14 results on '"Gottesman, S."'
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2. Sequence and structure of Clp P, the proteolytic component of the ATP-dependent Clp protease of Escherichia coli.
- Author
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Maurizi, M R, primary, Clark, W P, additional, Katayama, Y, additional, Rudikoff, S, additional, Pumphrey, J, additional, Bowers, B, additional, and Gottesman, S, additional
- Published
- 1990
- Full Text
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3. Clp P represents a unique family of serine proteases.
- Author
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Maurizi, M R, primary, Clark, W P, additional, Kim, S H, additional, and Gottesman, S, additional
- Published
- 1990
- Full Text
- View/download PDF
4. The ATP-dependent Clp protease of Escherichia coli. Sequence of clpA and identification of a Clp-specific substrate.
- Author
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Gottesman, S, primary, Clark, W P, additional, and Maurizi, M R, additional
- Published
- 1990
- Full Text
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5. Role of the heat shock protein DnaJ in the lon-dependent degradation of naturally unstable proteins.
- Author
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Jubete, Y, Maurizi, M R, and Gottesman, S
- Abstract
We have investigated the role of DnaJ in protein degradation by examining the degradation of intrinsically unstable proteins by Lon protease in vivo. In Escherichia coli, Lon protease is responsible for the rate-limiting step in degradation of highly unstable proteins such as SulA, RcsA, and lambdaN protein, as well as for about 50% of the rapid degradation of abnormal proteins such as canavanine-containing proteins. We found that Lon-dependent degradation of both SulA and lambdaN protein was unaffected in cells lacking functional DnaJ, whereas Lon-dependent turnover of canavanine-containing proteins was slower in dnaJ mutant cells. DnaJ also affected the slow SulA degradation seen in the absence of Lon. The rate of degradation of RcsA was reduced in dnaJ mutants, but both Lon-dependent and Lon-independent degradation was affected; abnormal, canavanine-containing proteins were similarly affected. Both the RcsA that accumulated in dnaJ mutant cells and the SulA that accumulated in lon dnaJ mutant cells was aggregated. The abnormal proteins that partitioned to the insoluble pellet became solubilized over time in dnaJ+ cells but not in dnaJ- cells. Therefore, the co-chaperone DnaJ is not essential for Lon-dependent degradation and may act in protein turnover only as an accessory factor helping to maintain substrates in a soluble form. Such an accessory factor is apparently necessary for abnormal proteins and for RcsA. The relative rates of degradation and aggregation of specific protein targets may determine the importance of the chaperone systems in turnover of a given protein.
- Published
- 1996
6. ATP-dependent degradation of CcdA by Lon protease. Effects of secondary structure and heterologous subunit interactions.
- Author
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Van Melderen, L, Thi, M H, Lecchi, P, Gottesman, S, Couturier, M, and Maurizi, M R
- Abstract
CcdA, the antidote protein of the ccd post-segregational killing system carried by the F plasmid, was degraded in vitro by purified Lon protease from Escherichia coli. CcdA had a low affinity for Lon (Km >/=200 microM), and the peptide bond turnover number was approximately 10 min-1. CcdA formed tight complexes with purified CcdB, the killer protein encoded in the ccd operon, and fluorescence and hydrodynamic measurements suggested that interaction with CcdB converted CcdA to a more compact conformation. CcdB prevented CcdA degradation by Lon and blocked the ability of CcdA to activate the ATPase activity of Lon, suggesting that Lon may recognize bonding domains of proteins exposed when their partners are absent. Degradation of CcdA required ATP hydrolysis; however, CcdA41, consisting of the carboxyl-terminal 41 amino acids of CcdA and lacking the alpha-helical secondary structure present in CcdA, was degraded without ATP hydrolysis. Lon cleaved CcdA primarily between aliphatic and hydrophilic residues, and CcdA41 was cleaved at the same peptide bonds, indicating that ATP hydrolysis does not affect cleavage specificity. CcdA lost alpha-helical structure at elevated temperatures (Tm approximately 50 degrees C), and its degradation became independent of ATP hydrolysis at this temperature. ATP hydrolysis may be needed to disrupt interactions that stabilize the secondary structure of proteins allowing the disordered protein greater access to the proteolytic active sites.
- Published
- 1996
7. The two-component, ATP-dependent Clp protease of Escherichia coli. Purification, cloning, and mutational analysis of the ATP-binding component.
