Renaturation of Bacillus thermoglucosidasius HrcA repressor by DNA and thermostability of the HrcA-DNA complex in vitro.
HrcA, a negative control repressor for chaperone expression from the obligate thermophile Bacillus thermoglucosidasius KP1006, was purified in a His-tagged form in the presence of 6 M urea but hardly renatured to an intact state due to extreme insolubility. Renaturation trials revealed that the addition of DNA to purified B. thermoglucosidasius HrcA can result in solubilization of HrcA free from the denaturing agent urea. Results from band shift and light scattering assays provided three new findings: (i) any species of DNA can serve to solubilize B. thermoglucosidasius HrcA, but DNA containing the CIRCE (controlling inverted repeat of chaperone expression) element is far more effective than other nonspecific DNA; (ii) B. thermoglucosidasius HrcA renatured with nonspecific DNA bound the CIRCE element in the molecular ratio of 2.6:1; and (iii) B. thermoglucosidasius HrcA binding to the CIRCE element was stable at below 50 degrees C whereas the complex was rapidly denatured at 70 degrees C, suggesting that the breakdown of HrcA is induced by heat stress and HrcA may act as a thermosensor to affect the expression of heat shock regulatory genes. These results will help to determine the nature of HrcA protein molecules.
Watanabe
K
K
Department of Applied Biochemistry, Kyoto Prefectural University, Shimogamo, Sakyo, Kyoto 606-8522, Japan. kwatanab@kpu.ac.jp
Yamamoto
T
T
Suzuki
Y
Y
D000595:Amino Acid Sequence; D001407:Bacillus / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins; D001483:Base Sequence; D018834:Chaperonin 60 / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins; D015964:Gene Expression Regulation, Bacterial; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D018869:Heat-Shock Response / Q000235:genetics; D008027:Light; D008969:Molecular Sequence Data; D020673:Protein Renaturation; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics / Q000502:physiology; D012097:Repressor Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D012542:Scattering, Radiation; D012995:Solubility; D013696:Temperature
D001426:Bacterial Proteins; D018834:Chaperonin 60; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D012097:Repressor Proteins