- 學位論文
Lon 蛋白酵素對類組織蛋白HU降解之探討
ATP-dependent degradation in Vitro of histone-like HU protein from Bacillus subtilis by Lon protease
摘要
HU蛋白，一種在原核生物中的類組織蛋白，跟細菌的類核物質相互關連，在細菌複製起始、調節轉錄、特定序列重組上扮演結構性的角色。為了能夠更加瞭解Lon酵素蛋白在調節HU蛋白上的角色，純化出來的Bt-Lon跟來自Bacillus subtilis 的HU蛋白定溫培養。Lon以ATP相關的方式有選擇性的分解Bs-HU，證實了Lon直接辨識並分解特定受質的能力。HU最初被分解的序列是在Leu44-Ile45 和Ala57-Arg58，也正是HU 的DNA結合區域所在。Plasmid DNA和雙股DNA均會增加Lon 分解HU的能力，但是plasmid DNA影響比較大。即使一開始HU的切點是在DNA的結合位，在HU剛開始分解的階段，HU的分解並不會影響HU的DNA結合能力。要到降解兩小時候，HU的DNA結合能力才開始下降。HU對DNA結構的改變，更可以被Lon酵素還原。 這是第一篇關於DNA結合蛋白HU是Lon蛋白酵素的一種生理受質，且Lon也麻ル圁U的分解可以參與基因調節。
並列摘要
HU, a prokaryotic histone-like protein, associates with bacterial nucleoids and plays architectural roles in replication initiation, transcription regulation and site-specific recombination. To gain an insight into the role of Lon protease in regulation of HU, the purified Bt-Lon was incubated with HU of Bacillus subtilis (Bs-HU). Lon selectively degraded Bs-HU in an ATP-dependent manner, thus confirming the ability of Lon to directly recognize and degrade specific substrates. Cleavage occurred at Leu44-Ile45 and Ala57-Arg58 peptide bonds in the early period of degradation, which are known to be in the DNA-binding motif of Bs-HU. Plasmid DNA and ds-DNA can both enhance proteolysis, with plasmid being better enhancer than ds-DNA . In addition, the degradation of Bs-HU by Bt-Lon in the early period does not influence its ability to bind single-stranded DNA even removing the DNA-binding motif; however, the degradation causes the loss of the DNA-binding ability after incubation with Bt-Lon for 2 h. Furthermore, the DNA conformation altered by Hu can be reformed from the degradation of HU by Bt-Lon. This is the first example to show that HU, a DNA binding protein, is a physiological substrate of Lon protease and Lon protease is probably involved in the regulation of gene expression by degradation of HU.