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Protein-induced bending of the simian virus 40 origin of replication   总被引:10,自引:0,他引:10  
A 3.5 S protein, isolated from mammalian nuclei, specifically binds to DNA fragments containing the simian virus 40 (SV40) origin of replication. Two distinct nucleoprotein complexes are formed, a complex with high electrophoretic mobility carrying probably only one protein molecule, and a complex with reduced electrophoretic mobility carrying probably two protein molecules per DNA fragment. Band shift competition as well as methylation interference assays locate the binding site of the protein in the A + T-rich "late" region of the origin between SV40 nucleotides 13 and 35. The late origin binding (LOB) protein and T antigen bind simultaneously to adjacent sites in the origin. Using circularly permuted DNA fragments of identical lengths we show that the LOB protein induces pronounced bending of the origin fragment. The bending center maps at the 5' end of the adenine tract with one bound protein molecule and at the 3' end when two LOB proteins are bound to one origin fragment.  相似文献   

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While many Type II restriction enzymes are dimers with a single DNA-binding cleft between the subunits, SfiI is a tetramer of identical subunits. Two of its subunits (a dimeric unit) create one DNA-binding cleft, and the other two create a second cleft on the opposite side of the protein. The two clefts bind specific DNA cooperatively to give a complex of SfiI with two recognition sites. This complex is responsible for essentially all of the DNA-cleavage reactions by SfiI: virtually none is due to the complex with one site. The communication between the DNA-binding clefts was examined by disrupting one of the very few polar interactions in the otherwise hydrophobic interface between the dimeric units: a tyrosine hydroxyl was removed by mutation to phenylalanine. The mutant protein remained tetrameric in solution and could bind two DNA sites. But instead of being activated by binding two sites, like wild-type SfiI, it showed maximal activity when bound to a single site and had a lower activity when bound to two sites. This interaction across the dimer interface thus enforces in wild-type SfiI a cooperative transition between inactive and active states in both dimers, but without this interaction as in the mutant protein, a single dimer can undergo the transition to give a stable intermediate with one inactive dimer and one active dimer.  相似文献   

5.
We have demonstrated that the gibbon ape leukemia virus (GALV) enhancer AP-1 element and the simian virus 40 AP-1 enhancer element bind different factors in HeLa nuclear extracts. A 39-kilodalton HeLa nuclear protein and the c-fos protein bind to the GALV element. Antibodies to c-fos abolish binding to the GALV AP-1 site. In contrast, anti-c-fos immunoglobulin fails to inhibit formation of the simian virus 40-specific complex from extracts of HeLa cells. Thus, AP-1-binding complexes are subject to compositional variation at different binding sites.  相似文献   

6.
Chaudhari P  Roy H 《Plant physiology》1989,89(4):1366-1371
Higher plant ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) cannot reassociate after dissociation, and its subunits do not assemble into active RuBisCO when synthesized in Escherichia coli. Newly synthesized subunits of RuBisCO are associated with a high molecular weight binding protein complex in pea chloroplasts. The immediate donor for large subunits which assemble into RuBisCO is a low molecular weight complex which may be derived from the high molecular weight binding protein complex. When the high molecular weight binding protein complex is diluted, it tends to dissociate, forming low molecular weight complexes. When the large subunit-binding protein complexes were examined after in organello protein synthesis, it was found that the low molecular weight complexes were more abundant when protein synthesis was carried out under hypotonic conditions. This increase in the assembly competent population of low molecular weight large subunit complexes can account for the increased amount of in vitro RuBisCO assembly which occurs under these conditions. The data indicate that the assembly of large subunits into RuBisCO is a function of the aggregation state of the large subunit binding protein complex during protein synthesis. This implies that the binding protein exerts its effects during or shortly after large subunit synthesis.  相似文献   

