共查询到20条相似文献,搜索用时 31 毫秒
1.
The DNA binding and structural properties of Xfin-31 (Lee, M.S., Gippert, G.P., Soman, K.V., Case, D.A. and Wright, P.E., 1989, Science 245, 635-637), a twenty five amino acid zinc finger peptide, in the reduced, oxidized and zinc complex forms, as well as the fourteen residue helical segment of the zinc finger (residues 12-25) have been compared using affinity coelectrophoresis (ACE) and circular dichroism (CD) spectroscopy. The zinc complex and oxidized peptides bind cooperatively to DNA although the cooperativity factor, omega, is more than 15-fold greater for the zinc complex. The reduced peptide in the absence of zinc and the helical segment do not bind cooperatively (omega = 1). Hence, the binding constant for singly contiguous sites (K omega) ranges over 100-fold for the various peptides even though the intrinsic binding constants (K) are similar. An increase in binding order and affinity for the other forms of Xfin-31 is correlated with an increasing similarity of the CD spectrum to that of the Xfin-31 zinc complex. The surprising DNA binding activity of the oxidized peptide may result from hydrophobic interactions between the amino-terminal loop formed by the Cys3-Cys6 disulfide bond and conserved hydrophobic residues in the carboxyl-terminal segment. Xfin-31 may be a particularly useful model for studying several poorly understood aspects of cooperative, non-specific DNA binding since it is small, has a stable, well-defined structure, and structures of zinc fingers bound to DNA have been determined. 相似文献
2.
3.
B. A. Krizek L. E. Zawadzke J. M. Berg 《Protein science : a publication of the Protein Society》1993,2(8):1313-1319
Most Cys2His2 zinc finger proteins contain tandem arrays of metal binding domains. The tandem nature of these arrays suggests that metal binding by these domains may not be independent but rather that metal binding may occur in a cooperative manner. This is especially true in light of the crystal structure of a three zinc finger array bound to DNA that revealed several types of interactions between domains. To address this question, peptides containing two tandem domains have been prepared. While metal binding studies do show that the two finger peptide has a metal ion affinity about threefold higher than that for a single domain peptide with the same sequence, additional studies reveal that this behavior is due to increased single site affinities in the context of the two domain peptide rather than to cooperativity. These studies indicate that domains of this type are independent of one another with regard to metal binding, at least in the absence of DNA. This observation has implications with regard to the question of whether the activities of proteins of this class might be modulated by available zinc concentrations. 相似文献
4.
The Cys(2)His(2)-type zinc finger is a common DNA binding motif that is widely used in the design of artificial zinc finger proteins. In almost all Cys(2)His(2)-type zinc fingers, position 4 of the α-helical DNA-recognition site is occupied by a Leu residue involved in formation of the minimal hydrophobic core. However, the third zinc finger domain of native Zif268 contains an Arg residue instead of the conserved Leu. Our aim in the present study was to clarify the role of this Arg in the formation of a stable domain structure and in DNA binding by substituting it with a Lys, Leu, or Hgn, which have different terminal side-chain structures. Assessed were the metal binding properties, peptide conformations, and DNA-binding abilities of the mutants. All three mutant finger 3 peptides exhibited conformations and thermal stabilities similar to the wild-type peptide. In DNA-binding assays, the Lys mutant bound to target DNA, though its affinity was lower than that of the wild-type peptide. On the other hand, the Leu and Hgn mutants had no ability to bind DNA, despite the similarity in their secondary structures to the wild-type. Our results demonstrate that, as with the Leu residue, the aliphatic carbon side chain of this Arg residue plays a key role in the formation of a stable zinc finger domain, and its terminal guanidinium group appears to be essential for DNA binding mediated through both electrostatic interaction and hydrogen bonding with DNA phosphate backbone. 相似文献
5.
