The interaction of VICK /VICR/DNA in Streptococci
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1. 2.
VICK/VICR/DNA 1... 1 1.1... 1 1.2... 1 1.3... 2... 2 1.4 VICR... 9 1.5... 10 2... 12 2.1... 12 2.2... 18 I
VICK/VICR/DNA 3... 28 3.1... 28 3.2... 32 3.3 VICK-VICR... 44 3.4 VICR-DNA... 50 3.5 VICK-DNA-VICR... 52 3.6 VICR... 57 4... 59 5... 62... 63... 64 II
VICK/VICR/DNA CONTENTS 1 Introduction... 1 1.1 Introduction of Streptococcus... 1 1.2 Drug-resistant problem of Pathogenic bacteria... 1 1.3 Two-component system... 2 1.3.1 An overview of Two-component system... 2 1.3.2 domain architecture and function of HK... 4 1.3.3 domain architecture and function of RR... 6 1.4 the phosphorylation of VICR... 9 1.5 Content and purpose of the research... 10 1.5.1 Content of the research... 10 1.5.2 Methods of the research... 10 1.5.3 purpose of the research... 11 2 Materials and methods... 12 2.1 Materials... 12 2.1.1 genomic DNA... 12 2.1.2 Plasmid and Bacteria... 12 2.1.3 Reagents... 12 2.1.4 Instruments... 14 2.1.5 Reagents Preparation... 15 2.2 Methods... 18 2.2.1 Methods of Restructuring plasmid... 18 2.2.2 the clone of mutation... 22 2.2.3 Protein expression and Purification... 24 2.2.4 protein-protein and protein-dna interaction... 26 2.2.5 preparation of Double-stranded DNA... 26 2.2.6 phosphorylation of VICR... 27 3 Results... 28 III
VICK/VICR/DNA 3.1 cloning work... 28 3.1.1 PCR amplification and recombinant plasmid... 28 3.1.2 clone of mutation... 32 3.2 Expression and Purification of Recombinant Proteins... 32 3.3 VICK-VICRR interaction... 44 3.4 VICR-DNA interaction... 50 3.5 VICK-VICR-DNA interaction... 52 3.6 phosphorylation of VICR... 57 4 Discussion... 59 5 Conclusion... 62 ACKNOWLEDGE... 63 References... 64 IV
VICK/VICR/DNA PH HK RR HK RR RR VICK/R YYCG/F VICK VICR. VICK VICR VICR/YYCF DNA VICK-VICR-DNA VICK DNA VICR VICK-VICR-DNA VICR VICK DNA ; ; V
VICK/VICR/DNA Abstract Streptococci are pathogens in many infectious diseases. Streptococcus pneumoniae is a major pathogenic bacterium in lobar pneumonia, and other infectious diseases. Streptococcus mutans causes tooth decay, dental arthritis and other oral diseases. Traditionally antibiotics are used to control these pathogenic bacteria. However, because of the antibiotic abuse, many bacteria have developed resistances in recent years, which has become a big threat in modern medicine, The two-component system (TCS) has been postulated as a target for new drug for nearly ten years. The TCS can sense the changes in environments, and regulate the expression of a specific set of proteins that enable bacteria to survive. This system does not exist in animals and therefore targeting TCS may have very limited side-effects A typical TCS system is comprised of two proteins (histidine kinase HK and response regulator RR). After sensing the stimuli, HK can go autophosphorylation, and then transfer the phosphate group to RR through its transphosphorylation activity, which in turn activates the RR to bind promoter regions and regulate target gene expression. In Streptococci, VICK/R is an essential TCS,also called WalR/K, is a homologoustcs of YYCG/F in Bacilli. In this thesis, I cloned and expressed the intracellular domains of VICK and full-length VICR from Streptococcus mutans and Streptococcus pneumoniae. I then investigated the interactions between VICK,VICR and DNA by native PAGE and EMSA. I found that VICK binds VICR and VICR is capable to bind a DNA probe of a promoter region of glucosyltransfease gene (gtfc). More importantly, VICK directly competes with VICR to bind DNA, inhibiting VICR activity. I also repeated these experiments using YYCF and YYCG from Bacillus subtilis. This is the first demonstration in TCS field that HK negetively regulates RR binding to DNA in vitro. We hypothesize that the phosphorylation of RR may play a key role in the dissociation from HK to bind DNA for transcriptional activation. We are currently testing this hypothesis. Key words: two component system; histidine kinase; response regulator VI
VICK/VICR/DNA [1] [2]. [3] [4] 35% [5] 1
VICK/VICR/DNA TCS( ) [6] TCS [7] 62 [8] TCS [6] [9] KinA ATP [10] TCS (HK)RR HK HK ATP HK HK 2
VICK/VICR/DNA [11, 12] RR RR [13] His-Asp 1 [8] [8] Fig1 Typical TCS singal pathway [8] RRHptHpt RR His-Asp-His-Asp 2 [8] [14] [15] 20% [16] [17] 3
VICK/VICR/DNA 2 TCS [8] Fig2 Atypical TCS singal pathway [8] HK 350 [16] HK HK HK HK H, N, G1, F G2 boxes [18] TMHAMP PAS DHP [19] 3 TM HAMP PAS DHP CA 1 9 30 90 190 273 450 3 VICK Fig3 The domain architecture of the histidine kinase VICK [20] Rcs [21] RcsF RcsCRcs [22] 4
VICK/VICR/DNA RcsFRcsC IgaAIgaA HK RcsC [23, 24] HAMP [25] NHAMP,VICK HAMP [26] HKHAMP 4 [27] HK HAMP HK [27-29] 4 HAMP [27] Fig4 the Model of typical HAMP domain [27] DHP DHP N1 H-Box [30] DHPHAMP HAMPDHP [31] HAMP [31] DHPDHP 5
VICK/VICR/DNA DHPHK [32] HKDHPHK EnvZDHPNMR HK TM0853DHP [30, 33] CA ATPATP350 ATPATPATP HK [34] CA5 β-αser/thr/tyr [33, 35] HKCA Hsp90DNAATPase [11] ATP N,G1,F,G2HK [36] RRNCDNA 5RRN D52CDNA DNARR [37] RR RRDNA [38] 5 RR [37] Fig5 The domain architecture of the response regulator RR [37] RR N βα 5 β α 6
VICK/VICR/DNA 5 β α [39] 60 DNA [40] DNA 15% RR 50% RR DNA [41] RR HK [42] PH PH HK RR [39] RR [43] RR RR BeF 3 [44] Thr/Ser Lys α4β5α5 RR [45] RR RR RR RR [46-48] Thr/Ser Lys PO 3 [39] RRDNADNA 7
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