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2.
Thermodynamics of specific and nonspecific DNA binding by two DNA-binding domains conjugated to fluorescent probes
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The complexes designed in this work combine the sequence-specific binding properties of helix-turn-helix DNA-binding motifs with intercalating cyanine dyes. Thermodynamics of the Hin recombinase and Tc3 transposase DNA-binding domains with and without the conjugated dyes were studied by fluorescence techniques to determine the contributions to specific and nonspecific binding in terms of the polyelectrolyte and hydrophobic effects. The roles of the electrostatic interactions in binding to the cognate and noncognate sequences indicate that nonspecific binding is more sensitive to changes in salt concentration, whereas the change in the heat capacity shows a greater sensitivity to temperature for the sequence-specific complexes in each case. The conjugated dyes affect the Hin DNA-binding domain by acting to anchor a short stretch of amino acids at the N-terminal end into the minor groove. In contrast, the N-terminal end of the Tc3 DNA-binding domain is bound in a well-ordered fashion to the DNA even in the absence of the conjugated dye. The conjugated dye and the DNA-binding domain portions of each conjugate bind noncooperatively to the DNA. The characteristic thermodynamic parameters of specific and nonspecific DNA binding by each of the DNA-binding domains and their respective conjugates are presented. 相似文献
3.
Joseph S. Xu Madeleine N. Hewitt Jaskeerat S. Gulati Matthew A. Cruz Hongli Zhan Shirley Liu Kathleen S. Matthews 《Protein science : a publication of the Protein Society》2018,27(4):839-847
The short 8–10 amino acid “hinge” sequence in lactose repressor (LacI), present in other LacI/GalR family members, links DNA and inducer‐binding domains. Structural studies of full‐length or truncated LacI‐operator DNA complexes demonstrate insertion of the dimeric helical “hinge” structure at the center of the operator sequence. This association bends the DNA ~40° and aligns flanking semi‐symmetric DNA sites for optimal contact by the N‐terminal helix‐turn‐helix (HtH) sequences within each dimer. In contrast, the hinge region remains unfolded when bound to nonspecific DNA sequences. To determine ability of the hinge helix alone to mediate DNA binding, we examined (i) binding of LacI variants with deletion of residues 1–50 to remove the HtH DNA binding domain or residues 1–58 to remove both HtH and hinge domains and (ii) binding of a synthetic peptide corresponding to the hinge sequence with a Val52Cys substitution that allows reversible dimer formation via a disulfide linkage. Binding affinity for DNA is orders of magnitude lower in the absence of the helix‐turn‐helix domain with its highly positive charge. LacI missing residues 1–50 binds to DNA with ~4‐fold greater affinity for operator than for nonspecific sequences with minimal impact of inducer presence; in contrast, LacI missing residues 1–58 exhibits no detectable affinity for DNA. In oxidized form, the dimeric hinge peptide alone binds to O1 and nonspecific DNA with similarly small difference in affinity; reduction to monomer diminished binding to both O1 and nonspecific targets. These results comport with recent reports regarding LacI hinge interaction with DNA sequences. 相似文献
4.
