Biomedical subjects

C Georgopoulos

Publications and source records attributed to C Georgopoulos.

At least 37 records · Page 2Linked to original sources

PubMed1997

Purification and biochemical properties of Saccharomyces cerevisiae Mdj1p, the mitochondrial DnaJ homologue.

The DnaK/DnaJ/GrpE heat shock proteins of Escherichia coli constitute the prototype DnaK chaperone machine. Various studies have shown that these three proteins work synergistically in a diverse array of biological functions, including protein folding and disaggregation, proteolysis, and transport across biological membranes. We have overexpressed and purified the mitochondrial Saccharomyces cerevisiae DnaJ homologue, Mdj1pDelta55, which lacks the mitochondrial presequence, and studied its biochemical properties in well defined in vitro systems. We find that Mdj1pDelta55 interacts with DnaK as judged both by an enzyme-linked immunosorbent assay, as well as stimulation of DnaK's weak ATPase activity in the presence of GrpE. In addition, Mdj1pDelta55 not only interacts with denatured firefly luciferase on its own, but also enables DnaK to bind to it in an ATP-dependent mode. Using co-immunoprecipitation assays we can demonstrate the presence of a stable Mdj1pDelta55-luciferase-DnaK complex. However, in contrast to DnaJ, Mdj1pDelta55 does not appear to interact well with certain seemingly folded proteins, such as the sigma32 heat shock transcription factor or the lambdaP DNA replication protein. Finally, Mdj1pDelta55 can substitute perfectly well for DnaJ in the refolding of denatured firefly luciferase by the DnaK chaperone machine. These studies demonstrate that Mdj1pDelta55 has conserved most of DnaJ's known biological properties, thus supporting an analogous functional role in yeast mitochondria.

Adenosine Triphosphatases↗

PubMed1997

The T/t common exon of simian virus 40, JC, and BK polyomavirus T antigens can functionally replace the J-domain of the Escherichia coli DnaJ molecular chaperone.

The N-terminal 70 residue "J-domain" of the Escherichia coli DnaJ molecular chaperone is the defining and highly conserved feature of a large protein family. Based upon limited, yet significant, amino acid sequence homology to the J-domain, the DNA encoding the T/t common exon of the simian virus 40 (SV40), JC, or BK polyoma virus T antigen oncoproteins was used to construct J-domain replacement chimeras of the E. coli DnaJ chaperone. The virally encoded J-domains successfully substituted for the bacterial counterpart in vivo as shown by (i) complementation for viability at low and high temperature of a hypersensitive bacterial reporter strain, and (ii) the restoration of bacteriophage lambda plaque forming ability in the same strain. The amino acid change, H42Q, in the SV40 T/t and the JC virus T/t exon, which is positionally equivalent to the canonical dnaJ259 H33Q mutation within the E. coli J-domain, entirely abolished complementing activity. These results strongly suggest that the heretofore functionally undefined viral T/t common exon represents a bona fide J-domain that preserves critical features of the characteristic domain fold essential for J-domain interaction with the ATPase domain of the Hsp70 family. This finding has implications for the regulation of DNA tumor virus T antigens by molecular chaperones.

Amino Acid Sequence↗

PubMed1997

Modulation of the Escherichia coli sigmaE (RpoE) heat-shock transcription-factor activity by the RseA, RseB and RseC proteins.

The sigma(E) (RpoE) transcription factor of Escherichia coli regulates the expression of genes whose products are devoted to extracytoplasmic activities. The sigma(E) regulon is induced upon misfolding of proteins in the periplasm or the outer membrane. Similar to other alternative sigma factors, the activity of sigma(E) is tightly regulated in E. coli. We have previously shown that sigma(E) is positively autoregulated at the transcriptional level. DNA sequencing, coupled with transcriptional analyses, have shown that sigma(E) is encoded by the first gene of a four-gene operon. The second gene of this operon, rseA, encodes an anti-sigma(E) activity. This was demonstrated at both the genetic and biochemical levels. For example, mutations in rseA constitutively increase sigma(E) activity. Consistent with this, overproduction of RseA leads to an inhibitory effect on sigma(E) activity. Topological analysis of RseA suggests the existence of one transmembrane domain, with the N-terminal part localized in the cytoplasm. Overproduction of this N-terminal domain alone was shown to inhibit sigma(E) activity. These observations were confirmed in vitro, because either purified RseA or only its purified N-terminal domain inhibited transcription from Esigma(E)-dependent promoters. Furthermore, RseA and sigma(E) co-purify, and can be co-immunoprecipitated, and chemically cross-linked. The sigma(E) activity is further modulated by the products of the remaining genes in this operon, rseB and rseC. RseB is a periplasmic protein, which negatively regulates sigma(E) activity and specifically interacts with the C-terminal periplasmic domain of RseA. In contrast, RseC is an inner membrane protein that positively modulates sigma(E) activity. Most of these protein-protein interactions were verified in vivo using the yeast two-hybrid system.

