大腸桿菌clpQ+clpY+及gspS+基因之研究：基因之調控及其基質辨識

Abstract

熱休克蛋白普遍存在於生物體內，作用為幫助生物抵抗環境衝擊。本文針對大腸桿菌熱休克蛋白ClpYQ (HslUV)的基因調控及基質辨識深入研究。遇到環境衝擊時，大腸桿菌會發生熱休克反應，此時折疊錯誤的蛋白促使細胞內熱休克基因表現，這些表現常由調控蛋白sigma factor，如RpoH (或稱σ32) 所調控。本文使用大腸桿菌熱休克基因clpQ+clpY+的啟動子與報導基因lacZ建構轉錄融合基因clpQ+::lacZ (op)及轉譯融合基因clpQ+::lacZ (pr)，利用噬菌體λRS45將融合基因clpQ+::lacZ帶入野生株及rpoS -，rpoH -，rpoH -rpoS -突變株後，偵測β-galactosi dase活性以瞭解clpQ+::lacZ的表現。當溫度由30℃升至42℃時，野生株及rpoS -突變株的β-galactosidase活性上升，但rpoH -及rpoH -rpoS -突變株則未見此現象。由β-galactosidase 活性分析與北方點墨法分析結果，可知clpQ+::lacZ轉錄的mRNA訊號強度與β-galactosidase活性成正比，且clpQ+::lacZ與clpQ+clpY+的表現相似。針對clpQ+clpY+啟動子上σ32 (rpoH) 可辨識的保守序列做C→T點突變，此突變使融合基因clpQm(c→t)::lacZ無法被σ32活化，β-galactosidase活性下降。經由遺傳分析結果證實，大腸桿菌clpQ+clpY+的啟動子可被σ32辨識。此外，clpQ+clpY+operon的五端未轉譯區域 (5’-UTR)，亦即轉錄起始處 (transcription start site) 與起始密碼 (start codon) 之間，長度為71 bp，此五端未轉譯區域帶有一段inverted repeat sequence (IR序列) 5’ CC CCGTAC TTTTGTACGGGG 3’，此IR序列普遍存在於腸道菌的clpQ+clpY+五端未轉譯區域中。藉由刪除此段IR序列，並與lacZ融合，分析融合基因clpQm2△40bp::lacZ的β-galactosidase 活性，以及此段序列缺失對ClpQ與ClpY間交互作用的影響，顯示IR所形成的stem-loop二級結構在clpQ+clpY+表現時，具有穩定mRNA的效果，本研究為ATP依賴型蛋白酶 (ATP-dependent protease)中，首次發現5’ stem-loop結構具有穩定下游mRNA的功能。在基質辨識的研究部分，ClpYQ以六元環方式組合，其中ClpY負責基質辨識，打開基質結構，並傳送至ClpQ進行分解。ClpY可分為三個作用區(domain)，N-terminal domain，I-intermediated domain及C-terminal domain，N domain具有ATPase的功能，C domain則與self-oligomerization及ClpQ的蛋白酶活性相關。本文使用酵母菌雙雜交系統，得知ClpY的I domain負責基質辨識，C domain則可與ClpQ作用，而I domain中的loop L2(175-209 aa)除了與基質結合外，並與後續的基質傳遞及分解相關。另外，本文亦研究大腸桿菌的酵素glutathionylspermidine synthetase之基因gspS，由於病原蟲Trypanosomatida會造成人類昏睡，發炎及死亡，而酵素TryS為此種病原蟲所特有，在人類細胞中不存在，所以TryS的研究對治療Trypanosomatida所造成的疾病極為重要，本文針對大腸桿菌中與TryS構造及功能皆相似的酵素GspS，其基因表現之調控做一初步的探討。大腸桿菌的gspS 長1860 bp，產物為glutathionylspermidine synthetase (GspS)，共有619個胺基酸，是一種具有雙重功能的酵素 (bifunctional enzyme)，可執行GSH和spermidine之間醯胺鍵的合成與分解。本研究確認gspS為一單獨的轉錄單位而非以操縱子形式存在，且GspS起始密碼上游序列具有啟動子功能，而在in vivo情況下，H2O2與BaeR都可誘導gspS之表現。

