The DnaK and DnaJ heat shock proteins function as the primary Hsp70 and Hsp40 homologues, respectively, of Escherichia coli. Intensive studies of various Hsp70 and DnaJ-like proteins over the past decade have led to the suggestion that interactions between specific pairs of these two types of proteins permit them to serve as molecular chaperones in a diverse array of protein metabolic events, including protein folding, protein trafficking, and assembly and disassembly of multisubunit protein complexes. To further our understanding of the nature of Hsp70-DnaJ interactions, we have sought to define the minimal sequence elements of DnaJ required for stimulation of the intrinsic ATPase activity of DnaK. As judged by proteolysis sensitivity, DnaJ is composed of three separate regions, a 9-kDa NH2-terminal domain, a 30-kDa COOH-terminal domain, and a protease-sensitive glycine- and phenylalanine-rich (G/F-rich) segment of 30 amino acids that serves as a flexible linker between the two domains. The stable 9-kDa proteolytic fragment was identified as the highly conserved J-region found in all DnaJ homologues. Using this structural information as a guide, we constructed, expressed, purified, and characterized several mutant DnaJ proteins that contained either NH2-terminal or COOH-terminal deletions. At variance with current models of DnaJ action, DnaJ1-75, a polypeptide containing an intact J-region, was found to be incapable of stimulating ATP hydrolysis by DnaK protein. We found, instead, that two sequence elements of DnaJ, the J-region and the G/F-rich linker segment, are each required for activation of DnaK-mediated ATP hydrolysis and for minimal DnaJ function in the initiation of bacteriophage lambda DNA replication. Further analysis indicated that maximal activation of ATP hydrolysis by DnaK requires two independent but simultaneous protein-protein interactions: (i) interaction of DnaK with the J-region of DnaJ and (ii) binding of a peptide or polypeptide to the polypeptide-binding site associated with the COOH-terminal domain of DnaK. This dual signaling process required for activation of DnaK function has mechanistic implications for those protein metabolic events, such as polypeptide translocation into the endoplasmic reticulum in eukaryotic cells, that are dependent on interactions between Hsp70-like and DnaJ-like proteins.

The proteins DnaK (hsp70) and GroEL (cpn60) from Escherichia coli are prototypes of two classes of molecular chaperones conserved throughout evolution. The analysis of transferred nuclear Overhauser effects in two-dimensional NMR spectra is ideally suited to determine chaperone-bound conformations of peptides. The peptide vsv-C (amino-acid sequence KLIGVLSSLFRPK) stimulates the ATPase of BiP and Hsc70 (ref. 3) and the intrinsic ATPase of DnaK. The affinity of the vsv-C peptide for DnaK is greatly reduced in the presence of ATP. Here we analyse transferred nuclear Overhauser effects and show that the peptide is in an extended conformation while bound to DnaK but is helical when bound to GroEL. NMR also indicates that the mobility of the peptide backbone is reduced more by binding to DnaK than by binding to GroEL, whereas the side chains are less mobile when bound to GroEL.

The Escherichia coli dnaK gene product, originally defined by mutations that blocked A phage DNA replication, is known to be necessary for E. coli viability. We have purified dnaK protein to homogeneity and have demonstrated that it possesses a weak DNA-independent ATPase activity, which results in the production of ADP and Pi. The proof that this ATPase activity is encoded by the dnaK+ gene relies primarily on the fact that the dnaK756 mutation results in the production of an ATPase activity with altered physical properties. The dnaK protein is phosphorylated in vitro and in vivo, probably as a result of an autophosphorylation reaction. The A 0 and P replication proteins were shown to interact in vitro with the dnaK protein. The ATPase activity of the dnaK protein was inhibited by purified A P protein and stimulated by purified A 0 protein. Moreover, the dnaK protein participates in the initiation of DNA synthesis in an in vitro DNA replication system that is dependent on the 0 and P proteins. Anti-dnaK protein immunoglobulin specifically inhibited DNA synthesis in this system.The study of host-virus interactions has been greatly facilitated through the isolation of host mutants that block viral growth. Through the use of indirect or direct selections, various investigators have uncovered five Escherichia coli genes whose products are necessary for bacteriophage A DNA replication: dnaB, dnaK, dnaJ, grpD, and grpE (1-4). Of these genes, only dnaB has been convincingly shown to be essential for E. coli DNA replication (5). Both the dnaK+ and dnaJ+ gene products are essential for bacterial growth as well, because some mutations in these genes block colony formation at 42°C. Both DNA and RNA bacterial metabolism are affected in dnaK-(4, 6) and dnaJ-mutants (7,8). The effect on RNA metabolism is unique and not shared by mutations in the rest of the dna genes of E. coli (5). Mutations in A phage that enable it to grow on dnaKhosts map in the P gene, which suggests an interaction between the host dnaK protein and the phage P protein (1, 2). The dnaK gene has been cloned into A phage (2), and its gene product has been identified by NaDodSO4/polyacrylamide gel electrophoresis (9) and shown to be identical to protein B66.0 (10), one of the heat shock proteins of E. coli (11)(12)(13). In this communication, we report that the dnaK+ protein (i) possesses a weak DNA-independent ATPase activity that is modulated in vitro by the A 0 and P proteins; (ii) is phosphorylated both in vitro and in vivo, probably as a result of an autophosphorylation reaction; and (iii) is active in an in vitro replication system (unpublished results) that is dependent on the A 0 and P proteins. METHODS AND MATERIALSStrains. The bacteria, bacteriophage, and plasmid strains used in this work have been described (2,9,10,14,15).Purification of dnaK' Protein. The details of the purification will be published elsewhere. Briefly, 12 g of wet paste of B178(pMOB45dnaK+) bacteria was lysed according to Shlomai and Kornberg (16). The supernatant was ...