Bacterial Proteins↗

PubMed1997

Positive control of the two-component RcsC/B signal transduction network by DjlA: a member of the DnaJ family of molecular chaperones in Escherichia coli.

The membrane-anchored DjIA protein represents the third member of the DnaJ 'J-domain' family of Escherichia coli that includes DnaJ and CbpA. DjIA possesses a J-domain at its extreme C-terminus but shares no additional homology with DnaJ. Our genetic analysis suggests that DjIA acts in concert with the RcsB/C two-component signal transduction system to augment induction of the cps (capsular polysaccharide) operon and synthesis of colanic acid mucoid capsule. The DjIA J-domain is essential for the observed stimulation of this pathway as deletion, or introduction of the mutation H233Q, within the highly conserved HPD tripeptide abolished all inducing activity. Deletion of the transmembrane anchor sequence also abolished all inducing activity. djIA is not an essential gene under all conditions tested, nor is it essential for mucoid capsule biosynthesis; however, strong overexpression leads to rapid loss of cell viability suggesting that the gene is normally tightly regulated. Northern analysis revealed that djIA message was extremely unstable but could be induced or stabilized in response to cold shock. The activation of the cps operon by DjIA is dependent upon both DnaK(Hsp70) and GrpE, and therefore we propose a role for DjIA, together with this chaperone machine, as a novel regulator of a two-component histidine kinase signal transduction pathway.

Amino Acid Sequence↗

PubMed1997

Structure-function analyses of the Ssc1p, Mdj1p, and Mge1p Saccharomyces cerevisiae mitochondrial proteins in Escherichia coli.

The DnaK, DnaJ, and GrpE proteins of Escherichia coli have been universally conserved across the biological kingdoms and work together to constitute a highly efficient molecular chaperone machine. We have examined the extent of functional conservation of Saccharomyces cerevisiae Ssc1p, Mdj1p, and Mge1p by analyzing their ability to substitute for their corresponding E. coli homologs in vivo. We found that the expression of yeast Mge1p, the GrpE homolog, allowed for the deletion of the otherwise essential grpE gene of E. coli, albeit only up to 40 degrees C. The inability of Mge1p to substitute for GrpE at very high temperatures is consistent with our previous finding that it specifically failed to stimulate DnaK's ATPase at such extreme conditions. In contrast to Mge1p, overexpression of Mdj1p, the DnaJ homolog, was lethal in E. coli. This toxicity was specifically relieved by mutations which affected the putative zinc binding region of Mdj1p. Overexpression of a truncated version of Mdj1p, containing the J- and Gly/Phe-rich domains, partially substituted for DnaJ function at high temperature. A chimeric protein, consisting of the J domain of Mdj1p coupled to the rest of DnaJ, acted as a super-DnaJ protein, functioning even more efficiently than wild-type DnaJ. In contrast to the results with Mge1p and Mdj1p, both the expression and function of Ssc1p, the DnaK homolog, were severely compromised in E. coli. We were unable to demonstrate any functional complementation by Ssc1p, even when coexpressed with its Mdj1p cochaperone in E. coli.

Bacterial Proteins↗

PubMed1996

Identification and characterization of HsIV HsIU (ClpQ ClpY) proteins involved in overall proteolysis of misfolded proteins in Escherichia coli.

