heldwein, E.E. and Brennan, R.G. (2001) Crystal structure of the transcription activator BmrR bound to DNA and a drug. Nature, 409, 378-382.
16.Godsey, M.H., Baranova, N.N., Neyfakh, A.A. and Brennan, R.G. (2001) Crystal structure of MtaN, a global multidrug transporter gene activator. J. Biol. Chem., 276, 47178-47184.
17.Kumaraswami, M., Newberry, K.J. and Brennan, R.G. (2010) Conformational plasticity of the coiled-coil domain of BmrR is required for bmr operator binding: the structure of unliganded BmrR. J. Mol. Biol., 398, 264-275.
18.Newberry, K.J. and Brennan, R.G. (2004) The structural mechanism for transcription activation by MerR family member multidrug transporter activation, N terminus. J. Biol. Chem., 279, 20356-20362.
19.Zeng, Q., Stalhandske, C., Anderson, M.C., Scott, R.A. and Summers, A.O. (1998) The core metal-recognition domain of MerR. Biochemistry, 37, 15885-15895.
20.Song, L., Caguiat, J., Li, Z., Shokes, J., Scott, R.A., Olliff, L. and Summers, A.O. (2004) Engineered single-chain, antiparallel, coiled coil mimics the MerR metal binding site. J. Bacteriol., 186, 1861-1868.
21.Helmann, J.D., Ballard, B.T. and Walsh, C.T. (1990) The MerR metalloregulatory protein binds mercuric ion as a tricoordinate, metal-bridged dimer. Science, 247, 946-948.
22.Shewchuk, L.M., Verdine, G.L., Nash, H. and Walsh, C.T. (1989) Mutagenesis of the cysteines in the metalloregulatory protein MerR indicates that a metal-bridged dimer activates transcription. Biochemistry, 28, 6140-6145.
23.Nascimento, A.M. and Chartone-Souza, E. (2003) Operon mer: bacterial resistance to mercury and potential for bioremediation of contaminated environments. Genet. Mol. Res., 2, 92-101.
24.Huang, C.C., Narita, M., Yamagata, T., Itoh, Y. and Endo, G. (1999) Structure analysis of a class II transposon encoding the mercury resistance of the Gram-positive Bacterium bacillus megaterium MB1, a strain isolated from minamata bay, Japan. Gene, 234, 361-369.
25.Brown, N.L., Misra, T.K., Winnie, J.N., Schmidt, A., Seiff, M. and Silver, S. (1986) The nucleotide sequence of the mercuric resistance operons of plasmid R100 and transposon Tn501: further evidence for mer genes which enhance the activity of the mercuric ion detoxification system. Mol. Genet. Genomic., 202, 143-151.
26.Brown, N.L., Ford, S.J., Pridmore, R.D. and Fritzinger, D.C. (1983) Nucleotide sequence of a gene from the Pseudomonas transposon Tn501 encoding mercuric reductase. Biochemistry, 22, 4089-4095.
27.Fox, B. and Walsh, C.T. (1982) Mercuric reductase. Purification and characterization of a transposon-encoded flavoprotein containing an oxidation-reduction-active disulfide. J. Biol. Chem., 257, 2498-2503.
28.Hobman, J.L. and Brown, N.L. (1997) bacterial mercury-resistance genes. Metal ions in biological systems, 34, 527-568.
29.Summers, A.O. (1986) Organization, expression, and evolution of genes for mercury resistance. Annu. Rev. Microbiol., 40, 607-634.
30.Ansari, A.Z., Bradner, J.E. and O''Halloran, T.V. (1995) DNA-bend modulation in a repressor-to-activator switching mechanism. Nature, 374, 371-375.
31.Frantz, B. and O''Halloran, T.V. (1990) DNA distortion accompanies transcriptional activation by the metal-responsive gene-regulatory protein MerR. Biochemistry, 29, 4747-4751.
32.O''Halloran, T.V., Frantz, B., Shin, M.K., Ralston, D.M. and Wright, J.G. (1989) The MerR heavy metal receptor mediates positive activation in a topologically novel transcription complex. Cell, 56, 119-129.
33.Lund, P.A. and Brown, N.L. (1989) Regulation of transcription in Escherichia coli from the mer and merR promoters in the transposon Tn501. J. Mol. Biol., 205, 343-353.
