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1.
The bacterial chaperone high-temperature protein G (HtpG), a member of the Hsp90 protein family, is involved in the protection of cells against a variety of environmental stresses. The ability of HtpG to form complexes with other bacterial proteins, especially those involved in fundamental functions, is indicative of its cellular role. An interaction between HtpG and DnaA, the main initiator of DNA replication, was studied both in vivo, using a bacterial two-hybrid system, and in vitro with a modified pull-down assay and by chemical cross-linking. In vivo, this interaction was demonstrated only when htpG was expressed from a high copy number plasmid. Both in vitro assays confirmed HtpG–DnaA interactions.  相似文献   

2.
Abstract We previously reported that plasmid DNA in Escherichia coli cells growing under aerobic conditions relaxed immediately and transiently after heat shock (Mizushima, T., Natori, S. and Sekimizu, K., Mol. Gen. Genet. (1993) 238, 1–5). We next examined DNA relaxation and induction of heat shock proteins after heat shock in cells growing under anaerobic conditions. DNA in these cells relaxed rapidly (2 min) after heat shock (42°C), as was the case with aerobically growing cells, but full superhelicity was not recovered. The relaxed state of DNA topology was maintained for 60 min after heat shock. Induction of DnaK and GroEL proteins, which was transient in aerobically growing cells, was continuous in anaerobically growing cells. Therefore, induction of heat shock proteins correlated with DNA relaxation in both aerobic and anaerobic conditions.  相似文献   

3.
Acid shock proteins of Escherichia coli   总被引:19,自引:0,他引:19  
Synthesis of total cellular proteins of Escherichia coli was studied after transfer of cultures from pH 6.9 to pH 4.3. Proteins induced by such an external pH shift down were identified by mono- and bi-dimensional electrophoresis. 30 to 45 min after an acid shift, a group of at least sixteen polypeptides was markedly induced. Four of these polypeptides corresponded to the well known heat shock proteins GroEL, DnaK, HtpG and HtpM. Their pH induction was RpoH-dependent. Three other pH-induced proteins were previously identified as stress proteins induced either by osmolarity or aerobiosis or low temperature (proteins 32 (defined in this paper), C70.0 and C62.7). Seven other proteins were specifically induced after an acid shift and were called acid shock proteins (ASP). The induction of one of these proteins was RpoH-dependent, whereas that of others was RpoH-independent.  相似文献   

4.
5.
Molecular beacons for detecting DNA binding proteins: mechanism of action   总被引:3,自引:0,他引:3  
New methodology for detecting sequence-specific DNA binding proteins has been recently developed (T. Heyduk, and E. Heyduk, Nat. Biotechnol. 20 (2002) 171). The central feature of this assay is protein-dependent association of two DNA fragments, each containing about half of a DNA sequence-defining the protein binding site. In this report we propose a physical model explaining the functioning of the assay. The model involves two linked equilibria: association between the two DNA fragments and binding of the protein exclusively to the complex between the two DNA fragments. Equilibrium and kinetic experiments provided evidence supporting the proposed model and showed that the model was sufficient to describe the behavior of the assay under a variety of conditions. Kinetic data identified the association between the two DNA half-sites as the rate-limiting step of the assay. Theoretical simulations based on the proposed model were used to investigate parameters important for the maximal sensitivity of the assay. Physical understanding of the assay will provide means for rational design of the assay for a variety of target proteins.  相似文献   

6.
DafA is encoded by the dnaK operon of Thermus thermophilus and mediates the formation of a highly stable complex between the chaperone DnaK and its co-chaperone DnaJ under normal growth conditions. DafA(Tth) contains 87 amino acid residues and is the only member of the DnaK(Tth) chaperone system for which no corresponding protein has yet been identified in other organisms and whose particular function has remained elusive. Here, we show directly that the DnaK(Tth)-DnaJ(Tth)-DafA(Tth) complex cannot represent the active chaperone species since DafA(Tth) inhibits renaturation of firefly luciferase by suppressing substrate association. Since DafA(Tth) must be released before the substrate proteins can bind we hypothesized that free DafA(Tth) might have regulatory functions connected to the heat shock response. Here, we present evidence that supports this hypothesis. We identified the 70S ribosome as binding target of free DafA(Tth). Our results show that the association of DafA(Tth) and 70S ribosomes does not require the participation of DnaK(Tth) or DnaJ(Tth). On the contrary, the assembly of DnaK(Tth)-DnaJ(Tth)-DafA(Tth) and ribosome-DafA(Tth) complexes seems to be competitive. These findings strongly suggest the involvement of DafA(Tth) in regulatory processes occurring at a translational level, which could represent a new mechanism of heat shock response as an adaptation to elevated temperature.  相似文献   