Heat shock response in Escherichia coli is autoregulated. Consistent with this, mutations in certain heat shock genes, such as dnaK, dnaJ, grpE or htrC lead to a higher constitutive heat shock gene expression at low temperatures. A similar situation occurs upon accumulation of newly synthesized peptides released prematurely from the ribosomes by puromycin. We looked for gene(s) which, when present in multicopy, prevent the constitutive heat shock response associated with htrC mutant bacteria or caused by the presence of puromycin. One such locus was identified and shown to carry the recently sequenced hslV hslU (clpQ clpY) operon. HslV/ClpQ shares a very high degree of homology with members of the beta-type subunit, constituting the catalytic core of the 20S proteasome. HslU/ClpY is 50% identical to the ClpX protein of E. coli, which is known to present large polypeptides to its partner, the ATP-independent proteolytic enzyme ClpP. We show that, in vivo, HslV and HslU interact and participate in the degradation of abnormal puromycylpolypeptides. Biochemical evidence suggests that HslV/ClpQ is an efficient peptidase whose activity is enhanced by HslU/CIpY in the presence of ATP.

ATP-Dependent Proteases↗

PubMed1996

Interplay of structure and disorder in cochaperonin mobile loops.

Protein-protein interactions typically are characterized by highly specific interfaces that mediate binding with precisely tuned affinities. Binding of the Escherichia coli cochaperonin GroES to chaperonin GroEL is mediated, at least in part, by a mobile polypeptide loop in GroES that becomes immobilized in the GroEL/GroES/nucleotide complex. The bacteriophage T4 cochaperonin Gp31 possesses a similar highly flexible polypeptide loop in a region of the protein that shows low, but significant, amino acid similarity with GroES and other cochaperonins. When bound to GroEL, a synthetic peptide representing the mobile loop of either GroES or Gp31 adopts a characteristic bulged hairpin conformation as determined by transferred nuclear Overhauser effects in NMR spectra. Thermodynamic considerations suggest that flexible disorder in the cochaperonin mobile loops moderates their affinity for GroEL to facilitate cycles of chaperonin-mediated protein folding.

Amino Acid Sequence↗

PubMed1996

Purification and biochemical properties of Saccharomyces cerevisiae's Mge1p, the mitochondrial cochaperone of Ssc1p.

Previous biochemical and genetic studies have demonstrated the universal conservation of the DnaK (Hsp70) chaperone machine. Its three members, DnaK, DnaJ, and GrpE, in Escherichia coli work synergistically to promote protein protection, disaggregation, and import into the various organelles. In the mitochondria of Saccharomyces cerevisiae the three corresponding members are designated as Ssc1p, Mdj1p, and Mge1p, respectively. The MGE1 gene was previously cloned by us and others, and its product has been shown to be absolutely essential for protein transport into mitochondria and hence cell viability. To better understand its biological role, we have proceeded to overexpress and purify the mature Mge1p in E. coli through the construction of the appropriate vector clone. Mge1p has been shown to functionally substitute for its E. coli GrpE counterpart in a variety of its biological functions, including suppression of the bacterial temperature-sensitive phenotype of the grpE280 mutation, formation of a stable complex with DnaK, stimulation of DnaK's ATPase activity, and the refolding of denatured luciferase by the DnaK/DnaJ chaperone proteins. Thus, the function of the GrpE homologues appears to be highly conserved across the biological kingdoms.

Adenosine Triphosphatases↗

PubMed1996

Structure-function analysis of the Escherichia coli GrpE heat shock protein.

We have isolated various missense mutations in the essential grpE gene of Escherichia coli based on the inability to propagate bacteriophage lambda. To better understand the biochemical mechanisms of GrpE action in various biological processes, six mutant proteins were overexpressed and purified. All of them, GrpE103, GrpE66, GrpE2/280, GrpE17, GrpE13a and GrpE25, have single amino acid substitutions located in highly conserved regions throughout the GrpE sequence. The biochemical defects of each mutant GrpE protein were identified by examining their abilities to: (i) support in vitro lambda DNA replication; (ii) stimulate the weak ATPase activity of DnaK; (iii) dimerize and oligomerize, as judged by glutaraldehyde crosslinking and HPLC size chromatography; (iv) interact with wild-type DnaK protein using either an ELISA assay, glutaraldehyde crosslinking or HPLC size chromatography. Our results suggest that GrpE can exist in a dimeric or oligomeric form, depending on its relative concentration, and that it dimerizes/oligomerizes through its N-terminal region, most likely through a computer predicted coiled-coil region. Analysis of several mutant GrpE proteins indicates that an oligomer of GrpE is the most active form that interacts stably with DnaK and that the interaction is vital for GrpE biological function. Our results also demonstrate that both the N-terminal and C-terminal regions are important for GrpE function in lambda DNA replication and its co-chaperone activity with DnaK.

