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The CarD-CarG complex controls various cellular processes in the bacterium Myxococcus xanthus including fruiting body development and light-induced carotenogenesis. The CarD N-terminal domain, which defines the large CarD_CdnL_TRCF protein family, binds to CarG, a zinc-associated protein that does not bind DNA. The CarD C-terminal domain resembles eukaryotic high-mobility-group A (HMGA) proteins, and its DNA binding AT hooks specifically recognize the minor groove of appropriately spaced AT-rich tracts. Here, we investigate the determinants of the only known CarD binding site, the one crucial in CarD-CarG regulation of the promoter of the carQRS operon (PQRS), a light-inducible promoter dependent on the extracytoplasmic function (ECF) σ factor CarQ. In vitro, mutating either of the 3-bp AT tracts of this CarD recognition site (TTTCCAGAGCTTT) impaired DNA binding, shifting the AT tracts relative to PQRS had no effect or marginally lowered DNA binding, and replacing the native site by the HMGA1a binding one at the human beta interferon promoter (with longer AT tracts) markedly enhanced DNA binding. In vivo, however, all of these changes deterred PQRS activation in wild-type M. xanthus, as well as in a strain with the CarD-CarG pair replaced by the Anaeromyxobacter dehalogenans CarD-CarG (CarDAd-CarGAd). CarDAd-CarGAd is functionally equivalent to CarD-CarG despite the lower DNA binding affinity in vitro of CarDAd, whose C-terminal domain resembles histone H1 rather than HMGA. We show that CarD physically associates with RNA polymerase (RNAP) specifically via interactions with the RNAP β subunit. Our findings suggest that CarD regulates a light-inducible, ECF σ-dependent promoter by coupling RNAP recruitment and binding to a specific DNA site optimized for affinity and position.  相似文献   

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K Kayukawa  Y Makino  S Yogosawa  T Tamura 《Gene》1999,234(1):139-147
RPB6 is one of the common subunits of all eukaryotic RNA polymerases and is indispensable for the enzyme function. Here, we isolated a rat cDNA encoding RPB6. It contained 127 amino acid (a.a.) residues. From alignment of RPB6 homologues of various eukaryotes, we defined two conserved regions, i.e. an N-terminal acidic region and a C-terminal core. In this study, we investigated in vitro phosphorylation of rat RPB6 by casein kinase II (CKII), a pleiotropic regulator of numerous cellular proteins. Three putative CKII-phosphorylated a.a. within rat RPB6 were assigned. We found that serines were phosphorylated by CKII in vitro. Mutagenesis studies provided evidence that a serine at a.a. position 2 was exclusively phosphorylated. Finally, an RPB6-engaged in-gel kinase assay clarified that CKII was a prominent protein kinase in rat liver nuclear extract that phosphorylates RPB6. Therefore, RPB6 was implied to be phosphorylated by CKII in the nucleus. We postulate that the N-terminal acidic region of the RPB6 subunit has some phosphorylation-coupled regulatory functions.  相似文献   

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The central region of the N-myc protein has a characteristic amino acid sequence EDTLSDSDDEDD, which is very similar to those of particular domains of adenovirus E1A, human papilloma virus E7, Simian virus 40 large T, c-myc and L-myc proteins. Domains of these three viral oncoproteins have recently been shown to be specific binding sites for the tumor-suppressor gene retinoblastoma protein. We have noted that the sequence of serine followed by a cluster of acidic amino acids is exactly the same as that of a typical substrate of casein kinase II (CKII). Therefore, we investigated whether these nuclear oncoproteins are phosphorylated by CKII. For this purpose, we fused the beta-galactosidase and N-myc genes including this domain and expressed it in Escherichia coli cells. Several mutant N-myc genes, containing single amino acid substitutions in this domain, were also used to produce fused proteins. Strong phosphorylation by CKII was detected with the fused protein of wild-type N-myc. However, no phosphorylation of beta-galactosidase itself was observed and the phosphorylations of fused mutant proteins were low. Another fused N-myc protein containing most of the C-terminal region downstream of this acidic region was not phosphorylated by CKII. Analysis of phosphorylation sites in synthetic peptides of this acidic region identified the major sites phosphorylated by CKII as Ser261 and Ser263. On two-dimensional tryptic mapping of phosphorylated N-myc proteins, major spots of in vitro-labeled and in-vivo-labeled N-myc proteins were detected in the same positions. These results suggest that two serine residues of the acidic central region of the N-myc protein are phosphorylated by CKII in vivo as well as in vitro. The functional significance of this acidic domain is discussed.  相似文献   

