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.     

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.     

E. coli recA protein-directed cleavage of phage lambda repressor requires polynucleotide

The recA protein mediates both genetic recombination and several cellular responses to DNA damage, including the induction of temperate bacteriophage. Indication of phage lambda results from proteolytic cleavage of lambda repressor directed by recA protein. We show here that this cleavage reaction requires both polynucleotide and ATP. We suggest that a stoichiometric complex of recA protein and DNA is active both to destroy repressors by proteolytic cleavage and to initiate pairing of this DNA to its homologous sequence in a DNA duplex ('strand invasion').     

Accurate prediction of the stability and activity effects of site-directed mutagenesis on a protein core.

Theoretical prediction of the structure, stability and activity of proteins, an important unsolved problem in molecular biology, would be of use for guiding site-directed mutagenesis and other protein-engineering techniques. X-ray diffraction studies have provided extensive structural information for many proteins, challenging theorists to develop reliable techniques able to use such knowledge as a base for prediction of mutants' characteristics. Here we report theoretical calculation of stabilization energies for 78 triple-site sequence variants of lambda repressor characterized experimentally by Lim and Sauer. The calculated energies correlate with the mutants' measured activities; active and inactive mutations are discriminated with 92% reliability. They correlate even more directly with the mutants' thermostabilities, correctly identifying two of the mutants to be more stable than the wild type.     

DNA arrangement in isometric phage heads.

DNA is wound tightly into phage heads in such a way that it tends to form layers concentric with the rigid protein shell. In P22 and wild-type lambda, DNA completely fills the internal volume, with a highly uniform local packing of adjacent segments; in lambda deletion mutants containing less than a full genome, the local packing distance increases correspondingly.     

Immunological activity of covalently linked T-cell epitopes.

Immune responses to proteins necessarily involve the recognition by T lymphocytes of a peptide or peptides derived from a protein complexed with a major histocompatibility antigen. The T-cell response of BALB/c mice to the bacteriophage lambda cI repressor protein (residues 1-102) is directed predominantly towards the epitope contained within a single peptide encompassing residues 12-26. Similar phenomena of immunodominance of a particular peptide have also been observed in other protein systems. The mechanisms that have been suggested to account for the focusing of the T-cell response are partial deletion in the T-cell repertoire, biased antigen processing, and competition for binding to the presenting molecule, the major histocompatibility complex encoded class II transplantation antigen. In a model system with a polypeptide containing two synthetically linked immunologically active epitopes, we now demonstrate the existence of a hierarchy between these epitopes, so that the immune response elicited is directed mainly towards the more immunogenic epitope, whereas the less immunogenic epitope elicits little or no T-cell reactivity. In addition, the same hierarchy of dominance is also apparent when the polypeptide is used to induce tolerance in the periphery in adult mice. The chimaeric peptide can induce tolerance only towards the more immunogenic epitope. These experiments indicate that the rules governing antigen processing and presentation that result in T-cell activation are apparently the same as the rules that govern the processes resulting in the induction of tolerance.     

Cloning of a representative genomic library of the human X chromosome after sorting by flow cytometry

A library of 50,000 recombinants representative of the human X chromosome has been constructed. Human X chromosomes were physically separated using a fluorescence-activated cell sorter. The DNA was purified from the chromosomes, digested to completion with the restriction enzyme EcoRI and cloned into the phage lambda gtWES.lambda B. The X-derived nature of the recombinants was confirmed by hybridization to rodent/human cell line DNA containing only the human X chromosome. Such libraries will be particularly useful for the investigation of genetic diseases such as Duchenne muscular dystrophy, where the basic defect has not been elucidated, and of neoplasia, where several specific chromosomal anomalies, particularly for the leukaemias, have been identified.     

用P11柱一步纯化色氨酸阻遏蛋白质

利用携带有克隆色氨酸阻遏蛋白基因的pJPR2质粒的大肠杆菌菌株,经过细菌培养、扩增及一系列生化方法,得到色氨酸阻遏蛋白的粗品,然后利用磷酸纤维素P11柱分离纯化得到单一的色氨酸阻遏蛋白,经SDS聚丙烯酰胺凝胶电泳鉴定和pRK9质粒中trpP/O片段保护实验证明,分离到的色氨酸阻遏蛋白是纯的,并具有生物活性。该方法和思维的建立,为研究核酸结合蛋白质的结构与功能提供了基础。     

解酚假单胞菌的分离及毒物降解的特点

在假单胞菌中有些株可以降解芳香类有机物,另一些株可以抗重金属.某些芳环化合物如苯酚等,及含汞的化合物都是可以造成环境污染的有毒物质.对假单胞菌降解水杨酸等芳香化合物的途径已有一些了解,但少有涉及代谢的调控问题.本文报道一株从化学工业污染区域新分离到的假单胞菌.这株菌不但可以降解水杨酸、苯甲酸及邻苯二酚,而且还能降解毒性较强的苯酚,并具有耐受一定浓度Hg~(2+)的能力,是一株有前途的去污染工程菌.对该菌降解混合芳香化合物的研究发现,邻苯二酚在这些芳香化合物降解代谢中是一个具有调节作用的物质.     

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.     

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.     

The chick ovomucoid gene contains at least six intervening sequences.

A 15-kilobase pair EcoRI chick DNA fragment, containing both the termination codon UGA and the 5'-portion of the structural ovomucoid gene, has been cloned in lambda phage Charon 4A by in vitro packaging. Restriction mapping and electron microscopic analyses of this cloned DNA have revealed that the structural ovomucoid gene sequences are separated by at least six intervening sequences.     

素环上非线性Lie中心化子

M是包含非平凡投影P的单位素环. 利用算子论方法证明了: 如果φ: M→M是非线性Lie中心化子, 则存在λ∈C及映射ξ: M→C满足ξ(［A,B］)=0(A,B∈M), 使得对任意的X∈M, 有φ(X)=λX+ξ(X)I.     

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.     

Sequence-induced DNA curvature at the bacteriophage lambda origin of replication

DNA replication in bacteriophage lambda begins at a unique origin between residues 39,000 and 39,200 of the lambda genome. This segment of DNA serves a dual function since it also lies within the coding sequence of the lambda replication initiator protein O which binds origin DNA. The lambda origin sequence contains four 19-base-pair (bp) segments (iterons) which have dyad symmetry, followed by a 40-bp A + T-rich zone of highly asymmetrical base composition. It was noted earlier that lambda origin DNA exhibits an anomalous electrophoretic mobility on gels; that is, the length of DNA as determined by DNA sequencing is approximately 20% less than is predicted from electrophoretic mobility. Recent studies of kinetoplast minicircle DNA (K-DNA) from the protozoan Leishmania tarentolae have led to the proposal that sequence-induced DNA curvature could account for such electrophoretic anomalies by alteration of the shape of the DNA molecule. We now present evidence that the lambda origin contains a static curve.