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Functional analysis of the regulatory protein LuxZ-R&R/DNA binding from Photobacterium leiognathi
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。
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'.
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