| Description |
The DnaK, DnaJ and GrpE Escherichia coli proteins have been shown to work together as a chaperone system which functions to help dissolve protein aggregates, such as heat inactivated RNA polymerase, and to aid in assembly and disassembly of protein structures, such as that found at the origin of lambda DNA replication. The DnaK protein is thought to be divided into two distinct domains, the amino-terminal ATPase domain, and the carboxyl-terminal substrate interaction domain. In the chaperone system, DnaK provides the major chaperoning activities, whereas DnaJ and GrpE act as DnaK's cohorts, helping DnaK to better interact with its substrates, and to be recycled. In order to better understand the interaction between the DnaK and GrpE proteins, single copy and multicopy mutations in dnaK that could function to allow E. coli growth in the absence of grpE were selected and characterized both genetically and biochemically. The single copy mutations, dnaK325 and dnaK332 are point mutations in the amino-terminal, ATPase domain of DnaK. The multicopy mutations result in truncations, from 94 to 248 amino acids from the carboxyl-terminal end of the DnaK protein. Genetic and biochemical characterization of these mutants indicate that the GrpE protein helps DnaK to interact with and to release its substrates through (a) maintaining a proper balance between the ATP- and ADP-bound forms of DnaK, and (b) influencing the conformation of DnaK. They further indicate that the carboxyl-terminus of the DnaK protein acts as a negative regulator of the amino-terminal ATPase activity, and that the amino-terminus must be involved in substrate interaction. |
