Regulation of clpQ+Y+ (hslV+U+) Gene Expression in Escherichia coli
نویسندگان
چکیده
The Escherichia coli ClpYQ (HslUV) complex is an ATP-dependent protease, and the clpQ⁺Y⁺ (hslV⁺U⁺) operon encodes two heat shock proteins, ClpQ and ClpY, respectively. The transcriptional (op) or translational (pr) clpQ⁺::lacZ fusion gene was constructed, with the clpQ⁺Y⁺ promoter fused to a lacZ reporter gene. The clpQ⁺::lacZ (op or pr) fusion gene was each crossed into lambda phage. The λlpQ⁺::lacZ⁺ (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 ° C to 42 ° C, the wild-type λclpQ⁺::lacZ⁺ (op) demonstrates an increased β-galactosidase (βGal) activity. However, the βGal 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 βGal activity. Similarly, the expression of the clpQ⁺::lacZ⁺ gene fusion was nearly identical to the clpQ⁺Y⁺ transcript under the in vivo condition. The clpQ(m1)::lacZ⁺, containing a point mutation in the -10 promoter region for RpoH binding, showed decreased βGal activity, independent of activation by RpoH. We conclude that RpoH itself regulates clpQ⁺Y⁺ gene expression. In addition, the clpQ⁺Y⁺ 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 clpQ(m2)Δ40::lacZ⁺, with a 40 bp deletion in the 5'UTR, showed a decreased βGal activity. In addition, from our results, it is suggested that this stem-loop structure is necessary for the stability of the clpQ⁺Y⁺ message.
منابع مشابه
Crystal structure of heat shock locus V (HslV) from Escherichia coli.
Heat shock locus V (HslV; also called ClpQ) is the proteolytic core of the ATP-dependent protease HslVU in Escherichia coli. It has sequence similarity with the beta-type subunits of the eukaryotic and archaebacterial proteasomes. Unlike these particles, which display 72-point symmetry, it is a dimer of hexamers with 62-point symmetry. The crystal structure of HslV at 3.8-A resolution, determin...
متن کاملStepwise activity of ClpY (HslU) mutants in the processive degradation of Escherichia coli ClpYQ (HslUV) protease substrates.
In Escherichia coli, ClpYQ (HslUV) is a two-component ATP-dependent protease composed of ClpY (HslU), an ATPase with unfolding activity, and ClpQ (HslV), a peptidase. In the ClpYQ proteolytic complex, the hexameric rings of ClpY (HslU) are responsible for protein recognition, unfolding, and translocation into the proteolytic inner chamber of the dodecameric ClpQ (HslV). Each of the three domain...
متن کاملThe Expression of Human Granulocyte Macrophage Colony Stimulating Factor by Heat-Induction in Escherichia coli
A self-regulated high-copy number plasmid containing chloramphenicol resistant gene, for the production of recombinant proteins under the regulation of bacteriophage ?pL promoter, was constructed. The designed 5024 base pair expression plasmid contained a heat sensitive repressor cI857 coding gene to regulate the function of ?pL promoter under heat shock induction. Using the constructed vector,...
متن کاملA COMPARATIVE STUDY BETWEEN EXPRESSION OF A SYNTHETIC GENE OF HUMAN BASIC FIBROBLAST GROWTH FACTOR (hbFGF) AND ITS RELATED cDNA IN ESCHERICHIA COLI
The gene encoding the human basic fibroblast growth factor (hbFGF) has been already chemically-synthesized and cloned in pET-3a expression vector (Pasteur Institute of Iran). In the present study, we compared the level of expression of this synthetic hbFGF and its related cDNA in Escherichia coli. The pBR322-cDNA of hbFGF supplied by Dr. Seno (from Molecular Biology Dept, Okaido prefectural uni...
متن کامل