7.
Chemical cross-linking of heteromeric glucocorticoid receptors   总被引:2,自引:0,他引:2  
M Rexin  W Busch  U Gehring 《Biochemistry》1988,27(15):5593-5601
Glucocorticoid receptors of wild-type and nti ("increased nuclear transfer") mutant S49.1 mouse lymphoma cells exist in extracts under low-salt conditions predominantly as high molecular weight species (Mr greater than or equal to 300,000). These receptor-hormone complexes are unable to bind to DNA. High salt (300 mM KCl) produces dissociated receptors of Mr 116,000 and 60-A Stokes radius (wild type) and Mr 60,000 and 38-A Stokes radius (nti mutant), both of which bind to DNA. We used reaction with bifunctional N-hydroxysuccinimide esters as well as oxidation with Cu2+/o-phenanthroline to stabilize the high molecular weight structures. These cross-linked complexes do not interact with DNA, but reductive cleavage again produces the dissociable receptor forms and restores their ability to bind to DNA. The protein modifying reagents iodoacetamide and diethyl pyrocarbonate also produce stabilized high molecular weight receptor complexes. Cross-linking of the high molecular weight receptor forms can also be achieved in intact cells. Immunochemical techniques were used to prove that the complexes cross-linked either in vivo or in cell extracts do contain the heat shock protein of Mr 90,000 as a common constituent. The data show that the high molecular weight receptor complexes are preexisting in intact cells and that dissociation generates DNA binding ability.  相似文献   

8.
The dissociation constants for the binding of oxidized and reduced wild-type cytochrome c(2) from Rhodobacter capsulatus and the lysine 93 to proline mutant of cytochrome c(2) to photosynthetic reaction centers (Rhodobacter sphaeroides) has been measured to high precision using plasmon-waveguide resonance spectroscopy. For the studies reported, detergent-solubilized photosynthetic reaction center was exchanged into a phosphatidylcholine lipid bilayer to approximate the physiological environment. At physiologically relevant ionic strengths ( approximately 100 mM), we found two binding sites for the reduced wild-type cytochrome (K(D) = 10 and 150 nM), with affinities that decrease with decreasing ionic strength (2-5-fold). These results implicate nonpolar interactions as an important factor in determining the dissociation constants. Taking advantage of the ability of plasmon-waveguide resonance spectroscopy to reslove the contribution of changes in mass and of structural anisotropy to cytochrome binding, we can demonstrate very different properties for the two binding sites. In contrast, the oxidized wild-type cytochrome only binds to a single site with a K(D) of 10 nM at high ionic strength, and this site has properties similar to the low-affinity site for binding the reduced cytochrome. The binding of oxidized cytochrome c(2) has a strong ionic strength response, with the affinity decreasing approximately 30-fold in going from high to low ionic strength. The K93P mutant binds to a single site in both redox states, which is similar, in terms of mass and structural anisotropy, to the oxidized wild-type site, with the affinity of the mutant oxidized state being approximately 30-fold weaker than that of the oxidized wild-type cytochrome at high ionic strength. Thus, reduced wild-type cytochrome can bind to both the high- and low-affinity sites, while the oxidized wild-type cytochrome and both redox states of the mutant cytochrome can only bind to the low-affinity site, possibly the consequence of the more stable structure of reduced wild-type cytochrome. In aggregate, the results are consistent with a model in which a transient conformational change in the region 88-102 in the cytochrome three-dimensional structure, the so-called hinge region, drives the dissociation of the oxidized cytochrome from the reaction center-cytochrome complex, facilitating turnover.  相似文献   

9.
Promiscuous mutant EcoRI endonucleases bind to the canonical site GAATTC more tightly than does the wild-type endonuclease, yet cleave variant (EcoRI(*)) sites more rapidly than does wild-type. The crystal structure of the A138T promiscuous mutant homodimer in complex with a GAATTC site is nearly identical to that of the wild-type complex, except that the Thr138 side chains make packing interactions with bases in the 5'-flanking regions outside the recognition hexanucleotide while excluding two bound water molecules seen in the wild-type complex. Molecular dynamics simulations confirm exclusion of these waters. The structure and simulations suggest possible reasons why binding of the A138T protein to the GAATTC site has DeltaS degrees more favorable and DeltaH degrees less favorable than for wild-type endonuclease binding. The interactions of Thr138 with flanking bases may permit A138T, unlike wild-type enzyme, to form complexes with EcoRI(*) sites that structurally resemble the specific wild-type complex with GAATTC.  相似文献   