7.
DnaK is a molecular chaperone of Escherichia coli that belongs to a family of conserved 70-kDa heat shock proteins. The Hsp70 chaperones are well known for their crucial roles in regulating protein homeostasis, preventing protein aggregation, and directing subcellular traffic. Given the complexity of functions, a chemical method for controlling the activities of these chaperones might provide a useful experimental tool. However, there are only a handful of Hsp70-binding molecules known. To build this area, we developed a robust, colorimetric, high-throughput screening (HTS) method in 96-well plates that reports on the ATPase activity of DnaK. Using this approach, we screened a 204-member focused library of molecules that share a dihydropyrimidine core common to known Hsp70-binding leads and uncovered seven new inhibitors. Intriguingly, the candidates do not appear to bind the hydrophobic groove that normally interacts with peptide substrates. In sum, we have developed a reliable HTS method that will likely accelerate discovery of small molecules that modulate DnaK/Hsp70 function. Moreover, because this family of chaperones has been linked to numerous diseases, this platform might be used to generate new therapeutic leads.  相似文献   

8.
The DNA and protein sequences of single-stranded DNA binding proteins (SSBs) encoded by the plP71a, plP231a, and R64 conjugative plasmids have been determined and compared to Escherichia coli SSB and the SSB encoded by F-plasmid. Although the amino acid sequences of all of these proteins are highly conserved within the NH2-terminal two-thirds of the protein, they diverge in the COOH-terminal third region. A number of amino acid residues which have previously been implicated as being either directly or indirectly involved in DNA binding are conserved in all of these SSBs. These residues include Trp-40, Trp-54, Trp-88, His-55, and Phe-60. On the basis of these sequence comparisons and DNA binding studies, a role for Tyr-70 in DNA binding is suggested for the first time. Although the COOH-terminal third of these proteins diverges more than their NH2-terminal regions, the COOH-terminal five amino acid residues of all five of these proteins are identical. In addition, all of these proteins share the characteristic property of having a protease resistant, NH2-terminal core and an acidic COOH-terminal region. Despite the high degree of sequence homology among the plasmid SSB proteins, the F-plasmid SSB appears unique in that it was the only SSB tested that neither bound well to poly(dA) nor was able to stimulate DNA polymerase III holoenzyme elongation rates. Poly [d(A-T)] melting studies suggest that at least three of the plasmid encoded SSBs are better helix-destabilizing proteins than is the E. coli SSB protein.  相似文献   

9.
Wu  Hong  Liu  Xiang-Qin 《Plant molecular biology》1997,34(2):339-343
The Guillardia theta chloroplast hlpA gene encodes a protein resembling bacterial histone-like protein HU. This gene was cloned and overexpressed in Escherichia coli cells, and the resulting protein product, HlpA, was purified and characterized in vitro. In addition to exhibiting a general DNA-binding activity, the chloroplast HlpA protein also strongly facilitated cyclization of a short DNA fragment in the presence of T4 DNA ligase, indicating its ability to mediate very tight DNA curvatures.  相似文献   

10.
Humidity has a large impact on the distribution and abundance of terrestrial invertebrates, but the molecular mechanisms governing drought resistance are not fully understood. Some attention has been given to the role of the heat shock response as a component of desiccation tolerance, but recent focus has been on the chaperone-like LEA (late embryogenesis abundant) proteins in anhydrobiotic animals. This study investigates the expression of putative LEA proteins as well as the heat shock protein Hsp70 during drought stress in soil and surface dwelling species of Collembola (springtails). In silico analysis of four EST candidates from two species of Collembola showed the presence of a Group 3 LEA protein in Megaphorura arctica. In common with other Group 3 LEA proteins, the new sequence is predicted to be 100% natively unfolded, with a strong degree of lysine and alanine periodicity and with a negative average hydrophobicity of −1.273. The sequence clusters with members of the Group 3 LEA in plants. Furthermore, cross-species Western blotting showed drought-induced expression of putative LEA proteins in six species of Collembola. In the surface dwelling species, Orchesella cincta, degree of dehydration and length of exposure correlated with level of putative LEA protein. Hsp70 was also found to increase in individuals of O. cincta and Folsomia candida that had been exposed to drought conditions for 6 days. These results show the presence of a LEA protein-coding region in Collembola, but also indicate that several proteins are involved in response to dehydration stress, including Hsp70.  相似文献   