- Author
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Katayama, Y, Gottesman, S, Pumphrey, J, Rudikoff, S, Clark, W P, and Maurizi, M R
- Abstract
The ATP-binding component (Component II, hereafter referred to as ClpA) of a two-component, ATP-dependent protease from Escherichia coli has been purified to homogeneity. ClpA is a protein with subunit Mr 81,000. It has an intrinsic ATPase activity and activates degradation of protein substrates only in the presence of a second component (Component I, hereafter referred to as ClpP), Mg2+, and ATP. The amount of ClpA varies by less than a factor of 2 in cells grown in different media and at temperatures from 30 to 42 degrees C. ClpA does not appear to be a heat-shock protein since its synthesis is not dependent on htpR. Antibodies against purified ClpA were used to identify lambda transducing phage bearing the clpA gene. The cloned gene contains a DNA sequence expected to code for the first 28 amino acids of ClpA, which were determined by protein sequencing of purified ClpA. The clpA gene in the phage was mutated by insertion of delta kan defective transposons and the mutations were transferred to E. coli by homologous recombination. The clpA gene was mapped to 19 min on the E. coli chromosome. Mutant cells with insertions early in the gene produce no ClpA protein detectable in Western blots, and extracts of such mutant cells have no detectable ClpA activity. clpA- mutants grow well under all conditions tested and are not defective in turnover of proteins during nitrogen starvation nor in the turnover of such highly unstable proteins as the lambda proteins O, N, and cII, or the E. coli proteins SulA, RcsA, and glutamate dehydrogenase. The degradation of abnormal canavanine-containing proteins is defective in clpA mutants especially in cells that also have a lon- mutation. Extracts of clpA- lon- cells have ATP-dependent casein degrading activity.
- Published
- 1988
- Full Text
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8. Purification of the bacteriophage lambda xis gene product required for lambda excisive recombination.
- Author
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Abremski, K and Gottesman, S
- Abstract
Excision of the lambda prophage from the chromosome of its Escherichia coli host requires the products of the two viral genes int and xis. This paper reports a purification of the lambda xis gene product using a complementation assay in which functional Xis must be added to purified Int and an E. coli-derived host factor extract. Excisive recombination between a left (attL) and right (attR) prophage attachment site cloned on the same plasmid DNA substrate occurred efficiently under these conditions. Purified Int and Xis together could not carry out excision in vitro unless an extract derived from the E. coli host was added; purified integration host factor satisfied this requirement. Xis appears to have a molecular weight of 8800 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. It possesses no detectable endonuclease or topoisomerase activities, does not appear to bind DNA to filters, and does not increase the ability of Int to bind DNA. The addition of Xis not only stimulated excisive recombination in vitro but also inhibited integrative recombination. Xis protected Int protein from heat inactivation, suggesting a possible interaction between the two proteins. In light of these observations, possible roles for Xis in recombination are discussed.
- Published
- 1982
- Full Text
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9. A multiple-component, ATP-dependent protease from Escherichia coli.
- Author
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Katayama-Fujimura, Y., Gottesman, S., and Maurizi, M.R.
- Abstract
A new ATP-dependent, casein-degrading proteolytic complex has been identified and partially purified from Escherichia coli. The proteolytic complex can be isolated from wild-type cells as well as from mutants in which the gene for the ATP-dependent Lon protease is deleted. The complex consists of at least two components (components I and II) that can be separated from each other (and from wild-type Lon protease) by phosphocellulose chromatography. Neither component has casein-degrading activity when added separately to assay solutions with or without ATP. Both components must be present simultaneously for casein degradation to occur. Of the nucleotides tested, only ATP activates the proteolytic complex, and the ATP must be present continuously for degradation to occur. Component II copurifies with an ATPase activity and binds to a Type 4 ATP affinity column. ATP protects component II from heat inactivation, suggesting that component II interacts with ATP. Proteolysis was not inhibited by any serine protease inhibitors but was inhibited by reagents such as the organomercurial Neohydrin and N-ethylmaleimide, which react with sulfhydryl groups. Our data provide convincing evidence that E. coli possesses a previously undescribed proteolytic system composed of at least two complementary components and absolutely dependent on ATP.
- Published
- 1987
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10. Structure of phosphorylated-like RssB, the adaptor delivering σ s to the ClpXP proteolytic machinery, reveals an interface switch for activation.