10.
The FLP recombination target (FRT) can be cut in half so that only one FLP protein binding site is present (a "half site"). FLP protein binds the half sites and joins them into dimeric, asymmetric head-to-head complexes held together chiefly by strong noncovalent interactions. These complexes react with full (normal) FRT sites to generate a variety of products. Analysis of these DNA species reveals that the reaction follows a well-defined reaction pathway that generally parallels the normal reaction pathway. The system is useful in analyzing early steps in recombination, since the identity of the products in a given recombination event unambiguously pinpoints the order in which the cleavage and strand exchange reactions occur. Two conclusions are derived from the present study: (i) Formation of the dimeric head-to-head complex of half sites is a prerequisite to further steps in recombination. (ii) The identity of the base pairs at positions 6 and -6 within the FRT site has a subtle effect in directing the first strand exchange event in the reaction to predominantly one of two possible cleavage sites. In addition, results are presented that suggest that a DNA-DNA pairing intermediate involving only two base pairs of the core sequence is formed prior to the first cleavage and strand exchange. DNA-DNA interactions may therefore not be limited to the isomerization step that follows the first strand exchange.  相似文献   

11.
Vanadium associates with serum transferrin of rats administered vanadyl(IV) sulfate or ammonium metavanadate(V) by gastric intubation. Low molecular weight species account for only 3% of the vanadium present in plasma. The element distributes between the two major isotransferrins in proportion to their concentrations. Rat apotransferrin binds both vanadium(IV) and vanadium(V), forming 2:1 metal-protein complexes in both instances. Although the two isotransferrins apparently differ in their physiological properties, they exhibit identical vanadyl(IV) (VO2+) EPR spectra, indicating identical or very similar metal binding sites for both proteins. In contrast to other transferrins, the two sites of the rat protein are spectroscopically indistinguishable and exhibit a VO2+ EPR spectrum similar to that of the C-terminal metal binding site of human serum transferrin. VO2+ EPR signals are observed with liver, spleen, and kidney tissue samples from animals maintained on a vanadium-supplemented diet. These signals arise from a specific intracellular VO2+ complex with the iron storage protein ferritin.  相似文献   

12.
The phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) and phaseolotoxin are produced by Pseudomonas syringae pv. phaseolicola at 18 degrees C but not at 28 degrees C. At 28 degrees C, the pathogen produces a protein(s) that binds (in vitro) to a 485-bp fragment (thermoregulatory region, TRR) from a heterologous clone from the pathogen genomic library, which in multiple copies overrides thermoregulation of phaseolotoxin production in wild-type cells (K. B. Rowley, D. E. Clements, M. Mandel, T. Humphreys, and S. S. Patil, Mol. Microbiol. 8:625-635, 1993). We report here that DNase I protection analysis of the 485-bp fragment shows that a single site is protected from cleavage by the protein in the 28 degrees C extract and that this site contains two repeats of a core motif G/C AAAG separated by a 5-bp spacer. Partially purified binding protein forms specific complexes with a synthetic oligonucleotide containing four tandem repeats of this motif. A 492-bp upstream fragment from argK encoding ROCT also forms specific complexes with the protein in the 28 degrees C crude extract, and a 260-bp subfragment from the TRR containing the binding site cross competes with the argk fragment, indicating that the same protein binds to nucleotides in both fragments. DNase I protection analysis of the fragment from argK revealed four separate protected sequence elements, with element III containing half of the core motif sequence (CTTTG), and the other elements containing similar sequences. Gel shift assays were done with DNA fragments from which one or all of the sites were removed as competitor DNAs against the argK probe. The results of these experiments confirmed that the binding sites (in argK) are necessary for the protein to bind to the argK fragment in a specific manner. Taken together, the results of studies presented here suggest that in cells of P. syringae pv. phaseolicola grown at high temperature argK may be negatively regulated by the protein produced at this temperature.  相似文献   