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Central to the chaperone function of Hsp70 stress proteins including Escherichia coli DnaK is the ability of Hsp70 to bind unfolded protein substrates in an ATP‐dependent manner. Mg2+/ATP dissociates bound substrates and, furthermore, substrate binding stimulates the ATPase of Hsp70. This coupling is proposed to require a glutamate residue, E175 of bovine Hsc70, that is entirely conserved within the Hsp70 family, as it contacts bound Mg2+/ATP and is part of a hinge required for a postulated ATP‐dependent opening/closing movement of the nucleotide binding cleft which then triggers substrate release. We analyzed the effects of dnaK mutations which alter the corresponding glutamate‐171 of DnaK to alanine, leucine or lysine. In vivo, the mutated dnaK alleles failed to complement the delta dnaK52 mutation and were dominant negative in dnaK+ cells. In vitro, all three mutant DnaK proteins were inactive in known DnaK‐dependent reactions, including refolding of denatured luciferase and initiation of lambda DNA replication. The mutant proteins retained ATPase activity, as well as the capacity to bind peptide substrates. The intrinsic ATPase activities of the mutant proteins, however, did exhibit increased Km and Vmax values. More importantly, these mutant proteins showed no stimulation of ATPase activity by substrates and no substrate dissociation by Mg2+/ATP. Thus, glutamate‐171 is required for coupling of ATPase activity with substrate binding, and this coupling is essential for the chaperone function of DnaK.

The 0 protein ofbacteriophage X localizes the initiation of DNA replication to a unique site on the A genome, oriX. By means of electron microscopy, we infer that the binding of 0 to oriX initiates a series of protein addition and transfer reactions that culminate in localized unwinding of the origin DNA, generating a prepriming structure for the initiation of DNA replication. We can define three stages to this prepriming reaction, the first two of which we have characterized previously. First, dimeric 0 protein binds to multiple DNA binding sites and self-associates to form a nucleoprotein structure, the O-some. Second, A P and host DnaB proteins interact with the O-some to generate a larger complex that includes additional DNA from an A+T-rich region adjacent to the 0 binding sites. Third, the addition ofthe DnaJ, DnaK, and Ssb proteins and ATP results in an origin-specific unwinding reaction, probably catalyzed by the helicase activity of DnaB. The unwinding reaction is unidirectional, proceeding "rightward" from the origin. The minimal DNA sequence competent for unwinding consists of two 0 binding sites and the adjacent A+T-rich region to the right of the binding sites. We conclude that the A 0 protein localizes and initiates a six-protein sequential reaction responsible for but preceding the precise initiation of DNA replication. Specialized nucleoprotein structures similar to the O-some may be a general feature of DNA transactions requiring extraordinary precision in localization and control.Bacteriophage X initiates DNA replication at a single replication origin, oriX (1-3). The DNA sequence of the oriX region has two major characteristics: four direct repeats of 18 base pairs (bp), each of which is an inverted repeat; and an adjacent region to the right of the repeats that is extremely rich in A+T (4-6). Two X proteins, 0 and P, are required for viral DNA replication (7-9). The 0 protein recognizes the origin, and P localizes the essential replication enzymes of Escherichia coli at this site by initiating a series of protein-protein interactions (3). The 0 protein binds to the four repeats (10, 11), probably recognizing each local inverted repeat as a dimer (ref. 12; unpublished work In this study, we show that the third stage ofthe prepriming reaction yields origin-specific unwinding of duplex DNA, presumably catalyzed by the helicase activity of DnaB (26). We infer that a locally unwound DNA structure associated with DnaB becomes the specific substrate for localized priming by DnaG. Thus, the 0 protein builds a nucleoprotein structure at the replication origin of A and initiates a series of protein assembly events culminating in the localized initiation of DNA replication. The DnaA protein of E. coli appears to act in a similar fashion to localize the initiation of bacterial DNA replication to the single site, oriC (27, 28 (18,30,32,33). Restriction enzymes were from New England Biolabs.DNA. The structure and preparation of plasmid pRLM4, which contains the replication origin of phage X, have ...