Tsodikov OV Saecker RM Melcher SE Levandoski MM Frank DE Capp MW Record MT 《Journal of molecular biology》1999,294(3):639-655
In our studies of lac repressor tetramer (T)-lac operator (O) interactions, we observed that the presence of extended regions of non-operator DNA flanking a single lac operator sequence embedded in plasmid DNA produced large and unusual cooperative and anticooperative effects on binding constants (Kobs) and their salt concentration dependences for the formation of 1:1 (TO) and especially 1:2 (TO2) complexes. To explore the origin of this striking behavior we report and analyze binding data on 1:1 (TO) and 1:2 (TO2) complexes between repressor and a single O(sym) operator embedded in 40 bp, 101 bp, and 2514 bp DNA, over very wide ranges of [salt]. We find large interrelated effects of flanking DNA length and [salt] on binding constants (K(TO)obs, K(TO2)obs) and on their [salt]-derivatives, and quantify these effects in terms of the free energy contributions of two wrapping modes, designated local and global. Both local and global wrapping of flanking DNA occur to an increasing extent as [salt] decreases. Global wrapping of plasmid-length DNA is extraordinarily dependent on [salt]. We propose that global wrapping is driven at low salt concentration by the polyelectrolyte effect, and involves a very large number (>/similar 20) of coulombic interactions between DNA phosphates and positively charged groups on lac repressor. Coulombic interactions in the global wrap must involve both the core and the second DNA-binding domain of lac repressor, and result in a complex which is looped by DNA wrapping. The non-coulombic contribution to the free energy of global wrapping is highly unfavorable ( approximately +30-50 kcal mol(-1)), which presumably results from a significant extent of DNA distortion and/or entropic constraints. We propose a structural model for global wrapping, and consider its implications for looping of intervening non-operator DNA in forming a complex between a tetrameric repressor (LacI) and one multi-operator DNA molecule in vivo and in vitro. The existence of DNA wrapping in LacI-DNA interactions motivates the proposal that most if not all DNA binding proteins may have evolved the capability to wrap and thereby organize flanking regions of DNA. 相似文献
5.
Many mutations that impact protein function occur at residues that do not directly contact ligand. To understand the functional contributions from the sequence that links the DNA-binding and regulatory domains of the LacI/GalR homologues, we have created a chimeric protein (LLhP), which comprises the LacI DNA-binding domain, the LacI linker, and the PurR regulatory domain. Although DNA binding site residues are identical in LLhP and LacI, thermodynamic measurements of DNA binding affinity show that LLhP does not discriminate between alternative DNA ligands as well as LacI. In addition, small-angle scattering experiments show that LLhP is more compact than LacI. When DNA is released, LacI shows a 20 A increase in length that was previously attributed to unfolding of the linker. This change is not seen in apo-LLhP, even though the linker sequences of the two proteins are identical. Together, results indicate that long-range functional and structural changes are propagated across the interface that forms between the linker and regulatory domain. These changes could be mediated via the side chains of several linker residues that contact the regulatory domains of the naturally occurring proteins, LacI and PurR. Substitution of these residues in LLhP leads to a range of functional effects. Four variants exhibit altered affinity for DNA, with no changes in selectivity or allosteric response. Another two result in proteins that bind operator DNA with very low affinity and no allosteric response, similar to LacI binding nonspecific DNA sequences. Two more substitutions simultaneously diminish affinity, enhance allostery, and profoundly alter DNA ligand selectivity. Thus, positions within the linker can be varied to modulate different aspects of repressor function. 相似文献
6.
The mechanism by which genetic regulatory proteins discern specific target DNA sequences remains a major area of inquiry. To explore in more detail the interplay between DNA and protein sequence, we have examined binding of variant lac operator DNA sequences to a series of mutant lactose repressor proteins (LacI). These proteins were altered in the C-terminus of the hinge region that links the N-terminal DNA binding and core sugar binding domains. Variant operators differed from the wild-type operator, O(1), in spacing and/or symmetry of the half-sites that contact the LacI N-terminal DNA binding domain. Binding of wild-type and mutant proteins was affected differentially by variations in operator sequence and symmetry. While the mutant series exhibits a 10(4)-fold range in binding affinity for O(1) operator, only a approximately 20-fold difference in affinity is observed for a completely symmetric operator, O(sym), used widely in studies of the LacI protein. Further, DNA sequence influenced allosteric response for these proteins. Binding of this LacI mutant series to other variant operator DNA sequences indicated the importance of symmetry-related bases, spacing, and the central base pair sequence in high affinity complex formation. Conformational flexibility in the DNA and other aspects of the structure influenced by the sequence may establish the binding environment for protein and determine both affinity and potential for allostery. 相似文献
7.
