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1.
Expression, purification, and functional characterization of the carboxyl-terminal domain fragment of bacteriophage 434 repressor. 总被引:2,自引:2,他引:0
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The repressor protein of bacteriophage 434 binds to DNA as a dimer of identical subunits. Its strong dimerization is mediated by the carboxyl-terminal domain. Cooperative interactions between the C-terminal domains of two repressor dimers bound at adjacent sites can stabilize protein-DNA complexes formed with low-affinity binding sites. We have constructed a plasmid, pCT1, which directs the overproduction of the carboxyl-terminal domain of 434 repressor. The protein encoded by this plasmid is called CT-1. Cells transformed with pCT1 are unable to be lysogenized by wild-type 434 phage, whereas control cells are lysogenized at an efficiency of 1 to 5%. The CT-1-mediated interference with lysogen formation presumably results from formation of heteromeric complexes between the phage-encoded repressor and the plasmid-encoded carboxyl-terminal domain fragment. These heteromers are unable to bind DNA and thereby inhibit the repressor's activity in promoting lysogen formation. Two lines of evidence support this conclusion. First, DNase I footprinting experiments show that at a 2:1 ratio of CT-1 to intact 434 repressor, purified CT-1 protein prevents the formation of complexes between 434 repressor and its OR1 binding site. Second, cross-linking experiments reveal that only a specific heterodimeric complex forms between CT-1 and intact 434 repressor. This latter observation indicates that CT-1 interferes with 434 repressor-operator complex formation by preventing dimerization and not by altering the conformation of the DNA-bound repressor dimer. Our other evidence is also consistent with this suggestion. We have used deletion analysis in an attempt to define the region which mediates the 434 repressor-CT-1 interaction. CT-1 proteins which have more than the last 14 amino acids removed are unable to interfere with 434 repressor action in vivo. 相似文献
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G B Koudelka 《Nucleic acids research》1998,26(2):669-675
In complexes of bacteriophage 434 binding sites with 434 repressor the central 4 bp of the 14 bp site are not contacted by the protein, although changes in these bases alter binding site affinity for the repressor. Our previous data suggested that the ability of the non-contacted central bases to be overtwisted in repressor-DNA complexes governs affinity of the binding site for 434 repressor. This idea was tested by examining the affinity of two central sequence variant 434 binding sites for 434 repressor as a function of binding site average twist. The 434 repressor preferred the relatively overwound binding site to the two more underwound forms. The greatest affinity enhancement resulting from increasing twist was observed with a binding site that is relatively underwound and more resistant to twisting deformation. Consistent with the idea that 434 repressor overtwists its binding site upon DNA binding, we show that 434 repressor is capable of binding to sites bearing a single base insertion in their center (a 15mer), but binds poorly to binding sites bearing central base deletions (12mer and 13mer). The N-terminal dimer interface plays a large role in determining 434 repressor central base preferences. Mutations in this interface eliminate central base discrimination and/or site size preferences. These mutations also lead to changes in the size of the repressor footprint on the various sized DNA sites that are consistent with their binding characteristics. 相似文献
4.
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
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The bacteriophage 434 repressor distinguishes between its six naturally occurring binding sites using indirect readout. In indirect readout, sequence-dependent differences in the structure and flexibility of non-contacted bases in a protein's DNA-binding site modulate the affinity of DNA for protein. The conformation and flexibility of a DNA sequence can be influenced by the interaction of the DNA bases or backbone with solution components. We examined the effect of changing the cation-type present in solution on the stability and structure of 434 repressor complexes with wild-type and mutant OR1 and OR3, binding sites that differ in their contacted and non-contacted base sequences. We find that the affinity of repressor for OR1, but not for OR3, depends remarkably on the type and concentration of monovalent cation. Moreover, the formation of a stable, specific repressor-OR1 complex requires the presence of monovalent cations; however, repressor-OR3 complex formation has no such requirement. Changing monovalent cation type alters the ability of repressor to protect OR1, but not OR3, from *OH radical cleavage. Altering the relative length of the poly(dA) x poly(dT) tract in the non-contacted regions of the OR1 and OR3 can reverse the cation sensitivity of repressor's affinities for these two sites. Taken together these findings show that cation-dependent alterations in DNA structure underlies indirect readout of DNA sequence by 434 repressor and perhaps other proteins. 相似文献
7.