In situ detection of a heat-shock regulatory element binding protein using a soluble synthetic enhancer sequence. 总被引:4,自引:1,他引:3
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)
A Harel-Bellan A T Brini D K Ferris P Robin W L Farrar 《Nucleic acids research》1989,17(11):4077-4087
In various studies, enhancer binding proteins have been successfully absorbed out by competing sequences inserted into plasmids, resulting in the inhibition of the plasmid expression. Theoretically, such a result could be achieved using synthetic enhancer sequences not inserted into plasmids. In this study, a double stranded DNA sequence corresponding to the human heat shock regulatory element was chemically synthesized. By in vitro retardation assays, the synthetic sequence was shown to bind specifically a protein in extracts from the human T cell line Jurkat. When the synthetic enhancer was electroporated into Jurkat cells, not only the enhancer was shown to remain undegraded into the cells for up to 2 days, but also it was shown to bind intracellularly a protein. The binding was specific and was modulated upon heat shock. Furthermore, the binding protein was shown to be of the expected molecular weight by UV crosslinking. However, when the synthetic enhancer element was co-electroporated with an HSP 70-CAT reporter construct, the expression of the reporter plasmid was consistently enhanced in the presence of the exogenous synthetic enhancer. 相似文献
6.
7.
Identification of a short sequence essential for actin binding by Dictyostelium ABP-120 总被引:14,自引:0,他引:14
Tryptic digestion of ABP-120, an actin cross-linking protein from Dictyostelium discoideum, generates a ladder of peptides differing in molecular mass by 13,000 daltons, indicating a structural repeat within the molecule. A number of peptides bind actin with the smallest having a molecular mass of 17,000 daltons (T17). Our sedimentation assays also show that a peptide of 14,000 daltons does not bind actin. Using the full-length cDNA sequence (Noegel, A., Rapp, S., Lottspeich, F., Schleicher, M., and Stewart, M. (1989) J. Cell Biol. 109, 607-618) and protein sequencing techniques, we have determined that T17 begins at residue 89 while T14 begins at residue 116. Therefore we have localized 27 amino acids which are essential for actin binding activity. This region is at the end of the molecule, distal from the repetitive beta-sheet region predicted from the cDNA sequence, and displays high sequence identity with regions in the N termini of ABP/filamin, dystrophin, beta-spectrin, and alpha-actinin. 相似文献
8.
A Meister S L Weinrich C Nelson W J Rutter 《The Journal of biological chemistry》1989,264(34):20744-20751
9.
Metal binding properties of single amino acid deletion mutants of zinc finger peptides: studies using cobalt(II) as a spectroscopic probe. 总被引:1,自引:0,他引:1
Peptides corresponding to Cys2His2 zinc finger domains from which one amino acid has been deleted have been synthesized and their metal-binding properties characterized. In contrast to earlier reports (Párraga, G., S. Horvath, L. Hood, E. T. Young, and R. E. Klevit. 1990. Proc. Natl. Acad. Sci. USA. 87:137-141.), such peptides do bind metal ions such as cobalt(II). A peptide with the sequence ProTyrLysCysProGluCysLysSerPheSerGlnLysSerAspLeuValLysHisGlnArgThrHis ThrGly (which corresponds to a previously characterized consensus zinc finger sequence from which a Gly residue immediately following the second Cys residue has been deleted) was found to form a 1:1 peptide to cobalt(II) complex with an absorption spectrum quite similar to those previously observed for zinc finger peptide-cobalt(II) complexes. The dissociation constant for this complex is 6 x 10(-6)M, a factor of 100 times higher than that for the parent peptide. A peptide with the sequence LysProTyrProCysGlyLeuCysArgCysPheThrArgArgAspLeuLeulleArgHisAlaGln - LyslleHisSerGlyAsnLeu corresponding to a similar mutation of the peptide ADR1 was also characterized. Spectroscopic studies with cobalt(II) revealed that this peptide forms both 1:1 and 2:1 peptide to cobalt(II) complexes. The absorption spectra of the two forms and the dissociation constants were determined via deconvolution methods. In contrast, the parent peptide ADR1a was found to form only a 1:1 complex under comparable conditions and this 1:1 complex was found to be more stable than that for the mutant. These results reveal that deletion mutations do adversely affect the stability of zinc finger peptide-metal complexes but that the effects are not as drastic as had been previously described. 相似文献
10.
