The products of the Escherichia coli dnaK, dnaJ, and grpE heat shock genes have been previously shown to be essential for bacteriophage A DNA replication at all temperatures and for bacterial survival under certain conditions. DnaK, the bacterial heat shock protein hsp7O analogue and putative chaperonin, possesses a weak ATPase activity. Previous work has shown that ATP hydrolysis allows the release of various polypeptides complexed with DnaK. Here we demonstrate that the ATPase activity of DnaK can be greatly stimulated, up to 50-fold, in the simultaneous presence of the DnaJ and GrpE heat shock proteins. The presence of either DnaJ or GrpE alone results in a slight stimulation of the ATPase activity of DnaK. The action of the DnaJ and GrpE proteins may be sequential, since the presence of DnaJ alone leads to an acceleration in the rate of hydrolysis of the DnaK-bound ATP. The presence of GrpE alone increases the rate of release of bound ATP or ADP without affecting the rate of hydrolysis. The stimulation of the ATPase activity of DnaK may contribute to its more efficient recycling, and it helps explaln why mutations in dnaK, dnaJ, or grpE genes often exhibit similar pleiotropic phenotypes.The Escherichia coli dnaK gene product, the prokaryotic analogue of hsp70, the eukaryotic 70-kDa heat shock protein, participates in a variety of basic cellular functions: (i) survival of bacteria under different stress conditions, (ii) initiation of bacteriophage A and E. coli oriC-dependent DNA replication, (iii) regulation of cell division, (iv) modulation of proteolysis, (v) protein phosphorylation, and (vi) transport of proteins across membranes (reviewed in refs. 1-3). Such a broad spectrum of action suggests involvement of the DnaK protein in some general mechanisms crucial for the survival of the cell. Pelham (4) has suggested that the heat shock proteins belonging to the hsp70 family are involved in binding to the hydrophobic domains of other proteins, exposed either naturally or as a result of stressful conditions. Such binding and release, following ATP hydrolysis, may allow the disassembly of "dead-end" protein structures formed under stress conditions. In support of this hypothesis, we have recently shown that the DnaK protein protects E. coli RNA polymerase from heat inactivation by preventing its aggregation. In addition, in an ATP-dependent reaction, the DnaK protein can also dissolve the RNA polymerase aggregates formed at high temperature, leading to a complete restoration of RNA polymerase activity (5). Early evidence that ATP may be involved in hsp70 function was the observation that the E. coli DnaK protein has a weak ATPase activity (6). It was subsequently shown that members of the mammalian hsp70 family of proteins bind tightly to ATP cross-linked to an agarose matrix (7) and that ATP is required for release of hsp70 protein from nuclei (8). Similar results were obtained when ATP was added to complexes of hsc70 (a constitutive member of the hsp70 family) and p53 (an anti-oncogenic protein) (9),...

The chaperone protein network controls both initial protein folding and subsequent maintenance of proteins in the cell. Although the native structure of a protein is principally encoded in its amino-acid sequence, the process of folding in vivo very often requires the assistance of molecular chaperones. Chaperones also play a role in a post-translational quality control system and thus are required to maintain the proper conformation of proteins under changing environmental conditions. Many factors leading to unfolding and misfolding of proteins eventually result in protein aggregation. Stress imposed by high temperature was one of the first aggregation-inducing factors studied and remains one of the main models in this field. With massive protein aggregation occurring in response to heat exposure, the cell needs chaperones to control and counteract the aggregation process. Elimination of aggregates can be achieved by solubilization of aggregates and either refolding of the liberated polypeptides or their proteolysis. Here, we focus on the molecular mechanisms by which heat-shock protein 70 (Hsp70), Hsp100 and small Hsp chaperones liberate and refold polypeptides trapped in protein aggregates.