11.
Abstract The purified MukB protein of Escherichia coli has DNA binding activity and nucleotide binding activity. We have isolated a mutation, mukB1013 , causing a substitution of valine at position 1379 to leucine. This mutant MukB protein was defective for DNA binding, while the ATP binding activity remained unaffected. A truncated MukB protein that is short of 109 amino acids from the C-terminus failed to bind DNA.  相似文献   

12.
General stress proteins in Bacillus subtilis   总被引:3,自引:0,他引:3  
Abstract Bacillus cells frequently faced with various adverse environmental factors in nature have evolved different adaptational strategies. The induction of stress proteins is an essential component of this adaptational network. In Bacillus subtilis there are two groups of stress proteins. The first group is factor specific, whereas the second group is induced by growth restrictive conditions in general. The relationship between the stringent response and the induction of stress proteins is discussed.  相似文献   

13.
Escherichia coli contains two thioredoxins, Trx1 and Trx2, and a thioredoxin-like protein, YbbN, which presents a strong homology in its N-terminal part with thioredoxin 1 and 2. YbbN, however, does not possess the canonical Cys-x-x-Cys active site of thioredoxins, but instead a Ser-x-x-Cys site. In addition to Cys-38, located in the SxxC site, it contains a second cysteine, Cys-63, close to Cys-38 in the 3D model. Cys-38 and Cys-63 undergo an oxidoreduction process, suggesting that YbbN functions with two redox cysteines. Accordingly, YbbN catalyzes the oxidation of reduced RNase and the isomerization of scrambled RNase. Moreover, upon oxidation, its oligomeric state changes from dimers to tetramers and higher oligomers. YbbN also possesses chaperone properties, promoting protein folding after urea denaturation and forming complexes with unfolded proteins. This is the first biochemical characterization of a member of the YbbN class of bacterial thioredoxin-like proteins, and in vivo experiments will allow to determine the importance of its redox and chaperone properties in the cellular physiology.  相似文献   

14.
The genes encoding the leucine binding proteins in E coli have been cloned and their DNA sequences have been determined. One of the binding proteins (LIV-BP) binds leucine, isoleucine, valine, threonine, and alanine, whereas the other (LS-BP) binds only the D- and L-isomers of leucine. These proteins bind their solutes as they enter the periplasm, then interact with three membrane components, livH, livG, and livM, to achieve the translocation of the solute across the bacterial cell membrane. Another feature of the binding proteins is that they must be secreted into the periplasmic space where they carry out their function. The amino acid sequence of the two binding proteins is 80% homologous, indicating that they are the products of an ancestral gene duplication. Because of these characteristics of the leucine binding proteins, we are using them as models for studying the relationships between protein structure and function.  相似文献   

15.
16.
Abstract The CbpA protein is an analog of the DnaJ molecular chaperone of Escherichia coli . The dnaJ cbpA double-null mutant exhibits severe defects in cell growth, namely, a very narrow temperature range for growth. To gain insight into the functions of CbpA as well as DnaJ, we isolated a multicopy suppressor gene that permits this dnaJ cbpA ~ mutant to grow normally at low temperatures. The suppressor gene was identified as rpoD , the gene that encodes the major σ 70. The biological implications of this finding are examined and discussed.  相似文献   

