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1.
The EPF family is a group of Cys2/His2zinc-finger proteins in petunia. In these proteins, characteristically long spacer regions have been found to separate the zinc fingers. Our previous DNA-binding studies demonstrated that two-fingered proteins (ZPT2-1 and ZPT2-2), which have spacers of different lengths, bind to two separate AGT core motifs in a spacing specific manner. To investigate the possibility that these proteins might distinguish between the target sequences on the basis of spacing between the core motifs, we screened petunia cDNA library for other proteins belonging to this family. Initial screening by PCR and subsequent cloning of full-length cDNAs allowed us to identify the genes for 10 new proteins that had two, three or four zinc fingers. Among the two-fingered proteins the spacing between zinc fingers varied from 19 to 65 amino acids. The variation in the length of spacers was even more extensive in three- and four-fingered proteins. The presence of such proteins is consistent with our hypothesis that the spacing between the core motifs might be important for target sequence recognition. Furthermore, comparison of diverse protein structures suggests that three- and two-fingered proteins might have resulted due to successive loss of fingers from a four-fingered protein during molecular evolution. We also demonstrate that a highly conserved motif (QALGGH) among the members of EPF family and other Cys2/His2 zinc-finger proteins in plants is critical for the DNA-binding activity.  相似文献   

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One, two or four copies of the 'helix-hairpin-helix' (HhH) DNA-binding motif are predicted to occur in 14 homologous families of proteins. The predicted DNA-binding function of this motif is shown to be consistent with the crystallographic structure of rat polymerase beta, complexed with DNA template-primer [Pelletier, H., Sawaya, M.R., Kumar, A., Wilson, S.H. and Kraut, J. (1994) Science 264, 1891-1903] and with biochemical data. Five crystal structures of predicted HhH motifs are currently known: two from rat pol beta and one each in endonuclease III, AlkA and the 5' nuclease domain of Taq pol I. These motifs are more structurally similar to each other than to any other structure in current databases, including helix-turn-helix motifs. The clustering of the five HhH structures separately from other bi-helical structures in searches indicates that all members of the 14 families of proteins described herein possess similar HhH structures. By analogy with the rat pol beta structure, it is suggested that each of these HhH motifs bind DNA in a non-sequence-specific manner, via the formation of hydrogen bonds between protein backbone nitrogens and DNA phosphate groups. This type of interaction contrasts with the sequence-specific interactions of other motifs, including helix-turn-helix structures. Additional evidence is provided that alphaherpesvirus virion host shutoff proteins are members of the polymerase I 5'-nuclease and FEN1-like endonuclease gene family, and that a novel HhH-containing DNA-binding domain occurs in the kinesin-like molecule nod, and in other proteins such as cnjB, emb-5 and SPT6.  相似文献   

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A diverse group of DNA-binding regulatory proteins share a common structural domain which is homologous to the sequence of a highly conserved and abundant chromosomal protein, HMG-1. Proteins containing this HMG-1 box regulate various cellular functions involving DNA binding, suggesting that the target DNA sequences share a common structural element. Members of this protein family exhibit a dual DNA-binding specificity: each recognizes a unique sequence as well as a common DNA conformation. The highly conserved HMG-1/-2 proteins may modulate the binding of other HMG-1 box proteins to bent DNA. We examine the structural and functional relationships between the proteins, identify their signature? and describe common features of their target DNA elements.  相似文献   

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In the axial elements of synaptonemal complexes (SCs) of the rat, major protein components have been identified, with relative electrophoretic mobilities (M rs) of 30 000-33 000 and 190 000. Using monoclonal anti-SC antibodies, we isolated cDNA fragments which encode the 190 000 M r component of rat SCs. The translation product predicted from the nucleotide sequence of the cDNA, called SCP2 (for synaptonemal complex protein 2), is a basic protein (pI = 8.0) with a molecular mass of 173 kDa. At the C-terminus, a stretch of approximately 50 amino acid residues is predicted to be capable of forming coiled-coil structures. SCP2 contains two clusters of S/T-P motifs, which are common in DNA-binding proteins. These clusters flank the central, most basic part of the protein (pI = 9.5). Three of the S/T-P motifs are potential target sites for p34(cdc2) protein kinase. In addition, SCP2 has eight potential cAMP/cGMP-dependent protein kinase target sites. The gene encoding SCP2 is transcribed specifically in the testis, in meiotic prophase cells. At the amino acid sequence and secondary structural level, SCP2 shows some similarity to the Red1 protein, which is involved in meiotic recombination and the assembly of axial elements of SCs in yeast. We speculate that SCP2 is a DNA-binding protein involved in the structural organization of meiotic prophase chromosomes.  相似文献   