Adenosine Triphosphatases↗

PubMed1996

NMR structure of the J-domain and the Gly/Phe-rich region of the Escherichia coli DnaJ chaperone.

The recombinant N-terminal 107-amino acid polypeptide fragment 2-108 of the DnaJ molecular chaperone of Escherichia coli, which contains the J-domain (residues 2 to 76) and the Gly/Phe-rich region (residues 77 to 108), was uniformly labeled with nitrogen-15 and carbon-13. The complete NMR solution structure of the J-domain was determined with the program DIANA on the basis of 682 nuclear Overhauser enhancement (NOE) upper distance limits and 180 dihedral angle constraints. It contains three well-defined helices comprising residues 6 to 10, 18 to 32 and 41 to 57, and a fourth helix, consisting of residues 61 to 68, which is well defined as a regular secondary structure but for which the location relative to the remainder of the molecule is not precisely determined. The helices II and III form an antiparallel helical coiled-coil. Helix I is approximately parallel to the plane defined by the helices II and III and runs from the carboxy-terminal end of the helix III to the center of helix II. Helix IV is positioned near the carboxy-terminal end of helix III and is on the same side of the coiled coil as helix I, but it is oriented approximately perpendicular to the plane of the helices II and III. This novel alpha-protein topology leads to formation of a hydrophobic core involving side-chains of all four helices. A strong correlation is seen between the extent of sequence-conservation of hydrophobic residues in the family of J-domain homologues, and the structural organization of the hydrophobic core in these proteins. The residues which have key roles for the specificity of the interaction of DnaJ-like proteins with their corresponding Hsp70 counterparts are located on the outer surfaces of the helices II and III, and in the loop connecting these two helices. Measurements of backbone amide proton exchange rates, 15N spin relaxation times and heteronuclear 15N {1H} NOEs provided additional insights into local conformational equilibria and internal rate processes in DnaJ(2-108). In the Gly/Phe-rich region, which is poorly ordered in the NMR solution structure and does not form a globular core, the polypeptide segment 90 to 103 differs from the segments 77 to 89 and 104 to 108 by reduced local flexibility. Considering that this same segment shows sequence conservation with corresponding segments in the Gly/Phe-rich regions of other DnaJ-like proteins, its reduced flexibility may be directly linked to the formation of the ternary DnaJ-DnaK-polypeptide complex.

Bacterial Proteins↗

PubMed1996

Structure-function analysis of the zinc finger region of the DnaJ molecular chaperone.

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.

Amino Acid Sequence↗

PubMed1996

In vivo protein folding: suppressor analysis of mutations in the groES cochaperone gene of Escherichia coli.

Our previous work has shown that the Escherichia coli groES14 and groES15 mutations result in reduced GroE chaperone machine function. By selecting for restoration of the ability of those mutant groES alleles to suppress the thermosensitivity of bacteria bearing the dnaA46 mutation, we isolated a number of intra- and extragenic suppressors that increase in vivo GroE chaperone function. One of the intragenic suppressors has been mapped to a segment that codes for the GroES mobile loop, previously shown to be indispensable for proper GroES/GroEL interaction. Two extragenic suppressors have been mapped to a groEL segment, previously identified by mutational analysis as coding for an important functional region of the GroEL protein. Our results should contribute to our eventual understanding of the structure-function relationships of the universally conserved GroE chaperone machine.

Bacterial Proteins↗

PubMed1996

Mutational analysis and properties of the msbA gene of Escherichia coli, coding for an essential ABC family transporter.