17.
The structure of the nucleotide-binding site of the Escherichia coli replication factor DnaC protein and the effect of the nucleotide cofactor on the protein structure have been examined using ultraviolet, steady-state, and time-dependent fluorescence spectroscopy. Emission spectra and quenching studies of the fluorescent nucleotide analogs, 3′-O-(N-methylantraniloyl)-5′-triphosphate (MANT-ATP) and 3′-O-(N-methylantraniloyl)-5′-diphosphate (MANT-ADP), bound to the DnaC protein indicate that the nucleotide-binding site forms a hydrophobic cleft on the surface of the protein. Fluorescence decays of free and bound MANT-ATP and MANT-ADP indicate that cofactors exist in two different conformations both, free and bound to the protein. However, the two conformations of the bound nucleotides differ in their solvent accessibilities. Moreover, there are significant differences in the solvent accessibility between ATP and ADP complexes. Specific binding of magnesium to the protein controls the structure of the binding site, particularly, in the case of the ATP complex, leading to additional opening of the binding site cleft. Both tyrosine and tryptophan residues are located on the surface of the protein. The tryptophans are clustered at a large distance from the nucleotide-binding site. However, in spite of a large spatial separation, binding of both cofactors induces significant and different changes in the structure of the environment of tryptophans, indicating long-range structural effects through the DnaC molecule. Moreover, only ATP induces changes in the distribution of the tyrosine residues on the surface of the protein. The data reveal that the nucleotide-DnaC protein complex is a sophisticated allosteric system, responding differently to the ATP and ADP binding.  相似文献   

18.
Molecular chaperones are involved in protein folding, protein targeting to membranes, and protein renaturation after stress. They interact specifically with hydrophobic sequences that are exposed in unfolded proteins, and buried in native proteins. We have studied the interaction of DnaK with native water-soluble proteins and membrane proteins. DnaK–native protein interactions are characterized by dissociation constants between 1 and 50 μM (compared with 0.01–1 μM for unfolded proteins). This affinity is within the range of most intracellular protein concentrations, suggesting that DnaK interacts with a greater number of native proteins than previously suspected. We found a correlation between the affinity of native proteins for DnaK and their affinity for hydrophobic-interaction chromatography adsorbents, suggesting that DnaK interacts with exposed hydrophobic groups in native proteins. The interaction between DnaK and membrane proteins is characterized by DnaK's high affinity for detergent-solubilized membrane proteins, and its lower affinity for membrane proteins inserted in lipid bilayers, suggesting that the chaperone can interact with the hydrophobic sequences of the former, while it cannot penetrate the hydrophobic core of lipid bilayers. Thus, the specificity of DnaK for hydrophobic sequences is involved in its interaction with not only unfolded proteins, but also native water-soluble proteins and membrane proteins. All proteins interact with DnaK according to their exposed hydrophobicity.  相似文献   

19.
Molecular chaperones are central to cellular protein homeostasis. In mammals, protein misfolding diseases and aging cause inflammation and progressive tissue loss, in correlation with the accumulation of toxic protein aggregates and the defective expression of chaperone genes. Bacteria and non-diseased, non-aged eukaryotic cells effectively respond to heat shock by inducing the accumulation of heat-shock proteins (HSPs), many of which molecular chaperones involved in protein homeostasis, in reducing stress damages and promoting cellular recovery and thermotolerance. We performed a meta-analysis of published microarray data and compared expression profiles of HSP genes from mammalian and plant cells in response to heat or isothermal treatments with drugs. The differences and overlaps between HSP and chaperone genes were analyzed, and expression patterns were clustered and organized in a network. HSPs and chaperones only partly overlapped. Heat-shock induced a subset of chaperones primarily targeted to the cytoplasm and organelles but not to the endoplasmic reticulum, which organized into a network with a central core of Hsp90s, Hsp70s, and sHSPs. Heat was best mimicked by isothermal treatments with Hsp90 inhibitors, whereas less toxic drugs, some of which non-steroidal anti-inflammatory drugs, weakly expressed different subsets of Hsp chaperones. This type of analysis may uncover new HSP-inducing drugs to improve protein homeostasis in misfolding and aging diseases.  相似文献   

20.
The thermoacidophilic archaeon Sulfolobus shibatae synthesizes a large amount of the 7-ku DMA binding proteins known as Ssh7. Our hybridization experiments showed that two Ssh7-encoding genes existed in the genome of S. shibatae. These two genes, designated ssh7a and ssh7b, have been cloned, sequenced and expressed in Escherichia coli. The two Ssh7 proteins differ only at three amino acid positions. In addition, the cis-regulatory sequences of the ssh7a and ssh7b genes are highly conserved. These results suggest the presence of a selective pressure to maintain not only the sequence but also the expression of the two genes. We have also found that there are two genes encoding the 7-ku protein in Sulfolobus solfataricus. Based on this and other studies, we suggest that the gene encoding the 7-ku protein underwent duplication before the separation of Sulfolobus species. Binding of native Ssh7 and recombinant (r)Ssh7 to short duplex DNA fragments was analyzed by electrophoretic mobility shift assays. Both n  相似文献   

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