The bacterial LacI/GalR family repressors such as lactose operon repressor (LacI), purine nucleotide synthesis repressor (PurR), and trehalose operon repressor (TreR) consist of not only the N-terminal helix-turn-helix DNA-binding domain but also the C-terminal ligand-binding domain that is structurally homologous to periplasmic sugar-binding proteins. These structural features imply that the repressor family evolved by acquiring the DNA-binding domain in the N-terminal of an ancestral periplasmic binding protein (PBP). Phylogenetic analysis of the LacI/GalR family repressors and their PBP homologues revealed that the acquisition of the DNA-binding domain occurred first in the family, and ligand specificity then evolved. The phylogenetic tree also indicates that the acquisition occurred only once before the divergence of the major lineages of eubacteria, and that the LacI/GalR and the PBP families have since undergone extensive gene duplication/loss independently along the evolutionary lineages. Multiple alignments of the repressors and PBPs furthermore revealed that repressors and PBPs with the same ligand specificity have the same or similar residues in their binding sites. This result, together with the phylogenetic relationship, demonstrates that the repressors and the PBPs individually acquired the same ligand specificity by homoplasious replacement, even though their genes are encoded in the same operon. 相似文献
8.
A rapid method to capture and screen for transcription factors by SELDI mass spectrometry. 总被引:12,自引:0,他引:12
Cameron E Forde Arlene D Gonzales Jean M Smessaert Gloria A Murphy Sharon J Shields J Patrick Fitch Sandra L McCutchen-Maloney 《Biochemical and biophysical research communications》2002,290(4):1328-1335
9.
Amino acid alterations were designed at the C terminus of the hinge segment (amino acids approximately 51-59) that links two functional domains within lactose repressor protein (LacI). Gly was introduced between Gly(58) and Lys(59) to generate Gly(58+1); Gln(60) was changed to Gly or Pro, and up to three additional glycines were inserted following Gln(60) --> Gly. All mutant proteins exhibited purification behavior, CD spectra, assembly state, and inducer binding properties similar to wild-type LacI and only small differences in trypsin proteolysis patterns. In contrast, significant differences were observed in DNA binding properties. Gly(58+1) exhibited a decrease of approximately 100-fold in affinity for O(1) operator, and sequential Gly insertion C-terminal to Gln(60) --> Gly resulted in progressively decreased affinity for O(1) operator, approaching nonspecific levels for insertion of >/=2 glycines. Where sufficient affinity for O(1) operator existed, decreased binding to O(1) in the presence of inducer indicated no disruption in the allosteric response for these proteins. Collectively, these results indicate that flexibility and/or spacing between the core and N-terminal domains did not significantly affect folding or assembly, but these alterations in the hinge domain profoundly altered affinity of the lactose repressor protein for its wild-type target sequence. 相似文献
10.
Repressor titration: a novel system for selection and stable maintenance of recombinant plasmids.
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S G Williams R M Cranenburgh A M Weiss C J Wrighton D J Sherratt J A Hanak 《Nucleic acids research》1998,26(9):2120-2124
The propagation of recombinant plasmids in bacterial hosts, particularly in Escherichia coli, is essential for the amplification and manipulation of cloned DNA and the production of recombinant proteins. The isolation of bacterial transformants and subsequent stable plasmid maintenance have traditionally been accomplished using plasmid-borne selectable marker genes. Here we describe a novel system that employs plasmid-mediated repressor titration to activate a chromosomal selectable marker, removing the requirement for a plasmid-borne marker gene. A modified E.coli host strain containing a conditionally essential chromosomal gene (kan) under the control of the lac operator/promoter, lac O/P, has been constructed. In the absence of an inducer (allolactose or IPTG) this strain, DH1 lackan , cannot grow on kanamycin-containing media due to the repression of kan expression by LacI protein binding to lac O/P. Transformation with a high copy-number plasmid containing the lac operator, lac O, effectively induces kan expression by titrating LacI from the operator. This strain thus allows the selection of plasmids without antibiotic resistance genes (they need only contain lac O and an origin of replication) which have clear advantages for use as gene therapy vectors. Regulation in the same way of an essential, endogenous bacterial gene will allow the production of recombinant therapeutics devoid of residual antibiotic contamination. 相似文献
11.
12.
Structural homology between rbs repressor and ribose binding protein implies functional similarity.