34.Moyle, H., Waldburger, C. and Susskind, M.M. (1991) Hierarchies of base pair preferences in the P22 ant promoter. J. Bacteriol., 173, 1944-1950.
35.Harley, C.B. and Reynolds, R.P. (1987) Analysis of E. coli promoter sequences. Nucleic Acids Res., 15, 2343-2361.
36.Huang, C.C., Narita, M., Yamagata, T. and Endo, G. (1999) Identification of three merB genes and characterization of a broad-spectrum mercury resistance module encoded by a class II transposon of Bacillus megaterium strain MB1. Gene, 239, 361-366.
37.Parkhill, J. and Brown, N.L. (1990) Site-specific insertion and deletion mutants in the mer promoter-operator region of Tn501; the nineteen base-pair spacer is essential for normal induction of the promoter by MerR. Nucleic Acids Res., 18, 5157-5162.
38.Kulkarni, R.D. and Summers, A.O. (1999) MerR cross-links to the alpha, beta, and sigma 70 subunits of RNA polymerase in the preinitiation complex at the merTPCAD promoter. Biochemistry, 38, 3362-3368.
39.Lee, I.W., Livrelli, V., Park, S.J., Totis, P.A. and Summers, A.O. (1993) In vivo DNA-protein interactions at the divergent mercury resistance (mer) promoters. II. Repressor/activator (MerR)-RNA polymerase interaction with merOP mutants. J. Biol. Chem., 268, 2632-2639.
40.Heltzel, A., Lee, I.W., Totis, P.A. and Summers, A.O. (1990) Activator-dependent preinduction binding of sigma-70 RNA polymerase at the metal-regulated mer promoter. Biochemistry, 29, 9572-9584.
41.Summers, A.O. (1992) Untwist and shout: a heavy metal-responsive transcriptional regulator. J. Bacteriol., 174, 3097-3101.
42.Guo, H.B., Johs, A., Parks, J.M., Olliff, L., Miller, S.M., Summers, A.O., Liang, L. and Smith, J.C. (2010) Structure and conformational dynamics of the metalloregulator MerR upon binding of Hg(II). J. Mol. Biol., 398, 555-568.
43.Song, L., Teng, Q., Phillips, R.S., Brewer, J.M. and Summers, A.O. (2007) 19F-NMR reveals metal and operator-induced allostery in MerR. J. Mol. Biol., 371, 79-92.
44.Ansari, A.Z., Chael, M.L. and O''Halloran, T.V. (1992) Allosteric underwinding of DNA is a critical step in positive control of transcription by Hg-MerR. Nature, 355, 87-89.
45.Chen, C.Y., Hsieh, J.L., Silver, S., Endo, G. and Huang, C.C. (2008) Interactions between two MerR regulators and three operator/promoter regions in the mercury resistance module of Bacillus megaterium. Biosci. Biotechnol. Biochem., 72, 2403-2410.
46.Huang, C.C., Narita, M., Yamagata, T., Phung le, T., Endo, G. and Silver, S. (2002) Characterization of two regulatory genes of the mercury resistance determinants from TnMERI1 by luciferase-based examination. Gene, 301, 13-20.
47.Otwinowski, Z. and Minor, W. (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol., 276, 307-326.
48.Terwilliger, T.C. (2003) Automated main-chain model building by template matching and iterative fragment extension. Acta Crystallogr. D, 59, 38-44.
49.Terwilliger, T.C. (2000) Maximum-likelihood density modification. Acta Crystallogr. D, 56, 965-972.
50.Terwilliger, T.C. and Berendzen, J. (1999) Discrimination of solvent from protein regions in native Fouriers as a means of evaluating heavy-atom solutions in the MIR and MAD methods. Acta Crystallogr. D, 55, 501-505.
51.Emsley, P. and Cowtan, K. (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr. D, 60, 2126-2132.
52.Adams, P.D., Afonine, P.V., Bunkoczi, G., Chen, V.B., Davis, I.W., Echols, N., Headd, J.J., Hung, L.W., Kapral, G.J., Grosse-Kunstleve, R.W. et al. (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D, 66, 213-221.
