Arrangement Of Binding Sites Research Articles

Differential DNA bending introduced by the Pseudomonas putida LysR-type regulator, CatR, at the plasmid-borne pheBA and chromosomal catBC promoters.

The plasmid-borne pheBA operon of Pseudomonas putida strain PaW85 allows growth of the host cells on phenol. The promoter of this operon is activated by the chromosomally encoded LysR-type regulator CatR, in the presence of the inducer cis,cis-muconate. cis,cis-muconate is an intermediate of catechol degradation by the chromosomally encoded ortho or beta-ketoadipate pathway. The catBC operon encodes two enzymes of the beta-ketoadipate pathway and also requires CatR and cis,cis-muconate for its expression. The promoters of the pheBA and catBC operons are highly homologous, and since both respond to CatR, it is likely that the pheBA promoter was recruited from the ancestral catBC promoter. Gel shift assays and DNase I footprinting have shown that the pheBA promoter has a higher binding affinity for CatR than the catBC promoter. Like the catBC promoter, the pheBA promoter forms two complexes (C1 and C2) with CatR in the absence of cis,cis-muconate, but only forms a single complex (C2) in the presence of cis,cis-muconate. Like the catBC promoter CatR repression binding site (RBS) and activation binding site (ABS) arrangement, the pheBA promoter demonstrates the presence of a 26 bp segment highly homologous to the RBS that is protected by CatR from DNase I digestion in the absence of the inducer. An additional 16 bp sequence, similar to the catBC promoter ABS, is protected only when the inducer cis-cis-muconate is present. The binding of CatR in absence of cis,cis-muconate bends the catBC and pheBA promoter regions to significantly different degrees, but CatR binding in the presence of cis,cis-muconate results in a similar degree of DNA bending. The evolutionary implications of the interactions of CatR with these two promoters are discussed.

Structure of the P22 att site. Conservation and divergence in the lambda motif of recombinogenic complexes.

We have defined the bacterial and viral DNA targets (att sites) of P22 site-specific recombination and characterized their interaction with integrase (Int) protein. The bacterial DNA target, attB, is approximately 27 base pairs and consists of two core type Int binding sites as inverted repeats. The top and bottom Int cleavage sites fall within the core type Int binding sites and are separated by a 7-base pair overlap region. A similar core region is found in the viral DNA target, attP, which is approximately 260 base pairs long and contains two IHF binding sites and five arm type binding sites for Int. The results suggest that P22 Int, like lambda Int, is a heterobivalent DNA-binding protein that is capable of forming complex higher order structures with recombinogenic function. Although P22 and lambda recombination involve very similar multiprotein interactions and core region structures, there are significant differences in the arrangements of distal protein binding sites. These differences are discussed in terms of the possible flexibility of the Int protein and the specificity with which the higher order complexes assemble and/or function.

Analysis of the DNA-binding affinity, Sequence Specificity and Context Dependence of the Glucocorticoid Receptor Zinc Finger Region

The glucocorticoid receptor binds with high affinity to glucocorticoid response elements (GREs), which commonly consist of imperfect DNA palindromes with hexameric core binding motifs separated by three base-pairs; the receptor dimerizes upon binding to these sequences, with one monomer occupying each core motif. To examine quantitatively the receptor-DNA interaction, and to characterize the effects of single-pair mutations and sequence context on this interaction, we have studied the binding of a purified glucocorticoid receptor fragment, T7X556, to a 30 bp oligonucleotide containing a complex arrangement of potential receptor binding sites (RBS): two consensus core motifs, RBSo and RBSa reside in the same orientation and overlap by one base-pair, and a third site, RBSb, a partial match to the consensus separated by three base-pairs and in opposite orientation from RBSa. Using four different footprinting reagents, we detected a receptor dimer bound to one face of the DNA double helix, making close contacts with two adjacent major grooves. One monomer bound with high affinity to RBSa and the other bound with lower affinity to RBSb; transfection studies revealed that both binding sites were necessary for GRE activity in vivo. We measured the affinity of the receptor interaction with RBSa, RBSb and non-specific sites, and showed that protein binding at RBSb was improved approximately 150-fold by cooperativity with RBSa. Remarkably, T7X556 failed to bind to the consensus RBSo sequence, even when it was mutated to match exactly RBSa; the preference for RBSa over RBSo was due neither to the presence of RBSb, nor to occlusion of RBSo by protein bound at RBSa. To examine the relative contribution of each core nucleotide to the binding reaction, we saturated RBSo and RBSa with point mutations. The results implied that four RBSa nucleotides, (or 5′-×G̱× ACA ̄ -3′), may make specific contacts with the protein, as certain mutations at these positions reduced binding drastically. Consistent with footprinting and transfection assays, equivalent mutations in RBSo had no effect on protein binding. Thus, these findings indicate that the consensus core motif alone is not sufficient to specify a functional RBS, and that flanking sequences create an appropriate context for protein binding.