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The deduced amino acid sequence of the rbs repressor, RbsR, of Escherichia coli is homologous over its C-terminal 272 residues to the entire sequence of the periplasmic ribose binding protein. RbsR is also homologous to a family of bacterial repressor proteins including LacI. This implies that the structure of the repressor consists of a two-domain binding protein portion attached to a DNA-binding domain having the four-helix structure of the LacI headpiece. The implications of these relationships to the mechanism of this class of repressors are discussed. 相似文献
13.
Fried MG Stickle DF Smirnakis KV Adams C MacDonald D Lu P 《The Journal of biological chemistry》2002,277(52):50676-50682
The osmotic stress technique was used to measure changes in macromolecular hydration that accompany binding of wild-type Escherichia coli lactose (lac) repressor to its regulatory site (operator O1) in the lac promoter and its transfer from site O1 to nonspecific DNA. Binding at O1 is accompanied by the net release of 260 +/- 32 water molecules. If all are released from macromolecular surfaces, this result is consistent with a net reduction of solvent-accessible surface area of 2370 +/- 550 A. This area is only slightly smaller than the macromolecular interface calculated for a crystalline repressor dimer-O1 complex but is significantly smaller than that for the corresponding complex with the symmetrical optimized O(sym) operator. The transfer of repressor from site O1 to nonspecific DNA is accompanied by the net uptake of 93 +/- 10 water molecules. Together these results imply that formation of a nonspecific complex is accompanied by the net release of 165 +/- 43 water molecules. The enhanced stabilities of repressor-DNA complexes with increasing osmolality may contribute to the ability of Escherichia coli cells to tolerate dehydration and/or high external salt concentrations. 相似文献
14.
Hiro-omi Tamura Shunji Natori Masuo Obinata 《Differentiation; research in biological diversity》1982,23(1-3):87-90
Abstract. The DNA-binding capacity of nuclear proteins of mouse cells was examined by the protein-blotting method. Under conditions in which the lac repressor specifically binds to the lac operator, the DNA-binding nuclear proteins from different tissues showed a tissue-specific distribution, suggesting that the species and amounts of nuclear proteins with DNA binding activity differ in different tissues.
When cloned eukaryotic genes were used for binding, eukaryotic DNA showed stronger binding than prokaryotic DNA. Competition experiments suggested that many nuclear proteins have different DNA binding properties from that of the prokaryotic repressor. 相似文献
When cloned eukaryotic genes were used for binding, eukaryotic DNA showed stronger binding than prokaryotic DNA. Competition experiments suggested that many nuclear proteins have different DNA binding properties from that of the prokaryotic repressor. 相似文献
15.
The DNA-binding capacity of nuclear proteins of mouse cells was examined by the protein-blotting method. Under conditions in which the lac repressor specifically binds to the lac operator, the DNA-binding nuclear proteins from different tissues showed a tissue-specific distribution, suggesting that the species and amounts of nuclear proteins with DNA binding activity differ in different tissues. When cloned eukaryotic genes were used for binding, eukaryotic DNA showed stronger binding than prokaryotic DNA. Competition experiments suggested that many nuclear proteins have different DNA binding properties from that of the prokaryotic repressor. 相似文献
16.
The effects of prior covalent cysteine modification or nonspecific DNA presence on the reaction of lac repressor protein with N-bromosuccinimide have been investigated. At low excesses, N-bromosuccinimide oxidation causes loss of operator DNA binding activity with simultaneous retention of inducer and nonspecific DNA binding activities. Cysteine and methionine are oxidized under the conditions utilized. Covalent modification of the cysteines of repressor prior to reaction decreased the observed loss of operator DNA binding capacity; the presence of nonspecific DNA partially prevented oxidation of the cysteines by N-bromosuccinimide, and concurrent protection of operator binding ability was observed. Methionine oxidation was observed in the cases where protection of the operator DNA binding capacity of repressor was seen. The region surrounding cysteine 107 was found to be influential in maintaining intact operator DNA binding function in repressor. This observation provides chemical evidence for the contribution of the core region of repressor in determining specificity of the protein in binding the lac operator. The protection from oxidation of cysteine residues in the core region by the presence of nonspecific DNA suggests that this binding influences the core region of the protein. 相似文献
17.
