Abstract
In this work we show that the GroEL (Hsp60 equivalent) chaperone protein can protected purified Escherichia coli RNA polymerase (RNAP) holoenzyme from heat inactivation better than the DnaK (Hsp70 equivalent) chaperone can. In this protection reaction, the GroES protein is not essential, but its presence reduces the amount of GroEL required. GroEL and GroES can also reactivate heat-inactivated RNAP in the presence of ATP. The mutant GroEL673 protein, with or without GroES, is incapable of reactivating heat-inactivated RNAP. GroEL673 can only protect RNAP, and this protecting ability is not stimulated by GroES. The mechanism by which the DnaJ and GrpE heat shock proteins contribute to DnaK's ability to reactivate heat-inactivated RNAP GroEL673 has also been investigated. We found that the DnaJ protein substantially reduces the levels of DnaK protein needed in this reactivation assay. However, the observed lag in reactivation is diminished only in the additional presence of the GrpE protein. Hence, DnaJ and GrpE are involved in both steps of this reactivation reaction (recognition of substrate and release of chaperone from the substrate-chaperone complex) while, in the case of the GroEL-dependent reaction, GroES is involved only during the release of chaperone from the substrate-chaperone complex.
Highlights
In this work we show that the GroEL (Hsp6Oequiv- throughout evolution, their eukaryotic and prokaryotic memalent) chaperone protein can protect purified Esche- bers being at least 50% identical at the amino acid sequence richia coli RNA polymerase (RNAP) holoenzyme from level [1]
GroEL673 can only protect RNAP, and this protecting ability is not stimulated by GroES.The mechanism by which the DnaJ and GrpE heat shock proteins contribute to DnaK’s ability to reactivate heat-inactivated RNAP GroEL673 has been investigated
Relative RNAP activity was calculated using the same assumption as for the reactivation assay. Both DnaK and GroEL ProtectRNAP from Heat Inactivation-We showed previously that theE. coli DnaK chaperone protein is capable of protecting RNAPfrom heat inactivation [17]
Summary
Dept. of Microbiology and Molecular Genetics, University of Texas, Medical School, P.O. GroES protein was eluted from the column with a 100-500 mM KC1 gradient in T-buffer Fractions containing GroES were dialyzed in I-buffer Demonstration of this invitro activity provides an important correlation with a previously observed phenotype noted in both dnaK and groE temperature-sensitive (Ts-) strains, that is, a reduction in global RNA synthesis at nonpermissive imidazole pH 6.9,20%glycerol, 1 mM EDTA, 1 D M dithiothreitol) and loaded onto a Bio-Rex 70 column (0.7cm X 15 cm, 4 ml/h) preequilibrated with I-buffer. The final concentration of GroES protein was 4 mg/ml
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