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SWI2/SNF2 chromatin-remodeling proteins mediate the mobilization of nucleosomes and other DNA-associated proteins. SWI2/SNF2 proteins contain sequence motifs characteristic of SF2 helicases but do not have helicase activity. Instead, they couple ATP hydrolysis with the generation of superhelical torsion in DNA. The structure of the nucleosome-remodeling domain of zebrafish Rad54, a protein involved in Rad51-mediated homologous recombination, reveals that the core of the SWI2/SNF2 enzymes consist of two alpha/beta-lobes similar to SF2 helicases. The Rad54 helicase lobes contain insertions that form two helical domains, one within each lobe. These insertions contain SWI2/SNF2-specific sequence motifs likely to be central to SWI2/SNF2 function. A broad cleft formed by the two lobes and flanked by the helical insertions contains residues conserved in SWI2/SNF2 proteins and motifs implicated in DNA-binding by SF2 helicases. The Rad54 structure suggests that SWI2/SNF2 proteins use a mechanism analogous to helicases to translocate on dsDNA.  相似文献   

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A gene from Xenopus laevis that is expressed specifically in the nervous system beginning at the stage of neural plate formation has been isolated and several cDNAs have been sequenced. The sequence of the predicted protein contains two copies of a presumed RNA-binding domain, each of which includes two short conserved motifs characteristic for ribonucleoproteins (RNPs), called the RNP-1 and RNP-2 consensus sequences. We name this gene Xenopus nrp-1, for nervous system-specific RNP protein-1. Sequence comparisons suggest that the nrp-1 protein is a heterogeneous nuclear RNP protein, but it is clearly distinct from previously reported hnRNP proteins such as the A1, A2/B1, and C1 proteins. nrp-1 RNA undergoes an alternative splicing event giving rise to two predicted protein isoforms that differ from each other by seven amino acids. In situ hybridization to tadpole brain shows that the nrp-1 gene is expressed in the ventricular zone where cell proliferation takes place. The occurrence of an RNP protein with nervous system-limited expression suggests that it may be involved in the tissue-specific control of RNA processing.  相似文献   

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A proximal promoter (-422/-13) of the bean seed storage protein beta-phaseolin gene contains cis-regulatory elements conferring spatial and temporal gene regulation. To correlate trans-acting elements with these cis-elements, we performed gel mobility shift and exonuclease III protection assays using bean seed nuclear proteins, and identified target sequences of four DNA-binding proteins associated with this promoter. Three CANNTG motifs, CACGTG (-248/-243), CACCTG (-163/-158), and CATATG (-100/-95), were determined as target sequences of the same DNA-binding protein designated CAN. Competition assays using oligonucleotides containing the wild-type or mutated CANNTG motif indicated that the CANNTG motif appears to be a preferred target sequence for CAN binding. Competition assays also demonstrated that DNA-binding protein AG-1 binds to AAAAAG(A/G)CAA (-356/-347, -191/-182), CA-1 binds to two CA-rich sequences (-201/-192, -175/-160), and that a TATA-box binding protein binds to either TATATAA (-43/-37) or TATAAA (-32/-27) or both. Based on these and other results, it is proposed that CACGTG motif (-248/-243) is a major cis-acting regulatory element conferring spatial and temporal control of the beta-phaseolin gene.  相似文献   

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A gene (AtTRP1) encoding a telomeric repeat-binding protein has been isolated from Arabidopsis thaliana. AtTRP1 is a single copy gene located on chromosome 5 of A. thaliana. The protein AtTRP1 encoded by this gene is not only homologous to the Myb DNA-binding motifs of other telomere-binding proteins but also is similar to several initiator-binding proteins in plants. Gel retardation assay revealed that the 115 residues on the C terminus of this protein, including the Myb motif, are sufficient for binding to the double-stranded plant telomeric sequence. The isolated DNA-binding domain of AtTRP1 recognizes each telomeric repeat centered on the sequence GGTTTAG. The almost full-length protein of AtTRP1 does not form any complex at all with the DNA fragments carrying four or fewer GGTTTAG repeats. However, it forms a complex with the sequence (GGTTTAG)(8) more efficiently than with the sequence (GGTTTAG)(5). These data suggest that the minimum length of a telomeric DNA for AtTRP1 binding consists of five GGTTTAG repeats and that the optimal AtTRP1 binding may require eight or more GGTTTAG repeats. It also implies that this protein AtTRP1 may bind in vivo primarily to the ends of plant chromosomes, which consist of long stretches of telomeric repeats.  相似文献   