- Author
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Brugger C, Schwartz J, Novick S, Tong S, Hoskins JR, Majdalani N, Kim R, Filipovski M, Wickner S, Gottesman S, Griffin PR, and Deaconescu AM
- Subjects
- Crystallography, X-Ray, Enzyme Activation, Hydrogen Deuterium Exchange-Mass Spectrometry, Phosphorylation, Protein Domains, DNA-Binding Proteins chemistry, DNA-Binding Proteins metabolism, Endopeptidase Clp chemistry, Endopeptidase Clp metabolism, Escherichia coli chemistry, Escherichia coli enzymology, Escherichia coli metabolism, Escherichia coli Proteins chemistry, Escherichia coli Proteins metabolism, Proteolysis, Sigma Factor chemistry, Sigma Factor metabolism, Transcription Factors chemistry, Transcription Factors metabolism
- Abstract
In enterobacteria such as Escherichia coli, the general stress response is mediated by σ
s , the stationary phase dissociable promoter specificity subunit of RNA polymerase. σs is degraded by ClpXP during active growth in a process dependent on the RssB adaptor, which is thought to be stimulated by the phosphorylation of a conserved aspartate in its N-terminal receiver domain. Here we present the crystal structure of full-length RssB bound to a beryllofluoride phosphomimic. Compared to the structure of RssB bound to the IraD anti-adaptor, our new RssB structure with bound beryllofluoride reveals conformational differences and coil-to-helix transitions in the C-terminal region of the RssB receiver domain and in the interdomain segmented helical linker. These are accompanied by masking of the α4-β5-α5 (4-5-5) "signaling" face of the RssB receiver domain by its C-terminal domain. Critically, using hydrogen-deuterium exchange mass spectrometry, we identify σs -binding determinants on the 4-5-5 face, implying that this surface needs to be unmasked to effect an interdomain interface switch and enable full σs engagement and hand-off to ClpXP. In activated receiver domains, the 4-5-5 face is often the locus of intermolecular interactions, but its masking by intramolecular contacts upon phosphorylation is unusual, emphasizing that RssB is a response regulator that undergoes atypical regulation., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2023
- Full Text
- View/download PDF
11. Lack of polyamines leads to cotranslational degradation of the general stress factor RpoS in Escherichia coli.
- Author
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Majdalani N, Chattopadhyay M, Keller C, and Gottesman S
- Subjects
- Stress, Physiological, Proteolysis, Open Reading Frames genetics, Escherichia coli genetics, Escherichia coli metabolism, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism, Gene Expression Regulation, Bacterial, Polyamines metabolism
- Abstract
The specialized sigma factor RpoS mediates a general stress response in Escherichia coli and related bacteria, activating promoters that allow cells to survive stationary phase and many stresses. RpoS synthesis and stability are regulated at multiple levels. Translation of RpoS is positively regulated by multiple small RNAs in response to stress. Degradation of RpoS, dependent upon the adaptor protein RssB, is rapid during exponential growth and ceases upon starvation or other stresses, increasing accumulation of RpoS. E. coli carrying mutations that block the synthesis of polyamines were previously found to have low levels of RpoS, while levels increased rapidly when polyamines were added. We have used a series of reporters to examine the basis for the lack of RpoS in polyamine-deficient cells. The polyamine requirement was independent of small RNA-mediated positive regulation of RpoS translation. Mutations in rssB stabilize RpoS and significantly bypassed the polyamine deficit, suggesting that lack of polyamines might lead to rapid RpoS degradation. However, rates of degradation of mature RpoS were unaffected by polyamine availability. Codon optimization in rpoS partially relieved the polyamine dependence, suggesting a defect in RpoS translation in the absence of polyamines. Consistent with this, a hyperproofreading allele of ribosomal protein S12, encoded by rpsL, showed a decrease in RpoS levels, and this decrease was also suppressed by either codon optimization or blocking RpoS degradation. We suggest that rpoS codon usage leads it to be particularly sensitive to slowed translation, due to either lack of polyamines or hyperproofreading, leading to cotranslational degradation. We dedicate this study to Herb Tabor and his foundational work on polyamines, including the basis for this study., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)
- Published
- 2023
- Full Text
- View/download PDF
12. Trouble is coming: Signaling pathways that regulate general stress responses in bacteria.
- Author
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Gottesman S
- Subjects
- Bacterial Proteins genetics, Bacterial Proteins metabolism, Escherichia coli metabolism, Host Factor 1 Protein metabolism, Protein Biosynthesis, RNA, Small Untranslated metabolism, Sigma Factor genetics, Sigma Factor metabolism, Bacteria metabolism, Signal Transduction, Stress, Physiological
- Abstract
Bacteria can rapidly and reversibly respond to changing environments via complex transcriptional and post-transcriptional regulatory mechanisms. Many of these adaptations are specific, with the regulatory output tailored to the inducing signal (for instance, repairing damage to cell components or improving acquisition and use of growth-limiting nutrients). However, the general stress response, activated in bacterial cells entering stationary phase or subjected to nutrient depletion or cellular damage, is unique in that its common, broad output is induced in response to many different signals. In many different bacteria, the key regulator for the general stress response is a specialized sigma factor, the promoter specificity subunit of RNA polymerase. The availability or activity of the sigma factor is regulated by complex regulatory circuits, the majority of which are post-transcriptional. In Escherichia coli , multiple small regulatory RNAs, each made in response to a different signal, positively regulate translation of the general stress response sigma factor RpoS. Stability of RpoS is regulated by multiple anti-adaptor proteins that are also synthesized in response to different signals. In this review, the modes of signaling to and levels of regulation of the E. coli general stress response are discussed. They are also used as a basis for comparison with the general stress response in other bacteria with the aim of extracting key principles that are common among different species and highlighting important unanswered questions.