DNA conformational change in Gal repressor-operator complex: involvement of central G-C base pair(s) of dyad symmetry. 总被引:3,自引:1,他引:2
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Gal repressor dimer binds to two gal operator sites, OE and OI, which are 16 bp long similar sequences with hyphenated dyad symmetries (11,12). Repressor occupation hinders the reactivity of the N7 atoms in the major groups of guanines, located at positions 1, 3 and 8, and the rotational 1', 3' and 8' of the symmetries. We have shown that Gal repressor binding to OE or OI DNA fragments increases the circular dichroism (CD) spectral peak in the 270 to 300 nm range. The CD change is similar to that observed for Lac repressor binding to its operator site (14). It is consistent with a DNA conformational change during complex formation between Gal repressor and OE and OI DNA. The CD spectral change was not observed when the central 8,8' G-C base pairs in the DNA-protein complex were replaced by A-T base pairs, whereas substitution of the 1,1' G-C base pairs do show the accompanying increase in the spectra during repressor binding. The absence of CD change of the Gal repressor complex with DNA mutated at the 8,8' base pairs suggest that the central G-C base pairs are required for the repressor induced conformational change. 相似文献
8.
Substituting an alpha-helix switches the sequence-specific DNA interactions of a repressor 总被引:18,自引:0,他引:18
It has been suggested that many DNA-binding proteins use an alpha-helix for specific sequence recognition. We have used amino acid sequence homologies to identify the presumptive DNA-recognition helices in two related proteins whose structures are unknown--the repressor and cro protein of bacteriophage 434. The 434 repressor and cro protein each bind to three similar sites in the rightward phage 434 operator, OR, and they make different contacts in each binding site, as revealed by the chemical probe dimethyl sulfate. We substituted the putative recognition alpha-helix of 434 repressor with the putative recognition alpha-helix of 434 cro protein to create a hybrid protein named repressor*. The specific DNA contacts made by repressor* are like those of 434 cro protein. 相似文献
9.
Dimerization specificity of P22 and 434 repressors is determined by multiple polypeptide segments. 总被引:1,自引:1,他引:0
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The repressor protein of bacteriophage P22 binds to DNA as a homodimer. This dimerization is absolutely required for DNA binding. Dimerization is mediated by interactions between amino acids in the carboxyl (C)-terminal domain. We have constructed a plasmid, p22CT-1, which directs the overproduction of just the C-terminal domain of the P22 repressor (P22CT-1). Addition of P22CT-1 to DNA-bound P22 repressor causes the dissociation of the complex. Cross-linking experiments show that P22CT-1 forms specific heterodimers with the intact P22 repressor protein, indicating that inhibition of P22 repressor DNA binding by P22CT-1 is mediated by the formation of DNA binding-inactive P22 repressor:P22CT-1 heterodimers. We have taken advantage of the highly conserved amino acid sequences within the C-terminal domains of the P22 and 434 repressors and have created chimeric proteins to help identify amino acid regions required for dimerization specificity. Our results indicate that the dimerization specificity region of these proteins is concentrated in three segments of amino acid sequence that are spread across the C-terminal domain of each of the two phage repressors. We also show that the set of amino acids that forms the cooperativity interface of the P22 repressor may be distinct from those that form its dimer interface. Furthermore, cooperativity studies of the wild-type and chimeric proteins suggest that the location of cooperativity interface in the 434 repressor may also be distinct from that of its dimerization interface. Interestingly, changes in the dimer interface decreases the ability of the 434 repressor to discriminate between its wild-type binding sites, O(R)1, O(R)2, and O(R)3. Since 434 repressor discrimination between these sites depends in large part on the ability of this protein to recognize sequence-specific differences in DNA structure and flexibility, this result indicates that the C-terminal domain is intimately involved in the recognition of sequence-dependent differences in DNA structure and flexibility. 相似文献
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The sequence of non-contacted bases at the center of the 434 repressor binding site affects the strength of the repressor-DNA complex by influencing the structure and flexibility of DNA (Koudelka, G. B., and Carlson, P. (1992) Nature 355, 89-91). We synthesized 434 repressor binding sites that differ in their central sequence base composition to test the importance of minor groove substituents and/or the number of base pair hydrogen bonds between these base pairs on DNA structure and strength of the repressor-DNA complex. We show here that the number of base pair H-bonds between the central bases apparently has no role in determining the relative affinity of a DNA site for repressor. Instead we find that the affinity of DNA for repressor depends on the absence or presence the N2-NH(2) group on the purine bases at the binding site center. The N2-NH(2) group on bases at the center of the 434 binding site appears to destabilize 434 repressor-DNA complexes by decreasing the intimacy of the specific repressor-DNA contacts, while increasing the reliance on protein contacts to the DNA phosphate backbone. Thus, the presence of an N2-NH(2) group on the purines at the center of a binding site globally alters the precise conformation of the protein-DNA interface. 相似文献
11.