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The architecture of single-stranded DNA-binding proteins, which play key roles in DNA metabolism, is based on different combinations of the oligonucleotide/oligosaccharide binding (OB) fold. Whereas the polypeptide serving this function in bacteria contains one OB fold, the eukaryotic functional homolog comprises a complex of three proteins, each harboring at least one OB fold. Here we show that unlike these groups of organisms, the Euryarchaeota has exploited the potential in the OB fold to re-invent single-stranded DNA-binding proteins many times. However, the most common form is a protein with two OB folds and one zinc finger domain. We created several deletion mutants of this protein based on its conserved motifs, and from these structures functional chimeras were synthesized, supporting the hypothesis that gene duplication and recombination could lead to novel functional forms of single-stranded DNA-binding proteins. Biophysical studies showed that the orthologs of the two OB fold/one zinc finger replication protein A in Methanosarcina acetivorans and Methanopyrus kandleri exhibit two binding modes, wrapping and stretching of DNA. However, the ortholog in Ferroplasma acidarmanus possessed only the stretching mode. Most interestingly, a second single-stranded DNA-binding protein, FacRPA2, in this archaeon exhibited the wrapping mode. Domain analysis of this protein, which contains a single OB fold, showed that its architecture is similar to the functional homologs thought to be unique to the Crenarchaeotes. Most unexpectedly, genes coding for similar proteins were found in the genomes of eukaryotes, including humans. Although the diversity shown by archaeal single-stranded DNA-binding proteins is unparalleled, the presence of their simplest form in many organisms across all domains of life is of greater evolutionary consequence.  相似文献   

17.
Structure of the HMG box motif in the B-domain of HMG1.   总被引:38,自引:7,他引:31       下载免费PDF全文
The conserved, abundant chromosomal protein HMG1 consists of two highly homologous, folded, basic DNA-binding domains, each of approximately 80 amino acid residues, and an acidic C-terminal tail. Each folded domain represents an 'HMG box', a sequence motif recently recognized in certain sequence-specific DNA-binding proteins and which also occurs in abundant HMG1-like proteins that bind to DNA without sequence specificity. The HMG box is defined by a set of highly conserved residues (most distinctively aromatic and basic) and appears to define a novel DNA-binding structural motif. We have expressed the HMG box region of the B-domain of rat HMG1 (residues 88-164 of the intact protein) in Escherichia coli and we describe here the determination of its structure by 2D 1H-NMR spectroscopy. There are three alpha-helices (residues 13-29, 34-48 and 50-74), which together account for approximately 75% of the total residues and contain many of the conserved basic and aromatic residues. Strikingly, the molecule is L-shaped, the angle of approximately 80 degrees between the two arms being defined by a cluster of conserved, predominantly aromatic, residues. The distinctive shape of the HMG box motif, which is distinct from hitherto characterized DNA-binding motifs, may be significant in relation to its recognition of four-way DNA junctions.  相似文献   

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Diverse mechanisms for DNA-protein recognition have been elucidated in numerous atomic complex structures from various protein families. These structural data provide an invaluable knowledge base not only for understanding DNA-protein interactions, but also for developing specialized methods that predict the DNA-binding function from protein structure. While such methods are useful, a major limitation is that they require an experimental structure of the target as input. To overcome this obstacle, we develop a threading-based method, DNA-Binding-Domain-Threader (DBD-Threader), for the prediction of DNA-binding domains and associated DNA-binding protein residues. Our method, which uses a template library composed of DNA-protein complex structures, requires only the target protein''s sequence. In our approach, fold similarity and DNA-binding propensity are employed as two functional discriminating properties. In benchmark tests on 179 DNA-binding and 3,797 non-DNA-binding proteins, using templates whose sequence identity is less than 30% to the target, DBD-Threader achieves a sensitivity/precision of 56%/86%. This performance is considerably better than the standard sequence comparison method PSI-BLAST and is comparable to DBD-Hunter, which requires an experimental structure as input. Moreover, for over 70% of predicted DNA-binding domains, the backbone Root Mean Square Deviations (RMSDs) of the top-ranked structural models are within 6.5 Å of their experimental structures, with their associated DNA-binding sites identified at satisfactory accuracy. Additionally, DBD-Threader correctly assigned the SCOP superfamily for most predicted domains. To demonstrate that DBD-Threader is useful for automatic function annotation on a large-scale, DBD-Threader was applied to 18,631 protein sequences from the human genome; 1,654 proteins are predicted to have DNA-binding function. Comparison with existing Gene Ontology (GO) annotations suggests that ∼30% of our predictions are new. Finally, we present some interesting predictions in detail. In particular, it is estimated that ∼20% of classic zinc finger domains play a functional role not related to direct DNA-binding.  相似文献   

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