Related target enhancers for dorsal and NF-kappa B signaling pathways.

Drosophila dorsoventral (DV) patterning and mammalian hematopoiesis are regulated by related signaling pathways (Toll, interleukin-1) and transcription factors (dorsal, nuclear factor-kappa B). These factors interact with related enhancers, such as the rhomboid NEE and kappa light chain enhancer, that contain similar arrangements of activator and repressor binding sites. It is shown that the kappa enhancer can generate lateral stripes of gene expression in transgenic Drosophila embryos in a pattern similar to that directed by the rhomboid NEE. Drosophila DV determinants direct these stripes through the corresponding mammalian cis regulatory elements in the kappa enhancer, including the kappa B site and kappa E boxes. These results suggest that enhancers can couple conserved signaling pathways to divergent gene functions.

Nucleotide binding sites on mitochondrial F1-ATPase. Electron spin resonance spectroscopy and photolabeling by azido-spin-labeled adenine nucleotides support an adenylate kinase-like orientation.

A spin-labeled photoaffinity ATP analog, 2-N3-2',3'-SL-ATP (2-N3-SL-ATP) was specifically loaded at catalytic (exchangeable) or noncatalytic (nonexchangeable) nucleotide-binding sites on nucleotide-depleted mitochondrial F1-ATPase. Photolysis of the enzyme complexes resulted in the specific modification of beta-Tyr-345 when the catalytic sites were occupied and beta-Tyr-368 when noncatalytic sites were filled. These are the same amino acid assignments that were made previously using 2-N3ATP. The results demonstrate that the attachment of a spin label moiety to the ribose ring does not prevent proper binding of the analog at both types of nucleotide sites on F1-ATPase and suggest that the probe can be used for investigations of the nucleotide-binding sites using ESR spectroscopy. Enzyme that is in complex with the 2-N3-SL-ATP exhibits an ESR spectrum that is typical for highly immobilized nitroxyl radicals both in the dark or after photolysis. Additional peaks in the high- and low-field regions arise due to dipolar spin interactions most likely involving a pair of catalytic and noncatalytic sites. The two sites are calculated to be approximately 15 A apart. This distance, obtained through ESR spectroscopy, combined with the finding that the 2 labeled amino acids are only 23 residues apart from each other, further supports an adenylate kinase-like arrangement of nucleotide binding sites on F1-ATPase where catalytic and noncatalytic sites are in close proximity (Vogel, P. D., and Cross, R. L. (1991) J. Biol. Chem. 266, 6101-6105).

Biochemical and spectroscopic characterization of the high molecular weight cytochrome c from Desulfovibrio vulgaris Hildenborough expressed in Desulfovibrio desulfuricans G200.

The gene of high molecular weight, multiheme cytochrome c (Hmc) from the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough has been overexpressed in Desulfovibrio desulfuricans G200. The recombinant protein has been purified. Its molecular weight (65,600), amino acid composition, and NH2-terminal sequence were found to be identical to those of the wild-type protein. The recombinant protein has been spectroscopically characterized (optical spectrum, EPR, circular dichroism) and compared to the wild-type protein. We have found 16 hemes per molecule by iron analysis and the pyridine hemochrome test. Both high- and low-spin features were observed in the EPR spectrum. A detailed spin quantitation analysis indicates 1 or 2 high-spin hemes and 14 or 15 low-spin hemes per molecule. The redox potentials of the hemes determined by voltammetric techniques gave an average of three different values, 0, -100, and -250 mV (versus NHE), for the wild-type and the recombinant cytochrome. The low potential values are similar to the values observed for the bis(histidinyl) coordinated hemes of cytochrome c3. A comparison of the arrangement of heme binding sites and coordinated histidines in the amino acid sequences of cytochrome c3 and Hmc has shown that the latter contains four domains, three of which are complete c3-like domains, while the fourth represents an incomplete c3-like domain which may contain His-Met coordinated hemes. These data are in agreement with the detailed study of the number and types of hemes reported in this paper.