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DNA topoisomerases are the enzymes responsible for controlling and maintaining the topological states of DNA. Type IA enzymes work by transiently breaking the phosphodiester backbone of one strand to allow passage of another strand through the break. The protein has to perform complex rearrangements of the DNA, and hence it is likely that different regions of the enzyme bind DNA with different affinities. In order to identify some of the DNA binding sites in the protein, we have solved the structures of several complexes of the 67 kDa N-terminal fragment of Escherichia coli DNA topoisomerase I with mono- and trinucleotides. There are five different binding sites in the complexes, one of which is adjacent to the active site. Two other sites are in the central hole of the protein and may represent general DNA binding regions. The positions of these sites allow us to identify different DNA binding regions and to understand their possible roles in the catalytic cycle.  相似文献   

17.
Kininogens serve dual functions by forming a scaffold for the assembly of the protein complex initiating the surface-activated blood coagulation cascade and as precursors for the kinin hormones. While rats have three kininogen genes, for mice, cattle, and humans only one gene has been described. Here, we present sequence and expression data of a second mouse kininogen gene. The two genes, kininogen-I and kininogen-II, are located in close proximity on chromosome 16 in a head-to-head arrangement. In liver and kidney, both genes are expressed and for each gene three alternative splice variants are synthesized. Two of them are the expected high and low molecular weight isoforms known from all mammalian kininogens. However, for both genes also a third, hitherto unknown splice variant was detected which lacks part of the high molecular weight mRNA due to splicing from the low molecular weight donor site to alternative splice acceptor sites in exon 10. The physiological functions of the six kininogen isoforms predicted by these findings need to be determined.  相似文献   

18.
Croce R  Canino G  Ros F  Bassi R 《Biochemistry》2002,41(23):7334-7343
The chlorophyll a/b-xanthophyll-protein CP26 complex belongs to the Lhc protein family. It binds nine chlorophylls and two xanthophylls per 26.6 kDa polypeptide. Determination of the characteristics of each binding site is needed for the understanding of functional organization of individual proteins belonging to the photosystem II supramolecular complex. The biochemical and spectroscopic features of native CP26 are presented here together with identification of pigment binding and energy transitions in different sites. The analysis has been performed via a new approach using recombinant CP26 complexes in which the chromophore content has been experimentally modified. Data were interpreted on the basis of homology with CP29 and LHCII complexes, for which detailed knowledge is available from mutation analysis. We propose that one additional Chl b is present in CP26 as compared to CP29 and that it is located in site B2. We also found that in CP26 three chlorophyll binding sites are selective for Chl a, one of them being essential for the folding of the pigment-protein complex. Two xanthophyll binding sites were identified, one of which (L1) is essential for protein folding and specifically binds lutein. The second site (L2) has lower selectivity and can bind any of the xanthophyll species present in thylakoids.  相似文献   

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Interactions of serine proteases with cultured fibroblasts   总被引:1,自引:0,他引:1  
This review summarizes the mechanisms by which several serine proteases, particularly urokinase, thrombin, and elastase, interact with cultured fibroblasts. Many of these studies were prompted by findings that interactions of these proteases with cells and the extracellular matrix are important in a number of physiologic and pathologic processes. Two main pathways have been identified for specific interactions of these proteases with fibroblasts. One involves surface binding sites for the free protease that appear to bind only one particular protease. An unusual feature collectively shared by the binding sites for urokinase, thrombin, and elastase is that the bound protease is not detectably internalized by the fibroblasts. The other pathway by which serine proteases interact with fibroblasts involves proteins named protease nexins (PNs). Three PNs have been identified. They are secreted by fibroblasts and inhibit certain serine proteases by forming a covalent complex with the protease catalytic site serine. The complexes then bind back to the fibroblasts via the PN portion of the complex and are internalized and degraded. Recent studies showing that the fibroblast surface and extracellular matrix accelerate the inactivation of thrombin by PN-1 support the hypothesis that the PNs control protease activity at and near the cell surface. The PNs differ from plasma protease inhibitors in their molecular properties, absence in plasma, site of synthesis, and site of clearance of the inhibitor:protease complexes.  相似文献   

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