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Biomedical subjects

M Zylicz

Publications and source records attributed to M Zylicz.

At least 19 recordsLinked to original sources
PubMed1992

Structural and functional relationships in DnaK and DnaK756 heat-shock proteins from Escherichia coli.

The secondary structures of DnaK and the mutant DnaK756 heat-shock proteins from Escherichia coli have been investigated by Fourier transform infrared spectroscopy. The analysis of infrared data showed that DnaK and DnaK756 proteins have different secondary structures that are not affected by the presence of ATP or beta, gamma-methyleneadenosine 5'-triphosphate. The infrared data indicate also that the tertiary structures of DnaK and DnaK756 proteins are different and that DnaK protein undergoes conformational changes in its tertiary structure not only during binding of ATP but also during ATP hydrolysis. Using fluorescence spectroscopy of a single tryptophan located in the N-terminal domain of DnaK protein and fluorescence of 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid, which interacts with hydrophobic domains of DnaK protein, we were able to distinguish between two conformational states of DnaK protein. After binding of triphosphonucleotides, the C-terminal domain of DnaK protein changes in tertiary structure in such a way that fewer hydrophobic segments are exposed on the surface of the protein. After ATP hydrolysis, the number of hydrophobic segments on the surface of the protein is further reduced, and moreover the tertiary structure of the N-terminal domain of the protein changes. These data are discussed in terms of structural and functional relationships of both DnaK and DnaK756 proteins.

Adenosine Triphosphate↗
PubMed1992

The DnaK chaperone modulates the heat shock response of Escherichia coli by binding to the sigma 32 transcription factor.

The heat shock response and the heat shock proteins have been conserved across evolution. In Escherichia coli, the heat shock response is positively regulated by the sigma 32 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 sigma 32 polypeptide to homogeneity. During the purification procedure, we found that a large fraction of the overexpressed sigma 32 polypeptide copurified with the universally conserved DnaK heat shock protein (the prokaryotic equivalent of the 70-kDa heat shock protein, HSP70). Further experiments established that purified sigma 32 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 sigma 32.

Bacterial Proteins↗
PubMed1991

The Escherichia coli DnaK chaperone, the 70-kDa heat shock protein eukaryotic equivalent, changes conformation upon ATP hydrolysis, thus triggering its dissociation from a bound target protein.

The DnaK protein of Escherichia coli and its eukaryotic hsp70 analogues are known to bind some polypeptides and to release or dissociate from them following ATP hydrolysis. Here we demonstrate that hydrolysis (and not simply binding) of nucleotide triphosphates leads to a change in the DnaK protein, from the "closed" to the "open" conformation. A conformational change is not observed with the mutant DnaK756 protein, which is always found in the open conformation. Although ATP is the preferred substrate, the hydrolysis of CTP, GTP, UTP, and dATP also results in DnaK's conversion from a closed to an open conformation. The ability of DnaK to hydrolyze various triphosphates correlates perfectly with its ability to release the bound denatured bovine pancreatic trypsin inhibitor polypeptide.

Adenosine Triphosphatases↗
PubMed1991

Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK.

The products of the Escherichia coli dnaK, dnaJ, and grpE heat shock genes have been previously shown to be essential for bacteriophage lambda DNA replication at all temperatures and for bacterial survival under certain conditions. DnaK, the bacterial heat shock protein hsp70 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 explain why mutations in dnaK, dnaJ, or grpE genes often exhibit similar pleiotropic phenotypes.

Adenosine Triphosphatases↗
PubMed1991

Role of the Escherichia coli grpE heat shock protein in the initiation of bacteriophage lambda DNA replication.

Initiation of replication of lambda DNA requires assembly of the proper nucleoprotein complex consisting of the lambda origin of replication-lambda O-lambda P-dnaB proteins. The dnaJ, dnaK and grpE heat shock proteins destabilize the lambda P-dnaB interaction in this complex permitting dnaB helicase to unwind lambda DNA near ori lambda sequence. First step of this disassembling reaction is the binding of dnaK protein to lambda P protein. In this report we examined the influence of dnaJ and grpE proteins on stability of the lambda P-dnaK complex. Our results show that grpE alone dissociates this complex, but both grpE and dnaJ together do not. These results suggest that, in the presence of grpE protein, dnaK protein has a higher affinity for lambda P protein complexed with dnaJ protein than in the situation where grpE protein is not used.