The htrB gene was discovered because its insertional inactivation interfered with Escherichia coli growth and viability at temperatures above 32.5 degrees C, as a result of accumulation of phospholipids. The msbA gene was originally discovered because when cloned on a low-copy-number plasmid vector it was able to suppress the temperature-sensitive growth phenotype of an htrB null mutant as well as the accumulation of phospholipids. The msbA gene product belongs to the superfamily of ABC transporters, a universally conserved family of proteins characterized by a highly conserved ATP-binding domain. The msbA gene is essential for bacterial viability at all temperatures. In order to understand the physiological role of the MsbA protein, we mutated the ATP-binding domain using random PCR mutagenesis. Six independent mutants were isolated and characterized. Four of these mutations resulted in single-amino-acid substitutions in non-conserved residues and were able to support cell growth at 30 degrees C but not at 43 degrees C. The remaining two mutations behaved as recessive lethals, and resulted in single-amino-acid substitutions in Walker motif B, one of the two highly conserved regions of the ATP-binding domain. Despite the fact that neither of these two mutant proteins can support E. coli growth, they both retained the ability to bind ATP in vitro. In addition, we present evidence to show that N-acetyl [3H]-glucosamine, a precursor of lipopolysaccharides, accumulates at the non-permissive temperature in the inner membrane of either htrB null or msbA conditional lethal strains. Translocation of the precursor to the outer membrane is restored by transformation with a plasmid containing the wild-type msbA gene. A possible role for MsbA as a translocator of lipopolysaccharides or its precursors is discussed.

ATP-Binding Cassette Transporters↗

PubMed1995

Both ambient temperature and the DnaK chaperone machine modulate the heat shock response in Escherichia coli by regulating the switch between sigma 70 and sigma 32 factors assembled with RNA polymerase.

In Escherichia coli individual sigma factors direct RNA polymerase (RNAP) to specific promoters. Upon heat shock induction there is a transient increase in the rate of transcription of approximately 20 heat shock genes, whose promoters are recognized by the RNAP-sigma 32 rather than the RNAP-sigma 70 holoenzyme. At least three heat shock proteins, DnaK, DnaJ and GrpE, are involved in negative modulation of the sigma 32-dependent heat shock response. Here we show, using purified enzymes, that upon heat treatment of RNAP holoenzyme the sigma 70 factor is preferentially inactivated, whereas the resulting heat-treated RNAP core is still able to initiate transcription once supplemented with sigma 32 (or fresh sigma 70). Heat-aggregated sigma 70 becomes a target for the joint action of DnaK, DnaJ and GrpE proteins, which reactivate it in an ATP-dependent reaction. The RNAP-sigma 32 holoenzyme is relatively stable at temperatures at which the RNAP-sigma 70 holoenzyme is inactivated. Furthermore, we show that formation of the RNAP-sigma 32 holoenzyme is favored over that of RNAP-sigma 70 at elevated temperatures. We propose a model of negative autoregulation of the heat shock response in which cooperative action of DnaK, DnaJ and GrpE heat shock proteins switches transcription back to constitutively expressed genes through the simultaneous reactivation of heat-aggregated sigma 70, as well as sequestration of sigma 32 away from RNAP.

DNA-Directed RNA Polymerases↗

PubMed1995

ATP hydrolysis is required for the DnaJ-dependent activation of DnaK chaperone for binding to both native and denatured protein substrates.

Using two independent experimental approaches to monitor protein-protein interactions (enzyme-linked immunosorbent assay and size exclusion high performance liquid chromatography) we describe a general mechanism by which DnaJ modulates the binding of the DnaK chaperone to various native protein substrates, e.g. lambda P, lambda O, delta 32, P1, RepA, as well as permanently denatured alpha-carboxymethylated lactalbumin. The presence of DnaJ promotes the DnaK for efficient DnaK-substrate complex formation. ATP hydrolysis is absolutely required for such DnaJ-dependent activation of DnaK for binding to both native and denatured protein substrates. Although ADP can stabilize such as an activated DnaK-protein complex, it cannot substitute for ATP in the activation reaction. In the presence of DnaJ and ATP, DnaK possesses the affinity to different substrates which correlates well with the affinity of DnaJ alone for these protein substrates. Only when the affinity of the DnaJ chaperone for its protein substrate is relatively high (e.g. delta 32, RepA) can a tertiary complex DnaK-substrate-DnaJ be detected. In the case that DnaJ binds weakly to its substrate (lambda P, alpha-carboxymethylated lactalbumin), DnaJ is only transiently associated with the DnaK-substrate complex, but the DnaK activation reaction still occurs, albeit less efficiently.

Adenosine Diphosphate↗

PubMed1995

The DnaJ chaperone catalytically activates the DnaK chaperone to preferentially bind the sigma 32 heat shock transcriptional regulator.