The heat shock response and the heat shock proteins have been conserved across evolution. In Escherichia coi, the heat shock response is positively regulated by the or32 transcriptional factor and negatively regulated by a subset of the heat shock proteins themselves. In an effort to understand the regulation of the heat shock response, we have purified the a 32 polypeptide to homogeneity. During the purification procedure, we found that a large fraction of the overexpressed a32 polypeptide copurifled with the universally conserved DoaK heat shock protein (the prokaryotic equivalent of the 70-kDa heat shock protein, HSP70). Further experiments established that purified a32 bound to DnaK and that this complex was disrupted in the presence of ATP. Consistent with the fact that dnaK756 mutant bacteria overexpress heat shock proteins at all temperatures, purified DnaK756 mutant protein did not appreciably bind to C32

Based on previous in vivo genetic analysis of bacteriophage lambda growth, we have developed two in vitro lambda DNA replication systems composed entirely of purified proteins. One is termed ‘grpE‐independent’ and consists of supercoiled lambda dv plasmid DNA, the lambda O and lambda P proteins, as well as the Escherichia coli dnaK, dnaJ, dnaB, dnaG, ssb, DNA gyrase and DNA polymerase III holoenzyme proteins. The second system includes the E.coli grpE protein and is termed ‘grpE‐dependent’. Both systems are specific for plasmid molecules carrying the ori lambda DNA initiation site. The major difference in the two systems is that the ‘grpE‐independent’ system requires at least a 10‐fold higher level of dnaK protein compared with the grpE‐dependent one. The lambda DNA replication process may be divided into several discernible steps, some of which are defined by the isolation of stable intermediates. The first is the formation of a stable ori lambda‐lambda O structure. The second is the assembly of a stable ori lambda‐lambda O‐lambda P‐dnaB complex. The addition of dnaJ to this complex also results in an isolatable intermediate. The dnaK, dnaJ and grpE proteins destabilize the lambda P‐dnaB interaction, thus liberating dnaB's helicase activity, resulting in unwinding of the DNA template. At this stage, a stable DNA replication intermediate can be isolated, provided that the grpE protein has acted and/or is present. Following this, the dnaG primase enzyme recognizes the single‐stranded DNA‐dnaB complex and synthesizes RNA primers. Subsequently, the RNA primers are extended into DNA by DNA polymerase III holoenzyme. The proposed model of the molecular series of events taking place at ori lambda is substantiated by the many demonstrable protein‐protein interactions among the various participants.

DnaJ is a molecular chaperone, which not only binds to its various protein substrates, but can also activate the DnaK cochaperone to bind to its various protein substrates as well. DnaJ is a modular protein, which contains a putative zinc finger motif of unknown function. Quantitation of the released Zn(II) ions, upon challenge with p-hydroxymercuriphenylsulfonic acid, and by atomic absorption showed that two Zn(II) ions interact with each monomer of DnaJ. Following the release of Zn(II) ions, the free cysteine residues probably form disulfide bridge(s), which contribute to overcoming the destabilizing effect of losing Zn(II). Supporting this view, infrared and circular dichroism studies show that the DnaJ secondary structure is largely unaffected by the release of Zn(II). Moreover, infrared spectra recorded at different temperatures, as well as scanning calorimetry, show that the Zn(II) ions help to stabilize DnaJ's tertiary structure. An internal 57-amino acid deletion of the cysteine-reach region did not noticeably affect the affinity of this mutant protein, DnaJDelta144-200, to bind DnaK nor its ability to stimulate DnaK's ATPase activity. However, the DnaJDelta144-200 was unable to induce DnaK to a conformation required for the stabilization of the DnaK-substrate complex. Additionally, the DnaJDelta144-200 mutant protein alone was unimpaired in its ability to interact with its final sigma32 transcription factor substrate, but exhibited reduced affinity toward its P1 RepA and lambdaP substrates. Finally, these in vitro results correlate well with the in vivo observed partial inhibition of bacteriophage lambda growth in a DnaJDelta144-200 mutant background.