Bacterial Proteins↗
PubMed1990

The E. coli dnaK gene product, the hsp70 homolog, can reactivate heat-inactivated RNA polymerase in an ATP hydrolysis-dependent manner.

Pelham previously proposed that the hsp70 family of heat shock proteins could prevent the formation and/or allow the dissolution of protein aggregates created during stress conditions. We confirmed this hypothesis by showing that the E. coli hsp70 homolog, the dnaK gene product, protects the host RNA polymerase enzyme from heat inactivation in an ATP-independent reaction. In addition, we show that heat-inactivated and aggregated RNA polymerase is both disaggregated and reactivated following simultaneous incubation with DnaK protein and hydrolyzable ATP. The DnaK756 mutant protein has lost the ability to disaggregate the inactivated RNA polymerase enzyme. Our results demonstrate that the DnaK protein contributes to E. coli's growth not only by protecting some enzymes from denaturation but also by reactivating some once they are misfolded or aggregated.

Adenosine Triphosphate↗
PubMed1990

Physical interactions between bacteriophage and Escherichia coli proteins required for initiation of lambda DNA replication.

The process of initiation of lambda DNA replication requires the assembly of the proper nucleoprotein complex at the origin of replication, ori lambda. The complex is composed of both phage and host-coded proteins. The lambda O initiator protein binds specifically to ori lambda. The lambda P initiator protein binds to both lambda O and the host-coded dnaB helicase, giving rise to an ori lambda DNA.lambda O.lambda P.dnaB structure. The dnaK and dnaJ heat shock proteins have been shown capable of dissociating this complex. The thus freed dnaB helicase unwinds the duplex DNA template at the replication fork. In this report, through cross-linking, size chromatography, and protein affinity chromatography, we document some of the protein-protein interactions occurring at ori lambda. Our results show that the dnaK protein specifically interacts with both lambda O and lambda P, and that the dnaJ protein specifically interacts with the dnaB helicase.

Bacterial Proteins↗
PubMed1990

The HtrA (DegP) protein, essential for Escherichia coli survival at high temperatures, is an endopeptidase.

As a preliminary step in the understanding of the function of the Escherichia coli HtrA (DegP) protein, which is indispensable for bacterial survival only at elevated temperatures, the protein was purified and partially characterized. The HtrA protein was purified from cells carrying the htrA gene cloned into a multicopy plasmid, resulting in its overproduction. The sequence of the 13 N-terminal amino acids of the purified HtrA protein was determined and was identical to the one predicted for the mature HtrA protein by the DNA sequence of the cloned gene. Moreover, the N-terminal sequence showed that the 48-kilodalton HtrA protein is derived by cleavage of the first 26 amino acids of the pre-HtrA precursor polypeptide and that the point of cleavage follows a typical target sequence recognized by the leader peptidase enzyme. The HtrA protein was shown to be a specific endopeptidase which was inhibited by diisopropylfluorophosphate, suggesting that HtrA is a serine protease.

Amino Acid Sequence↗
PubMed1990

Isolation and characterization of dnaJ null mutants of Escherichia coli.

