A phage repressor-operator complex at 7 A resolution

The crystal structure of a complex between the DNA-binding domain of phage 434 repressor and a synthetic 434 operator shows that the protein, very similar in conformation to gamma repressor, binds to B-form DNA with the second alpha-helix of a helix-turn-helix motif lying in the major groove.     

Dependence of the torsional rigidity of DNA on base composition

The Escherichia coli phage 434 repressor binds as a dimer to the operator of the DNA helix. Although the centre of the operator is not in contact with protein, the repressor binding affinity can be reduced at least 50-fold by changing the sequence there: operators with A.T base pairs near their centre bind the repressor more strongly than do operators with G.C base pairs at the same positions. To explain these observations, it has been proposed that the base composition at the centre of the operator affects the affinity of the operator for repressor by altering the ease with which operator DNA can undergo the torsional deformation necessary for complex formation. In this model, the variation in binding affinity would require the torsion constant to have specific values and to change in a sequence-dependent manner. We have now measured torsion constants for DNAs with widely different base compositions. Our results indicate that the torsion constants depend only slightly on the overall composition, and firmly delimit the range of values for each. Even the upper-limit values are much too small to account for the observed changes in affinity of the 434 repressor. These results rule out simple models that rely on substantial generic differences in torsion constant between A.T-rich sequences and G.C-rich sequences, although they do not rule out the possibility of particular sequences having abnormal torsion constants.     

DNA twisting and the effects of non-contacted bases on affinity of 434 operator for 434 repressor.

The bacteriophage 434 repressor regulates gene expression by binding with differing affinities to the six operator sites on the phage chromosome. The symmetrically arrayed outer eight base pairs (four in each half-site) of these 14-base-pair operators are highly conserved but the middle four bases are divergent. Although these four base pairs are not in contact with repressor, operators with A.T or T.A base pairs at these positions bind repressor more strongly than those bearing C.G or G.C, suggesting that these bases are important for the repressor's ability to discriminate between operators. There is evidence that the central base pairs influence operator function by constraining the twisting and/or bending of DNA. Here we show that there is a relationship between the intrinsic twist of an operator, as determined by the sequence of its central bases, and its affinity for repressor; an operator with a lower affinity is undertwisted relative to an operator with higher affinity. In complex with repressor, the twist of both high- and low-affinity operators is the same. These results indicate that the intrinsic twist of DNA and its twisting flexibility both affect the affinity of 434 operator for repressor.     

Structure of the repressor-operator complex of bacteriophage 434

The crystal structure of a specific complex between the DNA-binding domain of phage 434 repressor and a synthetic 434 operator DNA shows interactions that determine sequence-dependent affinity. The repressor recognizes its operators by its complementarity to a particular DNA conformation as well as by direct interaction with base pairs in the major groove.     

Effect of non-contacted bases on the affinity of 434 operator for 434 repressor and Cro

The repressor of phage 434 binds to six operator sites on the phage chromosome. A comparison of the sequences of these 14-base-pair (bp) operator sites reveals a striking pattern: at five of the six sites, the symmetrically arrayed outer eight base pairs (four in each half-site) are identical and the remaining site differs at only one position (Fig. 1b). In contrast, the sequences of the inner four base pairs are highly variable. Crystallographic analysis of the repressor-operator complex shows that at each half-site, the 'recognition alpha-helix' of the repressor is positioned in the major groove such that it could contact the outermost five base pairs, but not the innermost two (Fig. 1a). We show in this paper that the sequence of the central base pairs of the operator (two in each half-site) have a significant role in determining operator affinity for repressor, despite the evidence presented here and in the accompanying paper that these base pairs are not contacted by repressor. We also show that these central base pairs influence operator affinity for Cro, a second gene regulatory protein encoded by phage 434. We discuss the likely structural basis for this evidently indirect, but sequence-dependent, effect of the central base pairs of the operator on its affinity for the two regulatory proteins.     

Ethylation interference and X-ray crystallography identify similar interactions between 434 repressor and operator

In the crystal structure of a repressor-operator complex (the 434 repressor DNA-binding domain and its 14-base pair (bp) operator), Anderson et al. elsewhere in this issue identify six positions of likely contact between repressor protein and phosphates of the DNA backbone. At each of these positions, electron densities of protein and DNA merge. Experiments presented here indicate that intact 434 repressor approaches these phosphates very closely when it is bound to DNA in solution. Specifically, when any one of these phosphates is ethylated, repressor cannot bind to the modified operator. We also identify another position where ethylation has a significant but less dramatic effect on repressor binding, and note that in the structure, repressor closely approaches this phosphate. Our results strongly support the idea that the interactions between protein and the DNA phosphate backbone in the crystallized complex are the same as those made by intact repressor to operator DNA in solution. In addition, our results suggest that DNA is slightly bent by repressor binding.     