Sequence requirement for specific interaction of an enhancer binding protein (EBP1) with DNA. 总被引:16,自引:5,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)
Short DNA sequence motifs have been identified in viral and cellular enhancers which represent the binding sites for a variety of trans- acting factors. One such HeLa cell factor, EBP1, has been purified and shown to bind to sequences in the SV40 enhancer. The PRDII element in the human beta-interferon gene regulatory element (IRE) shows strong sequence similarity to the EBP1 binding site in the SV40 enhancer. We demonstrate here that EBP1 binds to its sites in the SV40 enhancer and IRE in a similar manner, making base specific contacts over one complete turn of the DNA double helix. Mutational analysis of the EBP1 sites in the IRE and SV40 enhancer has identified the DNA sequence requirements necessary for specific EBP1/DNA complex formation. In addition, 34 DNA sequences related to the EBP1 binding site were analysed for their ability to bind EBP1. Sequences constituting high affinity binding sites possess the sequence 5'-GG(N)6CC-3'. Single base pair changes in the region between the conserved Gs and Cs can generally be tolerated although it is clear that these intervening bases contribute to binding affinity. Mutations in the recognition site which could lead to gross structural changes in the DNA abolish EBP1 binding. 相似文献
11.
B K Chow V Ting F Tufaro R T MacGillivray 《The Journal of biological chemistry》1991,266(28):18927-18933
12.
13.
A complex regulatory DNA element associated with a major histocompatibility complex class I gene consists of both a silencer and an enhancer. 总被引:3,自引:4,他引:3
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
A novel regulatory element which contributes to the regulation of quantitative, tissue-specific differences in gene expression has been found between -771 and -676 bp upstream of the major histocompatibility complex (MHC) class I gene, PD1. Molecular dissection of this element reveals the presence of two overlapping functional activities: an enhancer and a silencer. Distinct nuclear factors bind to the overlapping enhancer and silencer DNA sequence elements within the regulatory domain. The levels of factors binding the silencer DNA sequence in different cell types are inversely related to levels of class I expression; in contrast, factors binding the enhancer DNA sequence can be detected in all cells. In cultured cell lines, inhibition of protein synthesis leads to the rapid loss of silencer complexes, with a concomitant increase in both enhancer complexes and MHC class I RNA. From these data, we conclude that a labile silencer factor competes with a constitutively expressed, stable enhancer factor for overlapping DNA-binding sites; the relative abundance of the silencer factor contributes to establishing steady-state levels of MHC class I gene expression. 相似文献
14.
S G Nadler J L Kapouch J I Elliott K R Williams 《The Journal of biological chemistry》1992,267(6):3750-3757
We have used synthetic peptides to study a conserved RNA binding motif in yeast poly(A)-binding protein. Two peptides, 45 and 44 amino acids in length, corresponding to amino and carboxyl halves of a 90-amino acid RNA-binding domain in the protein were synthesized. While the amino-terminal peptide had no significant affinity for nucleic acids, the carboxyl-terminal peptide-bound nucleic acids with similar characteristics to that for the entire 577 residue yeast poly(A)-binding protein. In 100 mM NaCl, the latter peptide retained over 50% of the intrinsic binding free energy of the protein, as well as, similar RNA versus DNA binding specificity. However, shuffling of the sequence of this 44 residue peptide had surprisingly little effect on its nucleic acid binding properties suggesting the overriding importance of amino acid composition as opposed to primary sequence. Deletion studies on the 44 residue peptide with the "correct" sequence succeeded in identifying amino acids important for conferring RNA specificity and for increasing our understanding of the molecular basis for nucleic acid binding by synthetic peptides. The shuffled peptide study, however, clearly indicates that considerable caution must be exercised before extrapolating results of structure/function studies on synthetic peptide analogues to the parent protein. 相似文献
15.
16.
17.
18.
19.