Coordination chemistry of lithium ion: a crystal and molecular structure review

There has been much recent interest in lithium and lithium ionophores. This growing interest in lithium is mainly due to the actual and potential applications of Li[sup +] in science, medicine, and technology. In the present review article, a tabulation is given, based on X-ray crystallographic data, of the bond lengths, geometry, coordination numbers, and solvent of crystallization of Li[sup +] complexes. The data included in the review extend from the earliest publications through February 1990. The authors have attempted in the text to draw the reader's attention to the various parameters involved in lithium-ligand interaction such as ligand coordination sites, ligand conformation changes, stereochemical arrangement of ligand binding sites, counterion effects, and solvent effects. In addition, discussion is included of lithium selective ionophores and of the involvement of Li[sup +] in biological cycles. Appropriate examples taken from the tables of data are used to illustrate the text. The compounds included in the review are listed by formula and abbreviation in Charts 1--18. The approach the authors chose for this review is a structural one, centered on the examination of the three-dimensional crystal structures of lithium salts and complexes. The basic assumption is that the structures of these lithium complexes more » in the solid state resemble their structures in solution, and that similar structure-selectivity relationships exist, at least to some extent, in both media. 190 refs. « less

Cloning, sequencing, and expression of the gene encoding the high-molecular-weight cytochrome c from Desulfovibrio vulgaris Hildenborough

By using a synthetic deoxyoligonucleotide probe designed to recognize the structural gene for cytochrome cc3 from Desulfovibrio vulgaris Hildenborough, a 3.7-kb XhoI genomic DNA fragment containing the cc3 gene was isolated. The gene encodes a precursor polypeptide of 58.9 kDa, with an NH2-terminal signal sequence of 31 residues. The mature polypeptide (55.7 kDa) has 16 heme binding sites of the form C-X-X-C-H. Covalent binding of heme to these 16 sites gives a holoprotein of 65.5 kDa with properties similar to those of the high-molecular-weight cytochrome c (Hmc) isolated from the same strain by Higuchi et al. (Y. Higuchi, K. Inaka, N. Yasuoka, and T. Yagi, Biochim. Biophys. Acta 911:341-348, 1987). Since the data indicate that cytochrome cc3 and Hmc are the same protein, the gene has been named hmc. The Hmc polypeptide contains 31 histidinyl residues, 16 of which are integral to heme binding sites. Thus, only 15 of the 16 hemes can have bis-histidinyl coordination. A comparison of the arrangement of heme binding sites and coordinated histidines in the amino acid sequences of cytochrome c3 and Hmc from D. vulgaris Hildenborough suggests that the latter contains three cytochrome c3-like domains. Cloning of the D. vulgaris Hildenborough hmc gene into the broad-host-range vector pJRD215 and subsequent conjugational transfer of the recombinant plasmid into D. desulfuricans G200 led to expression of a periplasmic Hmc gene product with covalently bound hemes.

Structural arrangement of tRNA binding sites on Escherichia coli ribosomes, as revealed from data on affinity labelling with photoactivatable tRNA derivatives

A systematic study of protein environment of tRNA in ribosomes in model complexes representing different translation steps was carried out using the affinity labelling of the ribosomes with tRNA derivatives bearing aryl azide groups scattered statistically over tRNA guanine residues. Analysis of the proteins crosslinked to tRNA derivatives showed that the location of the derivatives in the aminoacyl (A) site led to the labelling of the proteins S5 and S7 in all complexes studied, whereas the labelling of the proteins S2, S8, S9, S11, S14, S16, S17, S18, S19, S21 as well as L9, L11, L14, L15, L21, L23, L24, L29 depended on the state of tRNA in A site. Similarly, the location of tRNA derivatives in the peptidyl (P) site resulted in the labelling of the proteins L27, S11, S13 and S19 in all states, whereas the labelling of the proteins S5, S7, S9, S12, S14, S20, S21 as well as L2, L13, L14, L24, L27, L31, L32, L33 depended on the type of complex. The derivatives of tRNA f Met were found to crosslink to S1, S3, S5, S7, S9, S14 and L1, L2, L7/L12, L27. Based on the data obtained, a general principle of the dynamic functioning of ribosomes has been proposed: (i) the formation of each type of ribosomal complex is accompanied by changes in mutual arrangement of proteins — ‘conformational adjustment’ of the ribosome — and (ii) a ribosome can dynamically change its internal structure at each step of initiation and elongation; on the 70 S ribosome there are no rigidly fixed structures forming tRNA-binding sites (primarily A and P sites).

Subnucleosomes and their relationships to the arrangement of histone binding sites along nucleosome deoxyribonucleic acid.