Bacteriophage lambda requires the lambda O and P proteins for its DNA replication. The rest of the replication proteins are provided by the Escherichia coli host. Some of these host proteins, such as DnaK, DnaJ, and GrpE, are heat shock proteins. Certain mutations in the dnaK, dnaJ, or grpE gene block lambda growth at all temperatures and E. coli growth above 43 degrees C. We have isolated bacterial mutants that were shown by Southern analysis to contain a defective, mini-Tn10 transposon inserted into either of two locations and in both orientations within the dnaJ gene. We have shown that these dnaJ-insertion mutants did not grow as well as the wild type at temperatures above 30 degrees C, although they blocked lambda DNA replication at all temperatures. The dnaJ-insertion mutants formed progressively smaller colonies at higher temperatures, up to 42 degrees C, and did not form colonies at 43 degrees C. The accumulation of frequent, uncharacterized suppressor mutations allowed these insertion mutants to grow better at all temperatures and to form colonies at 43 degrees C. None of these suppressor mutations restored the ability of the host to propagate phage lambda. Radioactive labeling of proteins synthesized in vivo followed by immunoprecipitation or immunoblotting with anti-DnaJ antibodies demonstrated that no DnaJ protein could be detected in these mutants. Labeling studies at different temperatures demonstrated that these dnaJ-insertion mutations resulted in altered kinetics of heat shock protein synthesis. An additional eight dnaJ mutant isolates, selected spontaneously on the basis of blocking phage lambda growth at 42 degrees C, were shown not to synthesize DnaJ protein as well. Three of these eight spontaneous mutants had gross DNA alterations in the dnaJ gene. Our data provide evidence that the DnaJ protein is not absolutely essential for E. coli growth at temperatures up to 42 degrees C under standard laboratory conditions but is essential for growth at 43 degrees C. However, the accumulation of extragenic suppressors is necessary for rapid bacterial growth at higher temperatures.

Bacterial Proteins↗
PubMed1989

Initiation of lambda DNA replication with purified host- and bacteriophage-encoded proteins: the role of the dnaK, dnaJ and grpE heat shock proteins.

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.

Bacterial Proteins↗
PubMed1988

Initiation of lambda DNA replication reconstituted with purified lambda and Escherichia coli replication proteins.

Using highly purified bacteriophage lambda and E. coli replication proteins, we were able to reconstitute an in vitro system capable of replication ori lambda-containing plasmid DNA. The addition of a new E. coli factor, the grpE gene product, to this replication system reduced the level of dnaK protein required for efficient DNA synthesis by at least 10-fold, and also allowed the isolation of a stable DNA replication intermediate. Based on all available information, we propose a molecular mechanism for the action of the dnaK and grpE proteins during the prepriming reaction leading to lambda DNA synthesis.

Bacterial Proteins↗
PubMed1988

Purification and properties of the NusB protein of Escherichia coli.

Mutations in the nusB gene of Escherichia coli block transcriptional antitermination mediated by the N gene protein of bacteriophage lambda. We describe here two methods of overproducing the NusB protein in E. coli and a method of purifying NusB to apparent homogeneity on a large scale. Purified NusB directly stimulates transcriptional antitermination by the lambda N protein in vitro. It behaves as a monomer (Mr = 15,689) during gel permeation chromatography and gradient sedimentation. The number of NusB molecules in a wild type E. coli K12 cell ranges from about 3,000 to about 6,000 molecules/cell, depending on the growth medium, and is about 50-80% of the number of molecules of the core component of RNA polymerase in the cell. This implies that NusB has a major role in regulating chain elongation during the transcription of E. coli genes. Many E. coli strains with nusB mutations cannot grow at low temperature. However, a sup+ strain with the suppressible amber mutation nusBam115 can grow at 42 degrees C. Since such a strain does not produce NusB protein detectable by immunoprecipitation with anti-NusB, normal amounts of NusB are not essential for the survival of E. coli at 42 degrees C.

Bacterial Proteins↗
PubMed1988

Role of the Escherichia coli DnaK and DnaJ heat shock proteins in the initiation of bacteriophage lambda DNA replication.