The bacteriophage 434 repressor dimer preferentially undergoes autoproteolysis by an intramolecular mechanism
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Inactivation of the lambdoid phage repressor protein is necessary to induce lytic growth of a lambdoid prophage. Activated RecA, the mediator of the host SOS response to DNA damage, causes inactivation of the repressor by stimulating the repressor's nascent autocleavage activity. The repressor of bacteriophage lambda and its homolog, LexA, preferentially undergo RecA-stimulated autocleavage as free monomers, which requires that each monomer mediates its own (intramolecular) cleavage. The cI repressor of bacteriophage 434 preferentially undergoes autocleavage as a dimer specifically bound to DNA, opening the possibility that one 434 repressor subunit may catalyze proteolysis of its partner subunit (intermolecular cleavage) in the DNA-bound dimer. Here, we first identified and mutagenized the residues at the cleavage and active sites of 434 repressor. We utilized the mutant repressors to show that the DNA-bound 434 repressor dimer overwhelmingly prefers to use an intramolecular mechanism of autocleavage. Our data suggest that the 434 repressor cannot be forced to use an intermolecular cleavage mechanism. Based on these data, we propose a model in which the cleavage-competent conformation of the repressor is stabilized by operator binding. 相似文献
12.
Gel shift assays were used to examine the binding of the lactose (lac) repressor to polyoperator DNA molecules. Specific binding was differentiated from nonspecific DNA association by (i) equilibrating repressor-operator complexes below the nonspecific association constant and (ii) demonstrating the effects of the inducer isopropyl beta-D-thiogalactoside (IPTG) on the formation of repressor-operator complexes. With the linear polyoperator molecules, all eight operator sites could be simultaneously bound by distinct repressors. However, with circular molecules, the eight operator sites were saturable by repressor only in the nicked circular state and not in the covalently closed circular form. Under the experimental conditions used, there was no evidence of bifunctional repressor binding or loop formation. The results suggest that the conformational perturbation of DNA that occurs upon specific repressor binding was retained in topologically closed molecules and could modify other operator sites so as to make them unavailable for specific binding. 相似文献
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Interaction between the lac operator and the lac repressor headpiece: fluorescence and circular dichroism studies 总被引:1,自引:0,他引:1
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The interaction between the lac repressor headpiece and a small operator DNA fragment has been examined by fluorescence and circular dichroism (c.d.) measurements. Binding of the headpiece to the DNA fragment induces a strong quenching of the fluorescence of its tyrosine residues. Quantitative analysis of the fluorescence data demonstrates that, in a first step, two headpieces bind very strongly to the DNA fragment then weaker binding occurs. C.d. demonstrates that the binding induces conformational changes of the DNA. The c.d. change produced upon binding of the first two headpieces differs from that induced upon binding of two further headpieces . Binding of the second pair of headpieces is similar to non-specific binding to non-operator DNA. The conformation of the operator DNA in the presence of two headpieces differs drastically from that in presence of lac repressor. Addition of the core to the lac operator does not induce any conformational change of the nucleic acids. These results are discussed with respect to the relative roles of core and headpieces in the lac repressor-lac operator interaction. 相似文献
16.
The yeast two-hybrid system has been used to characterize many protein-protein interactions. A two-hybrid system for E. coli was constructed in which one hybrid protein bound to a specific DNA site recruits another to an adjacent DNA binding site. The first hybrid comprises a test protein, the bait, fused to a chimeric protein containing the 434 repressor DNA binding domain. In the second hybrid, a second test protein, the prey, is fused downstream of a chimeric protein with the DNA binding specificity of the lambda repressor. Reporters were designed to express cat and lacZ under the control of a low-affinity lambda operator. At low expression levels, lambda repressor hybrids weakly repress the reporter genes. A high-affinity operator recognized by 434 repressor was placed nearby, in a position that does not yield repression by 434 repressor alone. If the test proteins interact, the 434 hybrid bound to the 434 operator stabilizes the binding of the lambda repressor hybrid to the lambda operator, causing increased repression of the reporter genes. Reconstruction experiments with the fos and jun leucine zippers detected protein-protein interactions between either homodimeric or heterodimeric leucine zippers. 相似文献
17.