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We show that VP16 is phosphorylated by cellular kinases in vivo and in vitro and map the major sites of phosphorylation to be on serines towards the C-terminus, downstream of position 370 in both cases. Deletion of the acidic activation domain had no effect on phosphorylation, refining the sites to between position 370 and 411. Within VP16, the C-terminal boundary for complex formation with Oct-1 and HCF lies at position 388, and between 370 and 388 lies one serine, at position 375. This is a consensus casein kinase II (CKII) site and, using purified wild-type and mutant proteins, we show that it is the main CKII site in the body of the N-terminal complex-forming region. This site is also phosphorylated in nuclear extracts. Although other sites, mainly Ser411, are also phosphorylated by nuclear kinase(s), the single substitution of Ser375 to alanine abolishes CKII phosphorylation in vitro and virtually eliminates complex formation. This serine lies in a surface-exposed region of VP16 and, although complex formation is disrupted, other activities of the mutant are unaffected. Ser375 is also required in vivo where substitution to alanine abolishes transactivation, while replacement with threonine restores normal levels of activity.  相似文献   

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In prokaryotes, the principal signal transduction systems operating at the level of protein phosphorylation are the two-component systems. A number of hybrid histidine protein kinases in these systems contain several receiver domains, however, the function of these receiver domains is unknown. The RodK kinase in Myxococcus xanthus has an unconventional domain composition with a putative N-terminal sensor domain followed by a histidine kinase domain and three receiver domains. RodK is essential for the spatial coupling of the two morphogenetic events underlying fruiting body formation in M. xanthus, aggregation of cells into nascent fruiting bodies and the subsequent sporulation of these cells. RodK kinase activity is indispensable for RodK activity. By systematically substituting the conserved, phosphorylatable aspartate residues in the three receiver domains, genetic evidence is provided that each receiver domain is important for RodK function and that each receiver domain has a distinct function, which depends on phosphorylation. Biochemical analyses provided indirect evidence for phosphotransfer from the RodK kinase domain to the third receiver domain. This is the first example of a hybrid histidine protein kinase in which four signalling domains have been shown to be required for full activity.  相似文献   

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Smith AE  Buchmueller KL 《Biochemistry》2011,50(38):8107-8116
The molecular mechanism for the displacement of HMGA1 proteins from DNA is integral to disrupting their cellular function, which is linked to many metastatic cancers. Chemical shift and NOESY NMR experiments provide structural evidence for the displacement of an AT hook peptide (DNA binding motif of HMGA1 proteins) by both monomeric and dimeric distamycin. However, the displaced AT hook alters distamycin binding by weakening the distamycin:DNA complex, while slowing monomeric distamycin dissociation when AT hook is in excess. The central role of the AT hook was evaluated by monitoring full-length HMGA1a protein binding using fluorescence anisotropy. HMGA1a was effectively displaced by distamycin, but the cooperative binding exhibited by distamycin was eliminated by displaced HMGA1a. Additionally, these studies indicate that HMGA1a is displaced from the DNA by 1 equiv of distamycin, suggesting the ability to develop therapeutics that take advantage of the positively cooperative nature of HMGA1a binding.  相似文献   

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Phosphorylation of acidic ribosomal proteins P1/P2-P0 is a common phenomenon in eukaryotic organisms. It was found previously that in Trichosporon cutaneum, unlike in other yeast species, in addition to the two acidic ribosomal proteins, two other proteins of 15 kDa and 19 kDa of the small ribosomal subunit were phosphorylated. Here we describe two protein kinases: CKI and CKII, which are engaged in the modification of T. cutaneum ribosomal proteins. The acidic ribosomal proteins and the protein of 19 kDa were modified by CKII associated with ribosomes, while the protein of 15 kDa was modified by CKI. Protein kinase CKI was purified from cell-free extract (CKIC) and from ribosomal fraction (CKIR). The molecular mass of CKIC was established at 33 kDa while that of CKIR at 35-37 kDa. A protein of 40 kDa copurified with CKIR but not CKIC. Heparin significantly increased 40 kDa protein phosphorylation level by CKIR. Microsequencing analysis revealed the presence of CKI recognition motifs in the N-terminal fragment of the 40 kDa protein.  相似文献   