We examined the role of two Escherichia coli heat shock proteins, the dnaK and dnaJ gene products, during the initiation of lambda dv DNA replication in vitro. Using 14C-labeled lambda P protein we showed that the DnaK and DnaJ heat shock proteins function together to release lambda P protein from the preprimosomal complex consisting of lambda origin of replication-lambda O-lambda P-DnaB protein. Hydrolysis of ATP, catalyzed presumably by DnaK, is required during this reaction. Substitution of DnaK protein with that of the mutant DnaK756 protein blocks lambda P release. After DnaK and DnaJ action, the preprimosomal complex, isolated on Sepharose 4B, can support lambda dv DNA replication without any additional prepriming proteins. Using DnaK-affinity chromatography we showed that both lambda O and lambda P proteins bind to DnaK protein. The lambda P protein interacts with DnaK protein in a salt-resistant, hydrophobic manner, and ATP hydrolysis is necessary to elute at least part of lambda P protein from the DnaK-affinity column. The proposed mechanism of action of the prokaryotic DnaK and DnaJ heat shock proteins agrees with the hypothesis that Hsp70, the DnaK analogue of eukaryotes, uses ATP to disrupt hydrophobic aggregates [Pelham, H. R. B. (1986) Cell 46, 959-961].

Bacteriophage lambda↗
PubMed1987

The grpE protein of Escherichia coli. Purification and properties.

The grpE gene of Escherichia coli was first identified because a mutation in it, grpE280, prevented bacteriophage lambda DNA replication in vivo. Subsequent work resulted in the identification of the grpE protein in two-dimensional gels and its classification as a heat shock protein. Here we report the purification of the grpE protein. We show that overproduction of grpE occurs in dnaK 103 bacteria which do not produce a functional Mr 72,000 dnaK protein. The grpE protein was purified from this strain primarily by its specific retention on a dnaK affinity column. The interaction between these two proteins, which is stable in the presence of 2 M KCl, allowed other proteins to be washed from this column. grpE was then eluted by ATP, which disrupts the interaction. During purification, grpE activity was monitored by its ability to complement an in vitro lambda dv DNA replication system dependent on the lambda O and lambda P proteins. The effect of ATP on the dnaK-grpE complex was also observed during sedimentation of the two proteins in glycerol gradients. Purified grpE protein has a Mr of approximately 23,000 under both denaturing and native conditions, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sedimentation, respectively. However, in the presence of dnaK under native conditions, grpE cosediments with dnaK. When ATP is added to the gradient, the complex is disrupted, and the two proteins sediment independently as monomers.

Adenosine Triphosphate↗
PubMed1987

Enzymology of the pre-priming steps in lambda dv DNA replication in vitro.

We have examined some of the early pre-priming steps of bacteriophage lambda dv DNA replication in vitro. Previous experiments have shown that bacteriophage lambda replication requires host RNA polymerase-dependent RNA synthesis near or at the origin of replication (ori lambda) to initiate DNA synthesis. Using a crude Fraction II enzymatic system we have shown that during RNA polymerase action, at least the bacteriophage lambda O and lambda P replication proteins as well as the host dnaB protein must be present to initiate ori lambda-specific DNA replication. The presence of three other host initiation proteins, dnaG primase, dnaJ, and dnaK, is not required during RNA polymerase action. Because of the apparent absence of a requirement for the dnaJ and dnaK pre-priming proteins during the transcriptional activation step, we propose that the early events of lambda dv DNA replication, prior to action by the dnaG primase, can be divided into two recognizable steps: an early step which requires at least RNA polymerase, lambda O, lambda P, and dnaB, and a subsequent step which requires the action of at least the dnaJ and dnaK proteins.

Bacterial Proteins↗
PubMed1986

The nucleotide sequence of the Escherichia coli K12 dnaJ+ gene. A gene that encodes a heat shock protein.

The Escherichia coli dnaJ gene product is required for bacteriophage lambda DNA replication at all temperatures. It is also essential for bacterial viability in at least some conditions, since mutations in it result in temperature-sensitive bacterial growth. We have previously cloned the dnaJ gene and shown that its product migrates as a Mr 37,000 polypeptide under denaturing conditions. Here we present the primary DNA sequence of the dnaJ gene. It codes for a processed basic protein (63 basic and 51 acidic amino acids) composed of 375 amino acids totaling Mr 40,973. The predicted NH2-terminal amino acid sequence, overall amino acid composition, and isoelectric point agree well with those of the purified protein. We present evidence that the rate of expression of the dnaJ protein is increased by heat shock under the control of the htpR (rpoH) gene product.

Amino Acid Sequence↗