S L Grokhovski? A N Surovaia N Iu Sidorova H Votavova J Sponar 《Molekuliarnaia biologiia》1988,22(5):1315-1334
In the present communication, design, synthesis and DNA binding activities of the following two peptides are reported: Dns-Gly-Ala-Gln-Lys-Leu-Ala-Cly-Lys-Val-Gly-Thr-Lys-Val-Lys-Val-Gl y-Thr-Lys-Thr - Val-OH (I) and [(H-Ala-Lys-Leu-Ala-Thr-Lys-Ala-Gly-Val-Lys-Gln-Gln-Ser-Ile-Gln-Leu-Ile- Thr- Ala-Aca-Lys-Aca)2Lys-Aca]2Lys-Val-OH (II), where Aca = NH(CH2)5CO--; Dns is a residue of 5-dimethylaminonaphtalene-1-sulfonic acid. Peptide I contains a large fraction (ca.30%) of valyl and threonyl residues, which possess a high potential for beta structure formation. Peptide II contains four repeats of the amino acid sequence present in the presumed DNA binding helix-turn-helix unit of 434 Cro repressor. These four domains are linked in such a way that two domains can interact with two halves a 14 base pair long operator site on DNA. From CD studies we have found that peptide I is in a random coil conformation in the aqueous solution in the presence of 20% trifluoroethanol. By contrast, amino acid residues of peptide II assume alpha helical, beta and random coiled conformations under the same conditions. A change in the secondary structure of the two peptides upon binding to DNA is observed. The difference CD spectra obtained by subtracting the spectra of free DNA from the spectra of peptide I--DNA complexes gives rise to a beta-like pattern. The difference CD spectra obtained for complexes of peptide II with various natural and synthetic DNAs suggest that alpha-beta-transition takes place in the presumed helix-turn-helix repeat units of peptide II upon binding to DNA. Peptide I binds more strongly to poly(dG).poly(dC) than to poly(dA).poly(dT) and poly[d(GC)].poly[d(GC)]. The binding takes place in the minor DNA groove because minor groove binding antibiotic sibiromycin can displace peptide I from a complex with poly(dG).poly(dC). Analysis of footprinting diagramms shows that peptide I specifically protects phosphodiester bonds within operator sites OR1 and OR2 of phage lambda from nuclease cleavage. By contrast, peptide II does not react specifically with operators OR1, OR2 and OR3 of phage 434 although it forms very tight complexes with DNA which are stable in the presence of 1M NH4F. 相似文献
20.
The calmodulin binding domain of chicken smooth muscle myosin light chain kinase contains a pseudosubstrate sequence 总被引:22,自引:0,他引:22
B E Kemp R B Pearson V Guerriero I C Bagchi A R Means 《The Journal of biological chemistry》1987,262(6):2542-2548
Smooth muscle myosin light chain kinase contains a 64 residue sequence that binds calmodulin in a Ca2+-dependent manner (Guerriero, V., Jr., Russo, M. A., and Means, A. R. (1987) Biochemistry, in press). Within this region is a sequence with homology to the corresponding sequence reported for the calmodulin binding region of skeletal muscle myosin light chain kinase (Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, L., Walsh, K. A., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3187-3191). Inspection of these sequences reveals that they both share a similar number and spatial arrangement of basic residues with those present in the myosin light chain substrate. We have synthesized a 22-residue peptide corresponding to residues 480-501 (determined from the cDNA) of the smooth muscle myosin light chain kinase. This peptide, Ala-Lys-Lys-Leu-Ser-Lys-Asp-Arg-Met-Lys-Lys-Tyr-Met-Ala-Arg-Arg-Lys-Trp- Gln-Lys-Thr-Gly, inhibited calmodulin-dependent activation of the smooth muscle myosin light chain kinase with an IC50 of 46 nM. At saturating concentrations of calmodulin, the 22-residue peptide inhibited myosin light chain and synthetic peptide substrate phosphorylation competitively with IC50 values of 2.7 and 0.9 microM, respectively. An 11-residue synthetic peptide analog, corresponding to part of the calmodulin-binding sequence in skeletal muscle myosin light chain kinase, Lys-Arg-Arg-Trp-Lys-Lys-Asn-Phe-Ile-Ala-Val, also competitively inhibited synthetic peptide substrate phosphorylation with a Ki of 1 microM. The competitive inhibitory activity of the calmodulin binding regions is similar to the apparent Km of 2.7 microM for phosphorylation of the 23-residue peptide analog of the smooth muscle myosin light chain and raises the possibility that the calmodulin binding region of the myosin light chain kinase may act as a pseudosubstrate inhibitor of the enzyme. 相似文献