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We describe an unusual hybrid histidine protein kinase, which is important for spatially coupling cell aggregation and sporulation during fruiting body formation in Myxococcus xanthus. A rodK mutant makes abnormal fruiting bodies and spores develop outside the fruiting bodies. RodK is a soluble, cytoplasmic protein, which contains an N-terminal sensor domain, a histidine protein kinase domain and three receiver domains. In vitro phosphorylation assays showed that RodK possesses kinase activity. Kinase activity is essential for RodK function in vivo. RodK is present in vegetative cells and remains present until the late aggregation stage, after which the level decreases in a manner that depends on the intercellular A-signal. Genetic evidence suggests that RodK may regulate multiple temporally separated events during fruiting body formation including stimulation of early developmental gene expression, inhibition of A-signal production and inhibition of the intercellular C-signal transduction pathway. We speculate that RodK undergoes a change in activity during development, which is reflected in changes in phosphotransfer to the receiver domains.  相似文献   

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Nitric oxide synthase oxygenase domains (NOS(ox)) must bind tetrahydrobiopterin and dimerize to be active. New crystallographic structures of inducible NOS(ox) reveal that conformational changes in a switch region (residues 103-111) preceding a pterin-binding segment exchange N-terminal beta-hairpin hooks between subunits of the dimer. N-terminal hooks interact primarily with their own subunits in the 'unswapped' structure, and two switch region cysteines (104 and 109) from each subunit ligate a single zinc ion at the dimer interface. N-terminal hooks rearrange from intra- to intersubunit interactions in the 'swapped structure', and Cys109 forms a self-symmetric disulfide bond across the dimer interface. Subunit association and activity are adversely affected by mutations in the N-terminal hook that disrupt interactions across the dimer interface only in the swapped structure. Residue conservation and electrostatic potential at the NOS(ox) molecular surface suggest likely interfaces outside the switch region for electron transfer from the NOS reductase domain. The correlation between three-dimensional domain swapping of the N-terminal hook and metal ion release with disulfide formation may impact inducible nitric oxide synthase (i)NOS stability and regulation in vivo.  相似文献   

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Post-translational modification of proteins was examined during the life cycle of Myxococcus xanthus. A specific pattern of protein phosphorylation was observed in vegetative cells. When spore formation was induced by glycerol, significant changes in the pattern of protein phosphorylation were observed, including the phosphorylation of two membrane proteins. In in vitro experiments, the same membrane proteins were phosphorylated by ATP when the membrane preparation from cells treated with glycerol was used. Changes in the pattern of protein methylation were also observed during spore formation induced by glycerol or fruiting body formation. These results suggest that post-translational protein modification may be required for spore formation or fruiting body formation.  相似文献   

15.
Zhang W  Wu Q  Pwee KH  Jois SD  Kini RM 《Biochemistry》2003,42(21):6596-6607
Wheat HMGa protein is a typical member of the plant HMGA family. It has four AT hooks and a histone H1-like region. A panel of deletion mutants of HMGa was generated to study the role of different regions of HMGa in its binding to 4H (a synthetic DNA that mimics the in vivo structure of intermediates of homologous recombination and DNA repair) and linear DNAs. Although the histone H1-like region of HMGa does not bind to 4H or linear DNAs, it does enhance the binding. Mutants with any two adjacent AT hooks show specific binding to both 4H and linear P268 (and P31) with different binding affinities, which is partly due to the flanking regions between AT hooks. Conformational studies indicate that the alpha-helical content of HMGa increases significantly when it binds to 4H compared to that after binding to P31, linear DNA. In contrast, linear DNA, but not 4H, undergoes substantial conformational change when it binds to HMGa, indicating that linear DNA is relatively more flexible than 4H. A more significant difference in the affinities of binding of the mutants of HMGa to 4H was observed compared to their affinities of binding to linear DNA, P31. These differences could be due to the rigidity of the DNA and the characters of the AT hook regions in the mutants.  相似文献   

