共查询到20条相似文献,搜索用时 15 毫秒
1.
Early genetic analysis of alternate recombination pathways in Escherichia coli identified the RecE recombination pathway and the required exonuclease VIII encoded by the recE gene. Observations that not ail recombination events promoted by the RecE pathway require recA suggest the existence of an additional homologous pairing protein besides RecA in E. coli. Genetic and biochemical analysis of the recE gene region indicates there are two partially overlapping genes, recE and recT, encoding at least two proteins: exoVIII and the RecT protein. Biochemical analysis has shown that the RecT protein, in combination with exoVIII, promotes homologous pairing and strand exchange in reactions containing linear duplex DNA and homologous, circular, single-stranded DNA as substrates. This reaction occurs in the absence of any high-energy cofactor. These two proteins, RecT and exoVIII, appear to be members of a second class of homologous pairing proteins that are required in genetic recombination and differ from the class of homologous pairing proteins that includes RecA. Members of this second class of proteins appear to include both bacteriophage-encoded proteins and proteins from eukaryotes and their viruses. 相似文献
2.
3.
The doublesex gene of Drosophila melanogaster is the final member of a well characterized hierarchy of genes that controls somatic sex determination and differentiation. The male-specific and female-specific doublesex polypeptides occupy a terminal position in the hierarchy, and thus regulate those genes responsible for the development of sexually dimorphic characteristics of the fly. To investigate the molecular mechanism by which these two related proteins interact with specific target genes, we have identified and characterized their DNA binding domains. Using gel mobility shift experiments with sequentially deleted polypeptides, site-directed mutagenesis and spectrophotometric assays, we have shown that the two doublesex proteins share a common and novel zinc finger-related DNA binding domain distinct from any reported class of zinc binding proteins. We have further shown that of 10 null dsx alleles, six encode proteins deficient in DNA binding activity, and that three of these alleles are the result of mutations that alter cysteine and histidine residues in the metal binding domain. Our results provide evidence that both the male-specific and female-specific doublesex proteins share and depend upon the same DNA binding domain for function in vivo, suggesting that both proteins bind to, but differentially regulate, a common set of genes in both sexes. 相似文献
4.
The maize ID1 flowering time regulator is a zinc finger protein with novel DNA binding properties 总被引:11,自引:0,他引:11
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The INDETERMINATE protein, ID1, plays a key role in regulating the transition to flowering in maize. ID1 is the founding member of a plant-specific zinc finger protein family that is defined by a highly conserved amino sequence called the ID domain. The ID domain includes a cluster of three different types of zinc fingers separated from a fourth C2H2 finger by a long spacer; ID1 is distinct from other ID domain proteins by having a much longer spacer. In vitro DNA selection and amplification binding assays and DNA binding experiments showed that ID1 binds selectively to an 11 bp consensus motif via the ID domain. Unexpectedly, site-directed mutagenesis of the ID1 protein showed that zinc fingers located at each end of the ID domain are not required for binding to the consensus motif despite the fact that one of these zinc fingers is a canonical C2H2 DNA binding domain. In addition, an ID1 in vitro deletion mutant that lacks the extra spacer between zinc fingers binds the same 11 bp motif as normal ID1, suggesting that all ID domain-containing proteins recognize the same DNA target sequence. Our results demonstrate that maize ID1 and ID domain proteins have novel zinc finger configurations with unique DNA binding properties. 相似文献
5.
Ros is a chromosomally-encoded repressor containing a novel C2H2 zinc finger in Agrobacterium tumefaciens. Ros regulates the expression of six virulence genes and an oncogene on the Ti plasmid. Constitutive expression of these genes occurs in the spontaneous mutant 4011R derived from the octopine strain Ach-5, resulting in T-DNA processing in the absence of induction, and in the biosynthesis of cytokinin. Interestingly, the mutation in 4011R is an Arg to Cys conversion at amino acid residue 125 near the C-terminus well outside the zinc finger of Ros. Yet, Ros bearing this mutation is unable to bind to the Ros-box and is unable to complement other ros mutants. 相似文献
6.
Cys(2)-His(2)-type zinc finger proteins have a tandemly repeated array structure consisting of independent finger modules. They are expected to elevate the DNA binding affinity and specificity by increasing the number of finger modules. To investigate the relation between the number and the DNA binding affinity of the zinc finger, we have designed the two- to four-finger peptides by connecting the central zinc finger (finger 2) of Sp1 with the canonical linker sequence, Thr-Gly-Glu-Lys-Pro. Gel mobility shift assays reveal that the cognate three- and four-finger peptides, Sp1(zf222) and Sp1(zf2222), strongly bind to the predicted target sequences, but the two-finger peptide, Sp1(zf22), does not. Of special interest is the fact that the dissociation constant for Sp1(zf2222) binding to the target DNA is comparable to that for Sp1(zf222). The methylation interference, DNase I and hydroxyl radical footprintings, and circular permutation analyses demonstrate that Sp1(zf2222) binds to its target site with three successive zinc fingers and the binding of the fourth zinc finger is inhibited by DNA bending induced by the binding of the three-finger domain. The present results strongly indicate that the zinc finger protein binds to DNA by the three-finger domain as one binding unit. In addition, this information provides the basis for the design of a novel multifinger protein with high affinity and specificity for long DNA sequences, such as chromosomal DNAs. 相似文献
7.