Using molecular dynamics simulations in explicit solvent, we investigated the behavior of a 50-bp DNA sequence containing the 434 bacteriophage operators OR1 and OR2 separated by an 8-bp spacer. Two simulations of 1 ns each were carried out, with DNA alone and with DNA complexed to dimers of the R1-69 DNA binding domain of the phage 434 cI repressor protein at the OR1 and OR2 sites. Strong correlations among average structural parameters are observed between our simulations and available experimental data for the bound OR1/OR2 subsites. In the free state, some differences appear between the three relevant fragments (OR1, the spacer, and OR2). Unbound OR1 exhibits a large, shallow major groove into which the base atoms protrude and is also bent toward the major groove. This structure is maintained because structural fluctuations are weak. Unbound OR2 resembles canonical B-DNA although the structural parameters show greater fluctuations, essentially due to a malleable step (the innermost CpA/TpG), absent in OR1. Complexation with the proteins slightly alters the base positions but strongly modifies the sugar and backbone motions. The most crucial repressor effects are changes in the flexibility of the OR1/OR2 sites. Structural fluctuations are enhanced for OR1, conferring a favorable energetic contribution to the OR1 binding, whereas they are reduced for OR2. Therefore, both structural and dynamic properties of DNA suggest OR1 is the most attractive site for the repressor, which may explain the different binding association constants observed for the OR1 and OR2 sites. Finally, we also investigated the impact of the protein on the DNA backbone dynamics and find that direct or indirect interactions facilitate the DNA structural variations required for achieving complementarity with the protein. 相似文献
18.
Bending of synthetic bacteriophage 434 operators by bacteriophage 434 proteins. 总被引:5,自引:4,他引:1
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G B Koudelka 《Nucleic acids research》1991,19(15):4115-4119
The extent of DNA bending induced by 434 repressor, its amino terminal DNA binding domain (R1-69), and 434 Cro was studied by gel shift assay. The results show that 434 repressor and R1-69 bend DNA to the same extent. 434 Cro-induced DNA bends are similar to those seen with the 434 repressor proteins. On approximately 265 base pair fragments, the cyclic AMP receptor protein of Escherichia coli (CRP) produces larger mobility shifts than does 434 repressor. This indicates that the 434 proteins bend DNA to a much smaller extent than does CRP. The effects of central operator sequence on intrinsic and 434 protein-induced DNA bending was also examined by gel shift assay. Two 434 operators having different central sequences and affinities for 434 proteins display no static bending. The amount of gel shift induced by 434 repressor on these operators is identical, showing that the 434 repressor bends operators with different central sequences to the same extent. Hence, mutations in the central region of the operator do not influence the bent structure of the unbound or bound operator. 相似文献
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Bokinsky G Nivón LG Liu S Chai G Hong M Weeks KM Zhuang X 《Journal of molecular biology》2006,361(4):771-784
Like most cellular RNA enzymes, the bI5 group I intron requires binding by a protein cofactor to fold correctly. Here, we use single-molecule approaches to monitor the structural dynamics of the bI5 RNA in real time as it assembles with its CBP2 protein cofactor. These experiments show that CBP2 binds to the target RNA in two distinct modes with apparently opposite effects: a "non-specific" mode that forms rapidly and induces large conformational fluctuations in the RNA, and a "specific" mode that forms slowly and stabilizes the native RNA structure. The bI5 RNA folds though multiple pathways toward the native state, typically traversing dynamic intermediate states induced by non-specific binding of CBP2. These results suggest that the protein cofactor-assisted RNA folding involves sequential non-specific and specific protein-RNA interactions. The non-specific interaction potentially increases the local concentration of CBP2 and the number of conformational states accessible to the RNA, which may promote the formation of specific RNA-protein interactions. 相似文献
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We have examined the interaction between the cyclic AMP receptor protein (CAP) and a small DNA fragment containing its specific recognition sequence by circular dichroism spectroscopy. The binding of CAP to this fragment induces a B to "C-like" change in the CD spectrum, which is different from that observed for non-specific binding. A one-to-one (CAP dimer to DNA) binding stoichiometry was deduced from spectroscopic titration data, as was a non-specific binding site size of 17 bp/dimer. In addition, we have compared the non-specific binding affinity of CAP for the B and Z forms of synthetic DNA copolymers. A slight preference for the B form was found. These results do not support the recent specific suggestion that CAP binds to a left-handed form of DNA (1), but indicate more generally that an optically detectable conformational change takes place in DNA on binding CAP. 相似文献