The two major molecular chaperone families that mediate ATP-dependent protein folding and refolding are the heat shock proteins Hsp60s (GroEL) Hsp70s (DnaK). Clp proteins, like chaperones, highly conserved, present in all organisms, contain ATP polypeptide binding sites. We discovered ClpA, ATPase component of ClpAP protease, is a chaperone. ClpA performs function DnaK DnaJ vitro activation plasmid P1 RepA replication initiator protein. activated by conversion dimers to monomers. show...

The ssrA tag, an 11-aa peptide added to the C terminus of proteins stalled during translation, targets for degradation by ClpXP and ClpAP. Mutational analysis tag reveals independent, but overlapping determinants its interactions with ClpX, ClpA, SspB, a specificity-enhancing factor ClpX. ClpX interacts residues 9–11 at whereas ClpA recognizes positions 8–10 in addition 1–2 N terminus. SspB 1–4 7, N-terminal ClpX-binding determinants, determinants. As result, work together recognize...

The sigma(S) subunit of Escherichia coli RNA polymerase regulates the expression stationary phase and stress response genes. Control over activity is exercised in part by regulated degradation sigma(S). In vivo, requires ClpXP protease together with RssB, a protein homologous to regulator proteins. Using purified components, we reconstructed vitro demonstrate direct role for RssB delivering ClpXP. greatly stimulates Acetyl phosphate, which phosphorylates required. participates multiple...

ClpX and ClpA are molecular chaperones that interact with specific proteins and, together ClpP, activate their ATP-dependent degradation. The chaperone activity is thought to convert into an extended conformation can access the sequestered active sites of ClpP. We now show catalyze unfolding a green fluorescent protein fused recognition motif (GFP-SsrA). Unfolding GFP-SsrA depends on ATP hydrolysis. unfolded either by or treatment denaturants binds in presence adenosine 5′- O...

DnaK is a major heat shock protein of Escherichia coli and the homolog hsp70 in eukaryotes. We demonstrate mechanism by which another protein, DnaJ, render plasmid P1 initiator RepA 100-fold more active for binding to origin replication. Activation conversion dimers into monomers an ATP-dependent reaction monomer form binds with high affinity oriP1 DNA. Reversible chemical denaturants also convert simultaneously activate DNA binding. Increasing concentration converts deactivates RepA. Based...

The O protein of bacteriophage lambda localizes the initiation DNA replication to a unique site on genome, ori lambda. By means electron microscopy, we infer that binding initiates series addition and transfer reactions culminate in localized unwinding origin DNA, generating prepriming structure for replication. We can define three stages this reaction, first two which have characterized previously. First, dimeric binds multiple sites self-associates form nucleoprotein structure, O-some....

Purified E. coli dnaB and dnaC(D) gene products interact physically functionally in vitro. This interaction was demonstrated as follows: (a) A complex of isolated by gel filtration; ATP specifically required for isolation the complex. (b) The DNA-independent ribonucleoside triphosphatase activity associated with product inhibited product. (c) protected from inactivation N-ethyl-maleimide combination ATP; this protection specifically.

The E. coli proteins that catalyze the conversion of varphiX174 single-stranded DNA to duplex have now been purified extensively. reaction depends on dnaB, dnaC(D), dnaE, and dnaG gene products, elongation factors I II, binding protein, two additional proteins, replication X Y. synthesis by these requires viral DNA, dNTPs, Mg(+2), ATP. product synthesized is full-length linear DNA. has resolved into steps. first step involves interaction ATP with dnaB dnaC(D) Y in absence dNTPs. Subsequent...

The sigma(S) subunit of Escherichia coli RNA polymerase regulates the expression stationary phase and stress response genes. is highly unstable in exponentially growing cells, whereas its stability increases dramatically upon starvation or under certain conditions. degradation controlled by phosphorylatable adaptor protein RssB ClpXP protease. specifically directs to ClpXP. An unanswered question how RssB-mediated blocked conditions such as glucose phosphate starvation. We report here...

FtsZ is the major cytoskeletal protein in bacteria and a tubulin homologue. It polymerizes forms ring where constriction occurs to divide cell. We found that degraded by E. coli ClpXP, an ATP-dependent protease. In vitro, ClpXP degrades both protomers polymers; however, polymerized more rapidly than monomer. Deletion analysis shows N-terminal domain of ClpX important for polymer recognition C terminus contains signal. vivo, turned over slower clpX deletion mutant compared with WT strain....

Yeast Hsp104 and its bacterial homolog, ClpB, are Clp/Hsp100 molecular chaperones AAA+ ATPases. ClpB collaborate with the Hsp70 DnaK chaperone systems, respectively, to retrieve reactivate stress-denatured proteins from aggregates. The action of in promoting cell survival following heat stress is species-specific: cannot function bacteria act yeast. To determine regions necessary for this specificity, we tested chimeras vivo vitro. We show that middle domains dictate species-specificity at...

Elongation of a primed single-stranded DNA template catalyzed by E. coli polymerase III (DNA nucleotidyltransferase, deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) requires dnaZ protein and two other factors, elongation factors I III. The reaction occurs the following mechanism: (i) factor together catalyze transfer to template. This ATP or dATP in addition protein, III, template; it does not require (ii) binds complex with bind which is complexed I. binding...

Molecular chaperones are proteins that assist the folding, unfolding, and remodeling of other proteins. In eukaryotes, heat shock protein 90 (Hsp90) essential ATP-dependent molecular remodel activate hundreds client with assistance cochaperones. Escherichia coli , activity Hsp90 homolog, HtpG, has remained elusive. To explore mechanism action E. Hsp90, we used in vitro reactivation assays. We found promotes heat-inactivated luciferase a reaction requires prokaryotic Hsp70 chaperone system,...

Heat shock protein 90 (Hsp90) is a highly conserved ATP-dependent molecular chaperone that essential in eukaryotes. It required for the activation and stabilization of more than 200 client proteins, including many kinases steroid hormone receptors involved cell-signaling pathways. Hsp90 activity requires collaboration with subset cochaperones, Hsp70 chaperone. In higher eukaryotes, between indirect involves Hop, cochaperone interacts both Hsp70. Here we show yeast (Hsp82) (Ssa1), directly...

ClpA, a bacterial member of the Clp/Hsp100 chaperone family, is an ATP-dependent molecular and regulatory component ClpAP protease. To study mechanism binding unfolding proteins by ClpA translocation to ClpP, we used as model substrate fusion protein that joined recognition signal from RepA green fluorescent (GFP). degrades in vivo vitro. The binds specifically reaction requiring ATP but not hydrolysis. Binding alone sufficient destabilize native structure GFP portion protein. Upon...

ClpA, a member of the Clp/Hsp100 family ATPases, is molecular chaperone and, in combination with proteolytic component ClpP, participates ATP-dependent proteolysis. We investigated role ClpA protein degradation by ClpAP dissociating reaction into several discrete steps. In assembly step, ClpA-ClpP-substrate complexes assemble either ClpA-substrate interacting ClpP or ClpA-ClpP substrate; absence unable to bind substrates. Assembly requires ATP binding but not hydrolysis. discovered that...