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Myxococcus xanthus cells aggregate and develop into multicellular fruiting bodies in response to starvation. A new M. xanthus locus, designated dif for defective in fruiting, was identified by the characterization of a mutant defective in fruiting body formation. Molecular cloning, DNA sequencing and sequence analysis indicate that the dif locus encodes a new set of chemotaxis homologues of the bacterial chemotaxis proteins MCPs (methyl-accepting chemotaxis proteins), CheW, CheY and CheA. The dif genes are distinct genetically and functionally from the previously identified M. xanthus frz chemotaxis genes, suggesting that multiple chemotaxis-like systems are required for the developmental process of M. xanthus fruiting body formation. Genetic analysis and phenotypical characterization indicate that the M. xanthus dif locus is required for social (S) motility. This is the first report of a M. xanthus chemotaxis-like signal transduction pathway that could regulate or co-ordinate the movement of M. xanthus cells to bring about S motility.  相似文献   

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The flagellar filament-hook complex was removed from Campylobacter cells by shearing and was purified by differential solubilization and ultracentrifugation at pH 11 followed by cesium chloride buoyant density ultracentrifugation. Flagellar filaments were then dissociated in 0.2 M glycine-HCl (pH 2.2), and purified hooks were collected by ultracentrifugation. The hooks (105 by 24 nm) each displayed a conical protrusion at the proximal end, a concave cavity at the distal end, and helically arranged subunits. The apparent subunit molecular weight of the hook protein of seven of the eight Campylobacter strains studied was 92,500, while that of the other was 94,000. N-terminal amino acid analysis of the hook protein of two strains of Campylobacter coli and one strain of Campylobacter jejuni demonstrated that the first 15 residues were identical. Amino acid composition analysis showed that the Campylobacter hook protein contained 35.7% hydrophobic and 9.5% basic residues. Isoelectric focusing determined that the hook protein was acidic, with a pI of 4.9. Comparisons with the Salmonella and Caulobacter hook protein compositions and N-terminal amino acid sequences indicated that the Campylobacter protein was related, but more distantly than these two proteins were to each other. Immunochemical analysis with four different antisera and a panel of eight strains showed that serospecific epitopes were immunodominant. The Campylobacter hook proteins carried both cross-reactive and specific non-surface-exposed epitopes, as well as serospecific epitopes which were exposed on the surface of the assembled hook. One class of these surface-exposed hook epitopes was shared with serospecific flagellin epitopes and may involve posttranslational modification, while the second class of epitopes was hook specific and not shared with flagellin.  相似文献   

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The architectural factors HMGA are highly connected hubs in the chromatin network and affect key cellular functions. HMGA have a causal involvement in cancer development; in fact, truncated or chimeric HMGA forms, resulting from chromosomal rearrangements, lack the constitutively phosphorylated acidic C-terminal tail and display increased oncogenic potential, suggesting a functional role for this domain. HMGA belong to the intrinsically disordered protein category, and this prevents the use of classical approaches to obtain structural data. Therefore, we combined limited proteolysis, ion mobility separation-mass spectrometry (IMS-MS), and electrospray ionization-mass spectrometry (ESI-MS) to obtain structural information regarding full length and C-terminal truncated HMGA forms. Limited proteolysis indicates that HMGA acidic tail shields the inner portions of the protein. IMS-MS and ESI-MS show that HMGA proteins can assume a compact form and that the degree of compactness is dependent upon the presence of the acidic tail and its constitutive phosphorylations. Moreover, we demonstrate that C-terminal truncated forms and wild type proteins are post-translationally modified in a different manner. Therefore, we propose that the acidic tail and its phosphorylation could affect HMGA post-translational modification status and likely their activity. Finally, the mass spectrometry-based approach adopted here proves to be a valuable new tool to obtain structural data regarding intrinsically disordered proteins.  相似文献   

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