Probing co-operative DNA-binding in vivo. The lac O1:O3 interaction 总被引:10,自引:0,他引:10
The lac primary (O1) and weak upstream pseudo (O3) operators contained on a plasmid were footprinted in vivo in order to determine whether they act co-operatively in binding lac repressor in the cell. The occupancy at O3 by lac repressor was substantially reduced upon deletion of the lac primary operator, demonstrating co-operativity at a distance. Plots of operator occupancy versus active repressor concentration were obtained for each operator by treating the cells with different amounts of the lac inducer isopropyl-beta-D-thiogalactoside and probing lac repressor binding. This analysis can be used to obtain relative binding constants in vivo and demonstrates that O3 binds repressor only 10.3-fold less tightly than O1 in their co-operative interaction. The removal of DNA torsional tension in vivo by the use of coumermycin leads to the same loss of binding at O3 as does deleting O1. These in-vivo results are analogous to the in-vitro situation, where O3 binds repressor strongly in a DNA repression loop only on supercoiled templates. 相似文献
18.
M Brenowitz N Mandal A Pickar E Jamison S Adhya 《The Journal of biological chemistry》1991,266(2):1281-1288
The interaction of proteins bound to sites widely separated on the genome is a recurrent motif in both prokaryotic and eukaryotic regulatory systems. Lac repressor mediates the formation of "DNA loops" by the simultaneous interaction of a single protein tetramer with two DNA-binding sites. The DNA-binding properties of a Lac repressor mutant (LacIadi) deficient in the association of protein dimers to tetramers was investigated. The results of quantitative footprint and gel mobility-shift titrations suggest that the wild-type Lac repressor (LacI+) binds cooperatively to two operator sites separated by 11 helical turns on a linear DNA restriction fragment by the formation of a "looped complex." LacIadi binds to this two-site operator non-cooperatively and without formation of a looped complex. These results demonstrate that the dimer-tetramer association of LacI+ is directly responsible for its cooperative binding and its ability to mediate formation of a looped complex. The Iadi mutation disrupts the monomer-dimer as well as eliminating the dimer-tetramer association equilibria while the DNA binding affinity of LacIadi to a single site is unchanged relative to the wild-type protein. These results suggest that DNA binding and dimer-tetramer association are functionally unlinked. The similarity of the DNA-binding properties of LacIadi and Gal repressor, a protein believed to function by mediating the formation of a looped complex, are discussed. 相似文献
19.
20.
Use of difference boundary sedimentation velocity to investigate nonspecific protein-nucleic acid interactions 总被引:1,自引:0,他引:1
The difference boundary sedimentation velocity technique of Schachman and co-workers is demonstrated to be applicalbe to the measurement of binding constants (Kobsd) in the range 10(2)-10(5) M(-1) for the nonspecific interactions of proteins with DNA. The difference technique can reproducibly detect a 2% change in the sedimentation coefficient of the DNA upon binding ligands, corresponding to average extents of association as low as 10 molecules of protein (in the cases of Escherichia coli lac repressor and E. coli RNA polymerase) per molecule of bacteriophage T7 DNA. At these low binding densities, it is plausible to assume that the primary effect of ligand binding is on the buoyant mass of the complex and not on the frictional coefficient of the flexible DNA coil. Binding constants calculated by using this assumption agree well with literature values for the nonspecific interactions of RNase and lac repressor proteins with double-stranded DNA. Advantages of the method are that it is relatively rapid, requires the optical detection of the DNA only, and can be performed on small amounts of sample. The method appears useful for surveying (to an accuracy of +/-50% in Kobsd or +/-10% in log Kobsd) the effects of solution variables on Kobsd of protein-DNA interactions. Applications of the method to the nonspecific interactions of RNA polymerase core and holoenzymes with T7 DNA are discussed. 相似文献