TnMERI1汞抗性操作組轉錄調控因子MerR1之結構與功能解析

Structural studies of transcription regulator MerR1 from mercury resistance transposon TnMERI1

張家璐(Chia-Lu Chang)

指導教授 ： 詹迺立

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.10721

摘要

汞是自然界中的元素，廣泛存在於空氣、水、土壤之中；近年來，由於快速的工業發展，汞汙染對環境而言是一嚴重的威脅。汞對神經、消化、免疫系統以及肺、腎、皮膚、眼等多種器官均具有毒性，造成多種疾病的發生。微生物修復(biomedeiation)是一種有效且經濟地清除重金屬汙染的方式，一些生存於重金屬環境中的細菌發展出可提供重金屬抗性的操作組。　　
　 TnMERI1是最早從革蘭氏陽性菌Bacillus megaterium MB1中被發現的汞抗性操作組。細菌若在含有汞離子(Hg2+)或有機汞的環境下生長，必須利用汞抗性系統mer操作組攜帶之有機汞裂解酶與汞離子還原酶的活性，將有毒性的有機汞還原成沒有毒性且能以昇華方式離開細胞的汞原子。mer操作組之序列啟動子具有雙重對稱的特性(dyad symmetrical sequence)，並且在-35和-10兩個區域之間相隔了18~20個鹼基對；由於轉錄聚合酶(RNA polymerase)對 -35與 -10區域相隔17個鹼基對得啟動子呈現較強的轉錄活性，因此mer操作組的基礎轉錄活性較低。MerR1是負責mer操作組調控表達的轉錄因子。當汞離子(Hg2+)不存在時，MerR1雙聚體(dimer)與mer操作組結合並阻止轉錄聚合酶的結合，扮演抑制子(repressor)的角色。反之，在有汞離子(Hg2+)存在的情況下，汞離子會與MerR1雙聚體結合並且引發蛋白構型變化，造成mer操作組DNA扭曲，促使轉錄聚合酶能有效率地結合而起始轉錄作用。MerR1雙聚體可以和兩個汞離子(Hg+2)結合並且當作增強子(activator)來扭曲該段DNA的序列，使轉錄聚合酶(RNA polymerase)能結合上而轉錄作用得以起始。MerR1和汞離子(Hg2+)結合，每個結合位由三個半胱胺酸(cysteine)組成，其中一個單體提供Cys114和Cys123，而另一單體則提供Cys79。這幾個半胱胺酸(cysteine)若發生突變則會導致MerR1雙聚體無法與汞離子(Hg2+)結合，進而抑制mer操作組的表現。
　　本研究目前已找到適合晶體生長的環境條件，未來期望利用X-射線繞射原理分析，解出在有或無汞離子(Hg2+)存在條件下MerR1和DNA結合的結構，進而更清楚地了解MerR1調控mer操作組的機轉，以及MerR1與汞離子(Hg2+)的結合如何影響DNA之構型改變。

關鍵字

汞 ； TnMERI1 ； MerR1 ； mer操作組 ； X-射線繞射原理分析

並列摘要

Mercury is a naturally occurring element that is found in air, water and soil, and mercury pollution is a severe environmental threat due to rapid industrialization. Exposure to mercury may cause serious health problems. Mercury may have toxic effects on the nervous, digestive and immune systems, and on lungs, kidneys, skin and eyes. Mercury poisoning can result in several diseases. Bioremediation is an efficiently and ecological way to solve the problem. Mercury-resistant bacteria harbor the mer operon in their genome. The mer operon includes certain functional genes along with promoter, regulator, and operator. The mechanism involves the reduction of the highly reactive cationic form of mercury into metallic vapor. The genes, which are responsible for this resistance, are organized in an operon called mer operon. TnMERI1 was found as the first mercury resistance transposon identified from Gram-positive bacteria and was harbored by a Minamata bay sediment isolated bacterial strain which was designated as Bacillus megaterium MB1.
　　The operator/promoter region of mer operon is composed of pseudopalindromic promoter binding site and unnormal spaces, 18~20 bps, between -35 and -10 box. RNA polymerase only recognize 17 bp spaces. It need regulator MerR to distort DNA and then shorten the space. When Hg2+ is absent, MerR1 dimer bound to mer operon function as repressor. In the present of Hg2+, MerR1 dimer bound Hg2+ becomes activator to distort and bend DNA, then RNA polymerase binds to -10 and -35 box to start transcription of mer operon. The binding sites of Hg2+ is consisted of three cysteines, one monomer of MerR offers two cysteines ( cys114 and cys123) and the other one offers one cysteine (cys79). If one of those cysteines was mutated, MerR dimer can not bind to Hg2+, resulting to inhibit mer operon expression.
　　To understand the structural basis of MerR1-mediated transcriptional regulation and how mercury ion bind to MerR1, we try to solve the crystal structure of apo-MerR1 and Hg-MerR1 bound to DNA from Gram-positive bacteria Bacillus megaterium MB1. From our results, we get several conditions of crystallization, and we optimize the condition to get better quality of crystal for structural determination.

並列關鍵字

mercury ； TnMERI1 ； MerR1 ； mer operon ； X-ray crystallography

參考文獻

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