The main stress proteins of Escherichia coli function in an ordered protein-folding reaction. DnaK (heat-shock protein 70) recognizes the folding polypeptide as an extended chain and cooperates with DnaJ in stabilizing an intermediate conformational state lacking ordered tertiary structure. Dependent on GrpE and ATP hydrolysis, the protein is then transferred to GroEL (heat-shock protein 60) which acts catalytically in the production of the native state. This sequential mechanism of chaperone action may represent an important pathway for the folding of newly synthesized polypeptides.

All cells have stress response pathways that maintain homeostasis in each cellular compartment. In the Gram-negative bacterium Escherichia coli, the E pathway responds to protein misfolding in the envelope. The stress signal is transduced across the inner membrane to the cytoplasm via the inner membrane protein RseA, the anti-sigma factor that inhibits the transcriptional activity of E . Stress-induced activation of the pathway requires the regulated proteolysis of RseA. In this report we show that RseA is degraded by sequential proteolytic events controlled by the inner membrane-anchored protease DegS and the membrane-embedded metalloprotease YaeL, an ortholog of mammalian Site-2 protease (S2P). This is consistent with the mechanism of activation of ATF6, the mammalian unfolded protein response transcription factor by Site-1 protease and S2P. Thus, mammalian and bacterial cells employ a conserved proteolytic mechanism to activate membrane-associated transcription factors that initiate intercompartmental cellular stress responses.

Chaperones of the Hsp70 family bind to unfolded or partially folded polypeptides to facilitate many cellular processes. ATP hydrolysis and substrate binding, the two key molecular activities of this chaperone, are modulated by the cochaperone DnaJ. By using both genetic and biochemical approaches, we provide evidence that DnaJ binds to at least two sites on the Escherichia coli Hsp70 family member DnaK: under the ATPase domain in a cleft between its two subdomains and at or near the pocket of substrate binding. The lower cleft of the ATPase domain is defined as a binding pocket for the J-domain because (i) a DnaK mutation located in this cleft (R167H) is an allele-specific suppressor of the binding defect of the DnaJ mutation, D35N and (ii) alanine substitution of two residues close to R167 in the crystal structure, N170A and T173A, significantly decrease DnaJ binding. A second binding determinant is likely to be in the substrate-binding domain because some DnaK mutations in the vicinity of the substrate-binding pocket are defective in either the affinity (G400D, G539D) or rate (D526N) of both peptide and DnaJ binding to DnaK. Binding of DnaJ may propagate conformational changes to the nearby ATPase catalytic center and substrate-binding sites as well as facilitate communication between these two domains to alter the molecular properties of Hsp70.Molecular chaperones of the Hsp70 family are conserved proteins that modulate intracellular protein folding. By binding to unfolded or partially folded polypeptides, chaperones prevent misfolding and aggregation and promote folding, translocation, and the assembly and disassembly of multiprotein structures (1, 2). Both prokaryotes and eukaryotes have multiple Hsp70 proteins that function in diverse processes. Hsp70s have a highly conserved 44-kDa ATPase domain followed by a highly conserved 15-kDa peptide-binding domain and a less conserved 10-kDa C-terminal region. The structures of the two conserved domains have been determined separately by x-ray crystallography (3, 4). Hsp70s function in concert with a cochaperone, called DnaJ or Hsp40. For example, the Escherichia coli Hsp70 protein DnaK requires DnaJ to function in the initiation of bacteriophage DNA replication (5). DnaJ increases the ATPase activity and modulates substrate binding of Hsp70 (6) and is required for Hsp70 function in vivo. Cells contain multiple DnaJ family members, and, in some cases, a specific DnaJ is required for a particular Hsp70 to function (7). Despite the key role of DnaJ in Hsp70 function, little is known about the Hsp70 determinants that mediate binding to DnaJ. A very recent NMR study localizes one binding determinant to the ATPase domain of DnaK (8).E. coli DnaJ is comprised of a J-domain, a glycinephenylalanine rich segment, a cysteine rich segment, and a C-terminal region, of which the J-domain is the most important (9). The J-domain defines this family of proteins, and some members contain only this domain. The NMR structure of this domain has been determined (10, 11). A pri...