Previous studies have demonstrated that the Escherichia coli DnaK, DnaJ, and GrpE heat shock proteins participate in the initiation of bacteriophage lambda DNA replication by mediating the required disassembly of a preinitiation nucleoprotein structure that is formed at the phage replication origin. To gain some understanding in a simpler system of how the DnaJ and GrpE cochaperonins influence the activity of DnaK, we have examined the effect of the cochaperonins on the weak intrinsic ATPase activity of the molecular chaperone DnaK in the presence and absence of peptide effectors. We have found that random sequence peptide chains of 8 or 9 amino acid residues in length yield optimal (10-fold) activation of the DnaK ATPase, whereas peptides with 5 or fewer residues fail to stimulate the ATPase of this bacterial hsp70 homologue. Furthermore, we have discovered that those peptides that interact best with DnaK, as judged by their KA as activators of ATP hydrolysis by DnaK, also act as strong inhibitors of lambda DNA replication in vitro. The inhibitory effect of peptides on lambda DNA replication was overcome by increasing the concentration of DnaK in the replication system. Diminished inhibition was also found when the replication system was supplemented with GrpE cochaperonin, a protein known to increase the effectiveness of DnaK action in lambda DNA replication. These and other results suggest that the peptide-binding site of DnaK is required for its function in lambda DNA replication. Apparently, peptides sequester free DnaK protein and block lambda DNA replication by reducing the amount of DnaK that is free to mediate disassembly of nucleoprotein preinitiation structures. In related studies, we have found that DnaJ, like short peptides, activates the intrinsic ATPase activity of DnaK. DnaJ, however, is substantially more potent in this regard, since it activates DnaK at concentrations 1000-fold below those required for a peptide of random sequence. By itself, the GrpE cochaperonin has no effect on the peptide-independent ATPase activity of DnaK, but GrpE does vigorously stimulate the peptide-dependent ATPase of the DnaK chaperone. Under steady-state conditions, the Vmax of ATP hydrolysis by DnaK was elevated approximately 40-fold by the presence of GrpE and saturating levels of peptides.

The 0 protein of bacteriophage A is required for initiation of DNA replication at the A replicative origin designated onik. The binding sites for 0 protein are four direct repeats, each of which is an inverted repeat. By means of electron microscopy, we have found that phage A 0 protein utilizes these multiple binding sites to form a specific nucleoprotein structure in which the origin DNA is inferred to be folded or wound. The phage K 0 and P proteins and host DnaB protein interact at onA to generate a larger structure than that formed by 0 protein alone; P and DnaB proteins fail to form any observable complex when 0 protein is excluded from the reaction mixture. We conclude that the specialized nucleoprotein structure formed by phage K 0 protein and oniK provides for localized initiation of DNA replication by serving as the foundation for the assembly of the initial priming structure. Specialized nucleoprotein structures may be a general means to confer exceptional accuracy on DNA transactions requiring extraordinary precision.Replication by bacteriophage X initiates at a unique site designated oriX and proceeds bidirectionally (1-3). The DNA sequence in the ori region has two notable features: (i) four direct repeats of 18 base pairs (bp), each of which is an inverted repeat; and (ih) adjacent A+T-rich regions, one of which has partial homology to the DnaG priming site ofphage G4 (4-7). Two phage X proteins, 0 and P, are required for viral DNA replication (8)(9)(10). These proteins serve to direct the host replicative machinery to phage X through a sequence of interactions inferred from a variety of genetic and biochemical experiments (3). The 0 protein associates with the ori region (11-13), binding to the four direct repeats (14). 0 protein interacts with P protein (15-17), and P, with Escherichia coli DnaB protein (18-21). The addition of DnaB directs other host proteins, including DnaG, DnaJ, and DnaK, to the initiation of DNA replication at oriX (3,(21)(22)(23)(24)(25). Thus, the binding of 0 protein to oriA is the initiating event for a complex series of reactions, culminating in originspecific initiation of DNA replication.In this study, we used electron microscopy to examine the interactions that occur between the phage X origin of replication and X 0, X P, and host DnaB proteins. We find that 0 protein interacts with the four direct repeats in oriX to generate a specific nucleoprotein structure. If P and DnaB proteins are added in addition to 0 protein, we observe a larger and more asymmetric nucleoprotein structure, which we presume to be the second stage in the pathway to assembly of an initiating complex. We consider possible general functions for specialized nucleoprotein structures in DNA transactions requiring exceptional precision. MATERIALS AND METHODSProteins. Phage X 0 protein was purified by a modification of the procedure of Roberts and McMacken (13); the specific activity is 1 x 105 units/mg. Phage X P protein was purified as described by McMacken et al. (21); the specific activi...