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研究生:翁才軒
研究生(外文):Tsai-Hsuan Weng
論文名稱:克雷伯氏肺炎菌PmrA-DNA複合體的結構研究
論文名稱(外文):Structural Study of Klebsiella pneumoniae PmrA-DNA complex
指導教授:蕭傳鐙蕭傳鐙引用關係
指導教授(外文):Chwan-Deng Hsiao
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生命科學系暨基因體科學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:英文
論文頁數:34
外文關鍵詞:PmrADNACrystal structure
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革蘭氏陰性菌經由PmrA/PmrB two-component system來調控對多粘菌素(polymyxin)的抗性。當PmrA的N端receiver domain(RD)被組胺酸激酶(histidine kinase)PmrB磷酸化後,PmrA會形成dimer且其C端DNA-binding domain會辨認目標啟動子上的連續重複序列,活化並啟動對多粘菌素抗性基因的轉錄。
我們利用X光結晶學解出來自克雷伯氏肺炎菌(Klebsiella pneumoniae),以磷酸類似物BeF3−活化的全長PmrA與25bp DNA的複合體結晶結構,解析度為3.2 Å。RD形成的雙聚體以及DBD和DNA之間的交互作用和先前的研究一致,然而分子內的RD-DBD交互作用在兩個PmrA單體間是不對稱的:在其中一個單體,RD和DBD間作用的表面面積達到627.5 Å2;相反的,另一個PmrA單體的兩個domain之間並未有任何接觸。這種不對稱的交互作用可能在PmrA和DNA間的結合及之後的基因轉錄的活化上扮演重要的角色。
The PmrA/PmrB two-component system regulates polymyxin-resistance in Gram-negative bacteria. After phosphorylated by the histidine kinase PmrB, the N-terminal receiver domain (RD) of the response regulator PmrA then forms a dimer and the C-terminal DNA-binding domain (DBD) recognizes the tandem repeat sequences of target promoters, initiating transcription of polymyxin-resistant genes.
We solved the 3.2-Å resolution crystal structure of the BeF3−-activated full-length Klebsiella pneumoniae PmrA in complex with a 25-bp DNA. The dimerization of RD and the DBD-DNA interaction is consistent with previous study. However, the intramolecular RD-DBD interface of the two protomers is asymmetric: in one protomer the RD contacts the DBD with a buried surface area of 627.5 Å2, whereas in the other one the two domains are only connected by a linker. This asymmetric interaction may play a role in the PmrA-DNA binding and stabilize the transactivation loop in DBD, which interacts with the σ70 subunit and activates transcription.
Contents
ACKNOWLEDGEMENTS I
CONTENTS II
LIST OF TABLES IV
LIST OF FIGURES V
CHINESE ABSTRACT VI
ENGLISH ABSTRACT VII
CHAPTER 1: INTRODUCTION 1
1.1 Introduction to Two-Component Systems 1
1.2 PmrA/PmrB Two-Component System 1
1.3 Polymyxins—Treatment for Multidrug Resistant Bacteria 2
1.4 Structures of PmrA and Its Homologs 2
1.5 Specific Aims 3
CHAPTER 2: MATERIALS AND METHODS 5
2.1 BeF3−-activated PmrA protein 5
2.2 Preparation of PmrA-DNA complex 5
2.3 Crystallization screening for PmrA-DNA complex 5
2.4 Modification of crystallization condition for PmrA-DNA complex 6
2.5 Dehydration of the PmrA-DNA complex crystals 6
2.6 X-ray diffraction data collection 6
2.7 Structure determination 7
2.8 Structure Analysis 7
CHAPTER 3: RESULTS 8
3.1 Crystallization of PmrA-DNA complex 8
3.2 Overall structure of activated PmrA-DNA complex 8
3.3 RD dimer conformation 9
3.4 DBD-DNA interactions 9
3.5 DBD-DBD interactions 10
3.6 Asymmetric intramolecular RD-DBD interface 10
CHAPTER 4: DISCUSSION 11
4.1 The Crystal Structures of PmrA-DNA complex 11
4.2 DNA Bending in PmrA-DNA Complex 11
4.3 Transactivation of PmrA 12
4.4 Structural Comparison of PmrA and KdpE 12
CHAPTER 5: CONCLUSION 14
REFERENCES 15
TABLES 19
FIGURES 22

List of Tables
Table 1. pmra box DNAs used for PmrA-DNA co-crystals 19
Table 2. Data collection and refinement statistics for PmrA-DNA complexes 20
Table 3. RMSDs of the three PmrA-DNA complex structures 21
Table 4. Residues involved in PmrA-1 RD-DBD interactions 21

List of Figures
Figure 1. Structures of OmpR/PhoB family 22
Figure 2. SDS-PAGE analysis of purified PmrA 23
Figure 3. Crystals of PmrA-26bp and PmrA-25bp 23
Figure 4. Two PmrA-25bp complex in one asymmetric unit 24
Figure 5. Superposition of the three PmrA-DNA complex 24
Figure 6. Overall structure of PmrA-25bp complex 25
Figure 7. Superposition of PmrA-RD dimer and PmrA-25bp complex 25
Figure 8. Interactions between PmrA and DNA 26
Figure 9. DNA bending in PmrA-DNA complex 27
Figure 10. DBD-DBD interface 27
Figure 11. RD-DBD interactions within PmrA-1 28
Figure 12. Location of PmrA-binding site on pbgP promoter 29
Figure 13. Model of PmrA-DNA-RNAPH complex 30
Figure 14. Superposition of PmrA-DNA and KdpE-DNA 31
Figure 15. Sequence alignment of PmrA orthologs 32
Figure 16. Sequence alignment of OmpR/PhoB family members 33
Figure 17. Proposed model for transcription activation by PmrA 34
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