(1) Heat shock responses are typically observed in E. coli. Upon heat shock, protein misfolding leads to a cascade of intracellular protein synthesis, usually dependent on a sigma factor, i.e., σ32, for their gene expression. In this study, the transcriptional (op) or translational (pr) clpQ+::lacZ fusion gene was constructed, with the clpQ+clpY+ promoter fused to a lacZ reporter gene. The clpQ+::lacZ (op or pr) fusion gene was each crossed into lambda phage. The λclpQ+::lac (op), a transcriptional fusion gene, was used to form lysogens in the wild-type, rpoH - or/and rpoS - mutants. Upon shifting the temperature up from 30 ℃ to 42 ℃, the wild-type λclpQ+::lacZ(op) demonstrates an increased β-galactosidase activity. However, the β-galactosidase activity of clpQ+::lacZ(op) was decreased in the rpoH - and rpoH -rpoS - mutants but not in the rpoS - mutant. The levels of clpQ+::lacZ mRNA transcripts correlated well to their β-galactosidase activity. Similarly, the expression of the clpQ+::lacZ gene fusion was nearly identical to the clpQ+clpY+ transcript under the in vivo condition. The clpQm(c→t)::lacZ, containing a C to T point mutation in the -10 promoter region for RpoH binding, showed decreased β-galactosidase activity, independent of activation by RpoH. Thus, through a genetic analysis, the clpQ+clpY+ promoter is in vivo recognized by σ32. The transcriptional start point of the clpQ+clpY+ gene lies 71 bases upstream from the clpQ+ start codon. An untranslated region (UTR) upstream of this mRNA contains a 20 bp inverted repeat (IR) sequence 5’CCCCGTACTTTTGTAC GGGG3’, which is unique for the clpQ+clpY+ operon. In addition, from the wild bacterial genome, the 5’UTR of clpQ+clpY+ also exists in other bacterial species. The clpQ+clpY+ message carries a conserved 71 bp at the 5’ untranslated region (5’UTR) that is predicted to form the stem-loop structure by analysis of its RNA secondary structure. The clpQm2△40bp::lacZ, with a 40 bp deletion in the 5’UTR, showed a decreased β-galactosidase activity. In addition, from our results, it is suggested that this stem-loop structure is necessary for the stability of the clpQ+clpY+ message. It is noteworthy that this is the first example in the ATP dependent protease to demonstrate that the 5’ stem-loop structure itself participates in the stability of its downstream mRNA. (2) Regarding ClpY substrate recognition study, in the presence of ATP, the ClpYQ complex forms an active protease with an Y6Q6Q6Y6 configuration. ClpY binds, unfolds, and transfers the substrates outside the cylinder into a catalytic core where ClpQ degrades the substrates. The ClpY molecule is divided into three domains: the N-terminal domain, I-intermediate domain and C-terminal domain. The N domain has an ATPase activity, and the C domain is responsible for self-oligomerization of ClpY. Using the yeast two-hybrid system, we show that domain I of ClpY is responsible for recognition of its natural substrates while domain C is necessary for association with ClpQ. The loop 175-209 aa plays a role in substrate tethering. (3) In addition to clpQ+clpY+, gspS+ in Escherichia coli is included in this study. Parasitic Trypanosoma species cause serious tropical diseases such as kala-azar, African sleeping sickness, and Chagas diseases. Trypanothione synthetase (TryS) is a protein unique to Trypanosoma. However, Escherichia coli produce only the metabolic intermediate GspdSH by enzyme GspS, but not trypanothione. Evolutionary, TryS and GspS share the similarly functional domains. The gspS of E. coli, encoding a bifunctional enzyme GspS of 619 amino acids, is a gene with 1860 bp. GspS is responsible for the activities of amidase and synthetase between GSH and spermidine.In this study, we showed that gspS in E. coli is an unique transcriptional unit, and the singular promoter was present in the upstream region of the GspS structural gene. In addition, the gspS promoter is in vivo induced by H2O2 and BaeR.