Importance of DNA stiffness in protein-DNA binding specificity

From the first high-resolution structure of a repressor bound specifically to its DNA recognition sequence it has been shown that the phage 434 repressor protein binds as a dimer to the helix. Tight, local interactions are made at the ends of the binding site, causing the central four base pairs (bp) to become bent and overtwisted. The centre of the operator is not in contact with protein but repressor binding affinity can be reduced at least 50-fold in response to a sequence change there. This observation might be explained should the structure of the intervening DNA segment vary with its sequence, or if DNA at the centre of the operator resists the torsional and bending deformation necessary for complex formation in a sequence dependent fashion. We have considered the second hypothesis by demonstrating that DNA stiffness is sequence dependent. A method is formulated for calculating the stiffness of any particular DNA sequence, and we show that this predicted relationship between sequence and stiffness can explain the repressor binding data in a quantitative manner. We propose that the elastic properties of DNA may be of general importance to an understanding of protein-DNA binding specificity.     

Changing the binding specificity of a repressor by redesigning an alpha-helix

We replaced amino acids on the 'outside', or solvent-exposed, surface of the DNA recognition alpha-helix of 434 repressor with the corresponding amino acids from the recognition helix of P22 repressor. The binding specificity of the resulting hybrid protein, as measured in vivo and in vitro, was that of P22 repressor.     

Structure of the cro repressor from bacteriophage lambda and its interaction with DNA

The three-dimensional structure of the 66-amino acid cro repressor protein of bacteriophage lambda suggests how it binds to its operator DNA. We propose that a dimer of cro protein is bound to the B-form of DNA with the 2-fold axis of the dimer coincident with the 2-fold axis of DNA. A pair of 2-fold-related alpha-helices of the repressor, lying within successive major grooves of the DNA, seem to be a major determinant in recognition and binding. In addition, the C-terminal residues of the protein, some of which are disordered in the absence of DNA, appear to contribute to the binding.     

Cooperative tandem binding of met repressor of Escherichia coli

We present biochemical and genetic data to support the hypothesis that the Escherichia coli met repressor, MetJ, binds to synthetic and natural operator sequences in tandem arrays such that repression depends not only on the affinity of the DNA-protein interaction, but also on protein-protein contacts along the tandem array. This represents a novel form of regulatory switch. Furthermore, there seems to be homology between the organization of the met and trp operators.     

Structure of a phage 434 Cro/DNA complex

Comparison of the crystal structure of a complex of the phage 434 Cro protein and a synthetic DNA operator with the complex of the same operator and the 434 repressor DNA-binding domain shows different DNA conformations in the two structures. Binding of the protein determines the precise conformation of the DNA in each case.     

Neutron scattering studies of lac repressor

The lac repressor, a tetrameric protein of identical subunits [molecular weight (MW) 4 x 38,500], interacts specifically with the lac operator, preventing the expression of the structural genes of the lac operon. The presence of an inducer (such as isopropyl-beta-D-thiogalactoside; IPTG) which binds to the repressor, prevents operator binding by lowering the association constant by a factor of 10(3) (ref. 3). Genetic and biochemical analysis have shown that the major part--if not all--of the binding site for the lac operator is located in the 60 N-terminal residues of the protein. In certain conditions, limited trypsinolysis of the protein yields four N-terminal 'headpieces' each containing 51 or 59 residues, and a tetrameric core with full inducer binding activity. It was shown recently that this headpiece is able to bind nucleic acids, and interacts with the lac operator, giving the same pattern of sensitivity with respect to the methylation of the bases as does the intact repressor. We are studying the interaction of lac repressor with DNA by neutron scattering using contrast variation and discuss here measurements on the protein, its tryptic core and their complexes with IPTG. Our results demonstrate that the headpieces are located far (67 +/- 10 A) from the centre of mass of a somewhat elongated core, and that the inducer does not significantly change the radius of gyration of the protein.     

Three-dimensional crystal structures of Escherichia coli met repressor with and without corepressor

The three-dimensional crystal structure of met repressor, in the presence or absence of bound corepressor (S-adenosylmethionine), shows a dimer of intertwined monomers, which do not have the helix-turn-helix motif characteristic of other bacterial repressor and activator structures. We propose that the interaction of met repressor with DNA occurs through either a pair of symmetry-related alpha-helices or a pair of beta-strands, and suggest a model for binding of several dimers to met operator regions.     