8.
9.
10.
Cooperative binding of Agrobacterium tumefaciens VirE2 protein to single-stranded DNA. 总被引:13,自引:5,他引:8
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The VirE2 protein of Agrobacterium tumefaciens Ti plasmid pTiA6 is a single-stranded-DNA-binding protein. Density gradient centrifugation studies showed that it exists as a tetramer in solution. Monomeric VirE2 active in DNA binding could also be obtained by using a different protein isolation procedure. VirE2 was found to be thermolabile; brief incubation at 37 degrees C abolished its DNA-binding activity. It was insensitive to the sulfhydryl-specific reagent N-ethylmaleimide. Removal of the carboxy-terminal 37 residues of the 533-residue VirE2 polypeptide led to complete loss of DNA-binding activity; however, chimeric fusion proteins containing up to 125 residues of the VirE2 C terminus were inactive in DNA binding. In nuclease protection studies, VirE2 protected single-stranded DNA against degradation by DNase I. Analysis of the DNA-VirE2 complex by electron microscopy demonstrated that VirE2 coats a single-stranded DNA molecule and that the binding of VirE2 to its substrate is cooperative. 相似文献
11.
12.
A novel zinc finger coiled-coil domain in a family of nuclear proteins. 总被引:27,自引:0,他引:27
13.
The virulence (vir) genes of Agrobacterium tumefaciens Ti plasmids are positively regulated by virG in conjunction with virA and plant-derived inducing molecules. A procedure that utilizes both genetic selection and a genetic screen was developed to isolate mutations in virG that led to elevated levels of vir gene expression in the absence of virA and plant phenolic inducers. Mutants were isolated at a frequency of 1 in 10(7) to 10(8). Substitution mutations at two positions in the virG coding region were found to result in the desired phenotype. One mutant had an asparagine-to-aspartic acid substitution at residue 54, and the other contained an isoleucine-to-leucine substitution at residue 106. In both cases, the mutant phenotype required the presence of the active-site aspartic acid residue at position 52. Further analysis showed that no other substitution at residue 54 resulted in a constitutive phenotype. In contrast, several substitutions at residue 106 led to a constitutive phenotype. The possible roles of the residues at positions 54 and 106 in VirG function are discussed. 相似文献
14.
Tibbetts MD Shiozaki EN Gu L McDonald ER El-Deiry WS Shi Y 《Structure (London, England : 1993)》2004,12(12):2257-2263
The caspase-associated ring proteins (CARP1 and CARP2) are distinguished from other caspase regulators by the presence of a FYVE-type zinc finger domain. FYVE-type domains are divided into two known classes: FYVE domains that specifically bind to phosphatidylinositol 3-phosphate in lipid bilayers and FYVE-related domains of undetermined function. Here, we report the crystal structure of the N-terminal region of CARP2 (44-139) including the FYVE-type domain and its associated helical bundle at 1.7 A resolution. The structure reveals a cramped phosphoinositide binding pocket and a blunted membrane insertion loop. These structural features indicate that the domain is not optimized to bind to phosphoinositides or insert into lipid bilayers. The CARP2 FYVE-like domain thus defines a third subfamily of FYVE-type domains that are functionally and structurally distinct. Structural analyses provide insights into the possible function of this unique subfamily of FYVE-type domains. 相似文献
15.
16.
A conserved zinc finger domain in higher plants 总被引:3,自引:0,他引:3
17.
We present a quantitative, theoretical analysis of the recognition mechanisms used by two zinc finger proteins: Zif268, which selectively binds to GC-rich sequences, and a Zif268 mutant, which binds to a TATA box site. This analysis is based on a recently developed method (ADAPT), which allows binding specificity to be analyzed via the calculation of complexation energies for all possible DNA target sequences. The results obtained with the zinc finger proteins show that, although both mainly select their targets using direct, pairwise protein–DNA interactions, they also use sequence-dependent DNA deformation to enhance their selectivity. A new extension of our methodology enables us to determine the quantitative contribution of these two components and also to measure the contributions of individual residues to overall specificity. The results show that indirect recognition is particularly important in the case of the TATA box binding mutant, accounting for 30% of the total selectivity. The residue-by-residue analysis of the protein–DNA interaction energy indicates that the existence of amino acid–base contacts does not necessarily imply sequence selectivity, and that side chains without contacts can nevertheless contribute to defining the protein's target sequence. 相似文献
18.
19.