In Escherichia coli, the heat shock response is under the control of two alternative sigma factors: sigma 32 and sigma E. The sigma 32‐regulated response is well understood, whereas little is known about that of sigma E, except that it responds to extracytoplasmic immature outer membrane proteins. To further understand this response, we located the rpoE gene at 55.5′ and analyzed the role of sigma E. sigma E is required at high temperature, and controls the transcription of at least 10 genes. Some of these might contribute to the integrity of the cell since delta rpoE cells are more sensitive to SDS plus EDTA and crystal violet. sigma E controls its own transcription from a sigma E‐dependent promoter, indicating that rpoE transcription plays a role in the regulation of E sigma E activity. Indeed, under steady‐state conditions, the transcription from this promoter mirrors the levels of E sigma E activity in the cell. However, it is unlikely that the rapid increase in E sigma E activity following induction can be accounted for solely by increased transcription of rpoE. Based upon homology arguments, we suggest that a gene encoding a negative regulator of sigma E activity is located immediately downstream of rpoE and may function as the target of the E sigma E inducing signal.

FtsH, a member of the AAA family of proteins, is the only membrane ATP-dependent protease universally conserved in prokaryotes, and the only essential ATP-dependent protease in Escherichia coli. We investigated the mechanism of degradation by FtsH. Other well-studied ATP-dependent proteases use ATP to unfold their substrates. In contrast, both in vitro and in vivo studies indicate that degradation by FtsH occurs efficiently only when the substrate is a protein of low intrinsic thermodynamic stability. Because FtsH lacks robust unfoldase activity, it is able to use the protein folding state of substrates as a criterion for degradation. This feature may be key to its role in the cell and account for its ubiquitous distribution among prokaryotic organisms.

The heat shock response controls levels of chaperones and proteases to ensure a proper cellular environment for protein folding. In Escherichia coli, this response is mediated by the bacterial-specific transcription factor, 32 32 . We show that increasing the level of GroEL/S leads to a decrease in 32 activity in vivo and this effect can be eliminated by co-overexpression of a GroEL/S-specific substrate. We also show that depletion of GroEL/S in vivo leads to up-regulation of 32 by increasing the level of 32 . In addition, we show that changing the levels of GroEL/S during stress conditions leads to measurable changes in the heat shock response. Using purified proteins, we show that that GroEL binds to 32 and decreases 32 -dependent transcription in vitro, suggesting that this regulation is direct. We discuss why using a chaperone network to regulate 32 results in a more sensitive and accurate detection of the protein folding environment.

The introduction of a replication-inhibiting lesion into the DNA of Escherichia coli produces a marked elevation in mutation rate. The mutation pathway is a component of the induced, multigene SOS response. SOS mutagenesis is a tightly regulated process dependent on two RecA-mediated proteolytic events: cleavage of the LexA repressor to induce the UmuC and UmuD mutagenesis proteins, and cleavage of UmuD to UmuD' to activate the mutation pathway. To investigate the protein-protein interactions responsible for SOS mutagenesis, we have studied the interaction of UmuC, UmuD, and UmuD'. To probe intracellular interaction, we have used immunoprecipitation techniques with antibodies against UmuC or UmuD and UmuD'. We have found that antibody to UmuC precipitates UmuD' from cell extracts, and antibody to UmuD and UmuD' precipitates UmuC. Thus we conclude that UmuC probably associates tightly with UmuD' in cells. For biochemical studies, we have purified the UmuC and UmuD' proteins to use with the previously purified UmuD. UmuC associates strongly with an affinity column of UmuD and UmuD', eluting only under strongly dissociating conditions (2 M urea or 1.5 M KSCN). UmuC also associates efficiently with UmuD or UmuD' in solution, as judged by velocity sedimentation in a glycerol gradient. The likely stoichiometry is one UmuC with a dimeric UmuD or UmuD'. From these experiments and previous work, we infer that SOS mutagenesis depends on the action of the UmuC-UmuD' complex and probably RecA to rescue a stalled DNA polymerase III holoenzyme at the DNA lesion.

The subunit of eubacterial RNA polymerase is required throughout initiation, but how it communicates with core polymerase (␣ 2 ␤␤) is poorly understood. The present work addresses the location and function of the interface of with core. Our studies suggest that this interface is extensive as mutations in six conserved regions of 70 hinder the ability of to bind core. Direct binding of one of these regions to core can be demonstrated using a peptide-based approach. The same regions, and even equivalent residues, in 32 and 70 alter core interaction, suggesting that 70 family members use homologous residues, at least in part, to interact with core. Finally, the regions of that we identify perform specialized functions, suggesting that different portions of the interface perform discrete roles during transcription initiation.