- Published
- 2019
- Full Text
- View/download PDF
13. Bacterial small RNA-based negative regulation: Hfq and its accomplices.
- Author
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De Lay N, Schu DJ, and Gottesman S
- Subjects
- Endoribonucleases metabolism, Endoribonucleases physiology, Escherichia coli enzymology, Multienzyme Complexes metabolism, Multienzyme Complexes physiology, Polyribonucleotide Nucleotidyltransferase metabolism, Polyribonucleotide Nucleotidyltransferase physiology, RNA Helicases metabolism, RNA Helicases physiology, RNA Stability, RNA, Bacterial genetics, RNA, Bacterial metabolism, RNA, Bacterial physiology, RNA, Messenger genetics, RNA, Messenger metabolism, Salmonella enterica enzymology, Escherichia coli genetics, Escherichia coli Proteins physiology, Gene Expression Regulation, Bacterial, Host Factor 1 Protein physiology, RNA, Small Untranslated physiology, Salmonella enterica genetics
- Abstract
A large group of bacterial small regulatory RNAs (sRNAs) use the Hfq chaperone to mediate pairing with and regulation of mRNAs. Recent findings help to clarify how Hfq acts and highlight the role of the endonuclease RNase E and its associated proteins (the degradosome) in negative regulation by these sRNAs. sRNAs frequently uncouple transcription and translation by blocking ribosome access to the mRNA, allowing other proteins access to the mRNA. As more examples of sRNA-mediated regulation are studied, more variations on how Hfq, RNase E, and other proteins collaborate to bring about sRNA-based regulation are being found.
- Published
- 2013
- Full Text
- View/download PDF
14. Soluble beta 2-microglobulin-free class I heavy chains are released from the surface of activated and leukemia cells by a metalloprotease.
- Author
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Demaria S, Schwab R, Gottesman SR, and Bushkin Y
- Subjects
- Antigens, CD metabolism, Antigens, Differentiation, B-Lymphocyte metabolism, CD4 Antigens metabolism, Flow Cytometry, HLA-DR Antigens metabolism, Histocompatibility Antigens Class I biosynthesis, Histocompatibility Antigens Class I isolation & purification, Humans, Leukemia, Lymphocytic, Chronic, B-Cell enzymology, Macromolecular Substances, Phenanthrolines pharmacology, Receptors, Transferrin, Tetradecanoylphorbol Acetate pharmacology, Tumor Cells, Cultured, Zinc pharmacology, Histocompatibility Antigens Class I metabolism, Leukemia, B-Cell immunology, Leukemia, Lymphocytic, Chronic, B-Cell immunology, Leukemia, T-Cell immunology, Metalloendopeptidases metabolism, beta 2-Microglobulin isolation & purification
- Abstract
Regulation of the expression of major histocompatibility complex (MHC) class I heavy chains not associated with beta 2-microglobulin (beta 2m) on freshly isolated and in vitro cultured human B and T leukemia cells was analyzed. These beta 2m-free class I heavy chains originate from surface beta 2m-associated MHC class I molecules and are expressed as integral membrane glycoproteins on activated, but not resting, cells. We found that the levels of beta 2m-free class I heavy chains can be regulated by proteolytic cleavage and release into the medium of soluble molecules containing the extracellular domains. The release is mediated by a Zn(2+)-dependent, membrane-bound metalloprotease that does not cleave HLA-DR, CD4, and CD71 surface receptors and can be activated by phorbol myristate acetate. Specific cleavage by the metalloprotease occurs at a site close to the papain cleavage site in the alpha 3 domain of class I heavy chains. This site is not accessible to the metalloprotease in beta 2m-associated MHC class I molecules. The dissociation of beta 2m-associated MHC class I molecules and subsequent cleavage of beta 2m-free class I heavy chains may be partially responsible for controlling the levels of MHC class I molecules on the surface of activated cells.
- Published
- 1994
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