53.Painter, J. and Merritt, E.A. (2006) Optimal description of a protein structure in terms of multiple groups undergoing TLS motion. Acta Crystallogr. D, 62, 439-450.
54.Shewchuk, L.M., Verdine, G.L. and Walsh, C.T. (1989) Transcriptional switching by the metalloregulatory MerR protein: initial characterization of DNA and mercury (II) binding activities. Biochemistry, 28, 2331-2339.
55.Outten, C.E., Outten, F.W. and O''Halloran, T.V. (1999) DNA distortion mechanism for transcriptional activation by ZntR, a Zn(II)-responsive MerR homologue in Escherichia coli. J. Biol. Chem., 274, 37517-37524.
56.Veglia, G., Porcelli, F., DeSilva, T., Prantner, A. and Opella, S.J. (2000) The structure of the metal-binding motif GMTCAAC is similar in an 18-residue linear peptide and the mercury binding protein MerP. J. Am. Chem. Soc., 122, 2389-2390.
57.Steele, R.A. and Opella, S.J. (1997) Structures of the reduced and mercury-bound forms of MerP, the periplasmic protein from the bacterial mercury detoxification system. Biochemistry, 36, 6885-6895.
58.Dudev, T. and Lim, C. (2014) Competition among Metal Ions for Protein Binding Sites: Determinants of Metal Ion Selectivity in Proteins. Chem. Rev., 114, 538-556.
59.Wright, J.G., Tsang, H.T., Pennerhahn, J.E. and Ohalloran, T.V. (1990) Coordination Chemistry of the Hg-Merr Metalloregulatory Protein - Evidence for a Novel Tridentate Hg-Cysteine Receptor-Site. J. Am. Chem. Soc., 112, 2434-2435.
60.Utschig, L.M., Bryson, J.W. and O''Halloran, T.V. (1995) Mercury-199 NMR of the metal receptor site in MerR and its protein-DNA complex. Science, 268, 380-385.
61.Ralston, D.M. and O''Halloran, T.V. (1990) Ultrasensitivity and heavy-metal selectivity of the allosterically modulated MerR transcription complex. Pro. Natl. Acad. Sci. U.S.A., 87, 3846-3850.
62.Chen, P.R. and He, C. (2008) Selective recognition of metal ions by metalloregulatory proteins. Curr. Opin. Chem. Biol., 12, 214-221.
63.Ross, W., Park, S.J. and Summers, A.O. (1989) Genetic analysis of transcriptional activation and repression in the Tn21 mer operon. J. Bacteriol., 171, 4009-4018.
64.Shewchuk, L.M., Helmann, J.D., Ross, W., Park, S.J., Summers, A.O. and Walsh, C.T. (1989) Transcriptional switching by the MerR protein: activation and repression mutants implicate distinct DNA and mercury(II) binding domains. Biochemistry, 28, 2340-2344.
65.Parkhill, J., Lawley, B., Hobman, J.L. and Brown, N.L. (1998) Selection and characterization of mercury-independent activation mutants of the Tn501 transcriptional regulator, MerR. Microbiology, 144 ( Pt 10), 2855-2864.
66.Livrelli, V., Lee, I.W. and Summers, A.O. (1993) In vivo DNA-protein interactions at the divergent mercury resistance (mer) promoters. I. Metalloregulatory protein MerR mutants. J. Biol. Chem., 268, 2623-2631.
67.Miller, S.M. (1999) Bacterial detoxification of Hg(II) and organomercurials. Essays Biochem, 34, 17-30.
68.Murakami, K.S., Masuda, S., Campbell, E.A., Muzzin, O. and Darst, S.A. (2002) Structural basis of transcription initiation: an RNA polymerase holoenzyme-DNA complex. Science, 296, 1285-1290.
69.Campbell, E.A., Muzzin, O., Chlenov, M., Sun, J.L., Olson, C.A., Weinman, O., Trester-Zedlitz, M.L. and Darst, S.A. (2002) Structure of the bacterial RNA polymerase promoter specificity sigma subunit. Mol. Cell, 9, 527-539.
70.Caguiat, J.J., Watson, A.L. and Summers, A.O. (1999) Cd(II)-responsive and constitutive mutants implicate a novel domain in MerR. J. Bacteriol., 181, 3462-3471.

QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關論文
 
無相關期刊
 
無相關點閱論文