In Escherichia coli the heat shock response is under the positive control of the sigma 32 transcription factor. Three of the heat shock proteins, DnaK, DnaI, and GrpE, play a central role in the negative autoregulation of this response at the transcriptional level. Recently, we have shown that the DnaK and DnaJ proteins can compete with RNA polymerase for binding to the sigma 32 transcription factor in the presence of ATP, by forming a stable DnaJ-sigma 32-DnaK protein complex. Here, we report that DnaJ protein can catalytically activate DnaK's ATPase activity. In addition, DnaJ can activate DnaK to bind to sigma 32 in an ATP-dependent reaction, forming a stable sigma 32-DnaK complex. Results obtained with two DnaJ mutants, a missense and a truncated version, suggest that the N-terminal portion of DnaJ, which is conserved in all family members, is essential for this activation reaction. The activated form of DnaK binds preferentially to sigma 32 versus the bacteriophage lambda P protein substrate.

Adenosine Triphosphatases↗

PubMed1995

The ClpX heat-shock protein of Escherichia coli, the ATP-dependent substrate specificity component of the ClpP-ClpX protease, is a novel molecular chaperone.

All major classes of protein chaperones, including DnaK (the Hsp70 eukaryotic equivalent) and GroEL (the Hsp60 eukaryotic equivalent) have been found in Escherichia coli. Molecular chaperones enhance the yields of correctly folded polypeptides by preventing aggregation and even by disaggregating certain protein aggregates. Previously, we identified the ClpX heat-shock protein of E. coli because it enables the ClpP catalytic protease to degrade the bacteriophage lambda O replication protein. Here we report that ClpX alone possesses all the properties expected of a molecular chaperone protein. Specifically, it can protect the lambda O protein from heat-induced aggregation, disaggregate preformed lambda O aggregates, and even promote efficient binding of lambda O to its DNA recognition sequence. A lambda O-ClpX specific protein-protein interaction can be detected either by a modified ELISA assay or through the stimulation of ClpX's weak ATPase activity by lambda O. Unlike the behaviour of the major DnaK and GroEL chaperones, ClpX requires the presence of ATP or its non-hydrolysable analogue ATP-gamma-S for efficient interaction with other proteins including the protection of lambda O from aggregation. However, ClpX's ability to disaggregate lambda O aggregates requires hydrolysable ATP. We propose that the ClpX protein is a bona fide chaperone, whose biological role includes the maintenance of certain polypeptides in a form competent for proteolysis by the ClpP protease. Furthermore, our results suggest that the ClpX protein also performs typical chaperone protein functions independent of ClpP.

ATPases Associated with Diverse Cellular Activitie↗

PubMed1995

The rpoE gene encoding the sigma E (sigma 24) heat shock sigma factor of Escherichia coli.

Previous work has established that the transcription factor sigma E (sigma 24) is necessary for maintaining the induction of the heat shock response of Escherichia coli at high temperatures. We have identified the gene encoding sigma E using a genetic screen designed to isolate trans-acting mutations that abolish expression from either htrA or rpoHP3, two promoters recognized uniquely by sigma E-containing RNA polymerase. Such a screen was achieved by transducing strains carrying a single copy of either phtrA-lacZ or rpoHP3-lacZ fusions with mutagenized bacteriophage P1 lysates and screening for Lac- mutant colonies at 22 degrees C. Lac- mutants were subsequently tested for inability to grow at 43 degrees C (Ts- phenotype). Only those Lac- Ts- mutants that were unable to accumulate heat shock proteins at 50 degrees C were retained for further characterization. In a complementary approach, those genes which when cloned on a multicopy plasmid led to higher constitutive expression of the sigma E regulon were characterized and mapped. Both approaches identified the same gene, rpoE, mapping at 55.5 min on the E.coli genetic map and encoding a polypeptide of 191 amino acid residues. The wild-type and a mutant rpoE gene products were over-expressed and purified. It was found that the purified wild-type sigma E protein, when used in in vitro run-off transcription assays in combination with core RNA polymerase, was able to direct transcription from the htrA and rpoHP3 promoters, but not from known sigma 70-dependent promoters. In vivo and in vitro analyses of rpoE transcriptional regulation showed that the rpoE gene is transcribed from two major promoters, one of which is positively regulated by sigma E itself.

Bacterial Proteins↗