Micrococcal nuclease cleaves within nucleosomes at sites spaced about 10.4 base pairs (bp) apart. Cleavages at sites equivalent to 30-35 bp from the ends of 146-bp cores cause spontaneous loss of an H2a-H2b pair associated with 30-40 bp length DNA. Cleavages at certain other sites do not affect the nucleosome integrity unless a solvent perturbant such as urea is added. Chromatin moderately digested with micrococcal nuclease, when fractionated by sedimentation or electrophoresis in the presence of 3 M urea, yielded four previously unobserved subnucleosomes with the following histone/DNA compositions: (H3)/sub 2/(H4)/sub 2/(H2a)(H2b)/95-115 bp; (H3)(H4)/70-80 bp DNA; (H2a)(H2b)/50-60 bp DNA; and (H1)/60-70 bp DNA. All but the latter subnucleosome were also obtained upon DNase I digestion of purified nucleosome cores labeled on the 5' ends with /sup 32/P. Only subnucleosomes that retained H2a and H2b also retained labeled ends. These results show that H2a and H2b are paired on the terminal 30-40 bp of core DNA, as suggested from analyses of histone-DNA cross-link products by Mirzabekov and coworkers. Considerations of the orgins and compositions of subnucleosomes and of cross-linking data suggest an expanded model for the locations of histone binding sites along nucleosome core DNA. The principal features of this modelmore » are (i) strong electrostatic binding sites of H2a and H2b occur at positions approximately 20-30 bp from the core ends, (ii) strong electrostatic binding sites of H3 and H4 occur primarily on the central 40 bp of core DNA, (iii) strong nonelectrostatic, urea-sensitive binding sites of H3 and H4 occur at positions approximately 30-50 bp from the core ends, and (iv) urea-sensitive binding sites of H2a or H2b may occur on the terminal 10-20 bp of core DNA.« less

Studies on the arrangement of a glucose sensitive phlorizin binding site in the microvilli of isolated rat kidney brushborder

FEBS LettersVolume 25, Issue 2 p. 242-244 ArticleFree Access Studies on the arrangement of a glucose sensitive phlorizin binding site in the microvilli of isolated rat kidney brushborder L. Thomas, L. Thomas Max-Planck-Institut für Biophysik, 6 Frankfurt/M., GermanySearch for more papers by this authorR. Kinne, R. Kinne Max-Planck-Institut für Biophysik, 6 Frankfurt/M., GermanySearch for more papers by this author L. Thomas, L. Thomas Max-Planck-Institut für Biophysik, 6 Frankfurt/M., GermanySearch for more papers by this authorR. Kinne, R. Kinne Max-Planck-Institut für Biophysik, 6 Frankfurt/M., GermanySearch for more papers by this author First published: September 15, 1972 https://doi.org/10.1016/0014-5793(72)80494-0Citations: 11AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article.

Structure and Surface Binding: N and CO on Tungsten

The energetics of the interactions of N2 and CO with initially clean tungsten surfaces are analyzed to establish the dependence of chemisorption upon surface structure. For N2, calorimetric measurements yield a heat of adsorption of ∼90 kcal mole—1, constant over the range of coverages accessible at T=300°K. This suggests that the equilibrium binding sites for nitrogen are energetically equivalent. Desorption obeys bimolecular kinetics, with an activation energy of ∼81 kcal mole—1, unchanging to the highest surface concentrations (n∼250×1012 molecules cm—2). This constancy confirms the energetic equivalence of the surface sites filled by nitrogen, and demonstrates the presence of adatoms without lateral interactions. Alternative proposals for the occupation of binding sites of different strengths are examined and rejected as caused by experimental error. In contrast, evolution of CO takes place in distinct steps, with activation energies of 53, 75, and ∼100 kcal mole—1 for β material. Stepwise desorption stems from the arrangement of CO binding sites. These differ in strength, are widely separated from one another, and presumably consist of planes with different crystallographic orientation. Despite the constant heat of adsorption, the presence of an activation barrier to surface diffusion, amounting to ∼35 kcal mole—1 on the approaches to the (100), demonstrates that binding of nitrogen atoms does depend on surface structure. In the regions between equilibrium sites, a nitrogen adatom is less firmly bound. On the (110) planes, in which each tungsten atom has six nearest neighbors, the binding energy of a nitrogen adatom is lowered to such an extent that at room temperature these areas should be bare. This is confirmed by direct observation in the field ion microscope. Such ``structural exclusion'' arises from the high dissociation energy of N2, which precludes occupation of sites accessible to other, less strongly bonded molecules. For nitrogen atoms only a narrow band in the total spectrum of binding energies is available. Structural exclusion is also shown to be in accord with the following unique properties of nitrogen layers: (1) The limited concentration (∼40% of the total number of surface sites) achievable at room temperature; (2) The presence of an additional and heavily populated binding state (γ) at T<200°K; (3) The absence of interactions between adatoms.