Investigation of clpQ+clpY+ and gspS+ in Escherichia coli: gene regulation and substrate recognition

Autor: Hsiang-Yun Lien, 連湘芸
Druh dokumentu: 學位論文 ; thesis
Popis: 97
(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.
Databáze: Networked Digital Library of Theses & Dissertations
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