一种新的粒计算的覆盖模型

定义了新的Zoom-in算子,并讨论了它的性质.证明了Zoom-in算子与Zoom-out算子复合得到的（论域上的）一对近似算子恰为基于覆盖的第二型上、下近似算子.     

伴随对及其构造

先讨论伴随对定义及其等价条件,给出伴随对存在的一个充分条件;其次讨论40个模糊蕴涵算子和40个模糊圈乘算子的性质,并给出由模糊蕴涵算子构造模糊圈乘算子的方法;最后讨论40个算子对构成伴随对的情况.     

A mutant T4 lysozyme displays five different crystal conformations

Phage T4 lysozyme consists of two domains between which is formed the active-site cleft of the enzyme. The crystallographically determined thermal displacement parameters for the protein suggested that the amino terminal of the two domains undergoes 'hinge-bending' motion about an axis passing through the waist of the molecule. Such conformational mobility may be important in allowing access of substrates to the active site of the enzyme. We report here a crystallographic study of a mutant T4 lysozyme which demonstrates further the conformational flexibility of the protein. A mutant form of the enzyme with a methionine residue (Met 6) replaced by isoleucine crystallizes with four independent molecules in the crystal lattice. These four molecules have distinctly different conformations. The mutant protein can also crystallize in standard form with a structure very similar to the wild-type protein. Thus the mutant protein can adopt five different crystal conformations. The isoleucine for methionine substitution at the intersection of the two domains of T4 lysozyme apparently enhances the hinge-bending motion presumed to occur in the wild-type protein, without significantly affecting the catalytic activity or thermal stability of the protein.     

实数编码遗传算法离散重组算子分析

用组合数学分析了实数编码遗传算法的一点交叉、多点交叉和均匀交叉等三种离散重组算子的组合能力，算子的组合能力算子组合出新染色体数目的大小衡量，分析表明，对同一父染色体对交，一点交叉最多可组合出2(n-1)个新的染色体，多点交叉为2C^kn-1个，均匀交叉为2（2^n-1-1)个，函数优化实验研究表明，在算法中采用何种离散重组算子较为合适与算子的组合能力有关，也与优化问题有关。     

第六型覆盖粒计算模型

通过覆盖粗糙集上的邻域关系定义了一种新的Zoom-in算子,并讨论了它的性质及其与已有的Zoom-in算子之间的关系.讨论了新定义的Zoom-in算子与Zoom-out算子之间的不同复合而产生的不同的近似算子的性质.进一步建立了这些算子与拓扑空间及Galois联络之间的联系.特别地,证明了2个算子在论域上复合得到的近似算子,恰是基于覆盖的第六型粗糙近似算子.     

Crystal structure of the lambda repressor and a model for pairwise cooperative operator binding

Bacteriophage lambda has for many years been a model system for understanding mechanisms of gene regulation. A 'genetic switch' enables the phage to transition from lysogenic growth to lytic development when triggered by specific environmental conditions. The key component of the switch is the cI repressor, which binds to two sets of three operator sites on the lambda chromosome that are separated by about 2,400 base pairs (bp). A hallmark of the lambda system is the pairwise cooperativity of repressor binding. In the absence of detailed structural information, it has been difficult to understand fully how repressor molecules establish the cooperativity complex. Here we present the X-ray crystal structure of the intact lambda cI repressor dimer bound to a DNA operator site. The structure of the repressor, determined by multiple isomorphous replacement methods, reveals an unusual overall architecture that allows it to adopt a conformation that appears to facilitate pairwise cooperative binding to adjacent operator sites.     

Direct homeodomain-DNA interaction in the autoregulation of the fushi tarazu gene.

A major problem in the elucidation of the molecular mechanisms governing development is the distinction between direct and indirect regulatory interactions among developmental control genes. In vivo studies have indicated that the Drosophila segmentation gene fushi tarazu (ftz) directly or indirectly autoregulates its expression. Here we describe a generally applicable experimental approach which establishes a direct in vivo interaction of the homeodomain protein ftz with the ftz cis-autoregulatory control region. In vitro studies have shown that the DNA-binding specificity of the ftz homeodomain can be changed by a single amino-acid substitution in the recognition helix (Gln 50----Lys). Whereas wild-type ftz homeodomain binds preferentially to a CCATTA motif, the mutant homeodomain (ftzQ50K) recognizes a GGATTA motif. We now find that the in vivo activity of an ftz autoregulatory enhancer element is reduced by mutations of putative ftz-binding sites to GGATTA. This down-regulatory effect is specifically suppressed in vivo by the DNA-binding specificity mutant ftzQ50K. These results establish a direct positive autoregulatory feedback mechanism in the regulation of this homeobox gene.