A model is proposed for the structure of stereospecific sites in regulatory proteins. On its basis a possible code is suggested that governs the binding of regulatory proteins at specific control sites on DNA. Stereospecific sites of regulatory proteins are assumed to contain pairs of antiparallel polypeptide chain segments which form a right-hand twisted antiparallel beta-sheet, with single-stranded regions at the ends of the beta-structure. The model predicts that binding reaction between a regulatory protein and double-helical DNA is a cooperative phenomenon and is accompanied by significant structural alteration at the stereospecific site of the protein. Half of hydrogen bonds normally existing in beta-structure are broken upon complex formation with DNA and a new set of hydrogen bonds is formed between polypeptide amide groups and DNA base pairs. In a stereospecific site, one chain (t-chain) is attached through hydrogen bonds to the carbonyl oxygens of pyramides and N3 adenines lying in one DNA strand, while the second polypeptide chain (g chain) is hydrogen bonded to the 2-amino groups of guanine residues lying in the opposite DNA strand. The amide groups serve as specific reaction sites being hydrogen bond acceptors in g-chain and hydrogen bond donors in t-chain. The single-stranded portions of t- and g-chains lying in neighbouring subunits of regulatory protein interact with each other forming deformed beta-sheets. The recognition of regulatory sequences by proteins is based on the structural complementarity between stereospecific sites of regulatory proteins and base pairs sequences at the control sites. An essential feature of these sequences is the asymmetrical distribution of guanine residues between the two DNA strands. The code predicts that there are six fundamental amino acid residues (serine, threonine, asparagine, histidine, glutamine and cysteine) whose sequence in stereospecific site determines the base pair sequence to which a given regulatory protein would bind preferentially. The code states a correspondence between four amino acid residues at the stereospecific site of regulatory protein with the two residues being in t- and g-segments, respectively, and AT(GC) base pair at the control site. It is thus possible to determine which amino acid residues in the repressor and which base pairs in the operator DNA are involved in specific interactions with each other, as exemplified by lac repressor binding to lac operator.

Amino Acid Sequence↗

PubMedDate not supplied

[Effect of DNA local conformation on the affinity and binding specificity of bis-netropsins to DNA].

The interaction of short nucleotide duplexes with bis-netropsins, in which netropsin fragments are linked in the tail-to-tail orientation via cis-diammineplatinum group (<--Nt-Pt(NH3)-Nt-->) or aliphatic pentamethylene chain (<--Nt-(CH2)5-Nt-->), has been studied. Both the bis-netropsins have been shown to bind to DNA oligomer 5'-CCTATATCC-3' (I) as a hairpin with parallel orientation of netropsin fragments in 1:1 stoichiometry. Monodentate binding has been detected upon binding of bis-netropsins to other duplexes of sequences 5'-CCXCC-3'--where X = TTATT (II), TTAAT (III), TTTTT (IV), and AATTT (V)--along with the binding of bis-netropsins as a hairpin. The formation of dimeric antiparallel motif between the halves of two bound bis-netropsin molecules has been observed in the complexes of <--Nt-(CH2)5-Nt--> with DNA oligomers IV and V. The ratio of binding constant of bis-netropsin as a hairpin (K2) to monodentate binding constant (K1) has been shown to correlate with the width and/or conformational lability of DNA in the binding site. The share of bis-netropsin bound as a hairpin decreases in the order: TATAT > TTATT > TTAAT > TTTTT > AATTT, whereas the contribution of monodentate binding rises. The minimal strong binding site for <--Nt-Pt(NH3)-Nt--> and <--Nt-(CH2)5-Nt--> binding as a hairpin has been found to be DNA duplex 5'-CGTATACG-3'.

Anti-Bacterial Agents↗

PubMedDate not supplied

[Structure of the complexes of distamycin type antibiotics and actinomycin D with DNA: new data on the localization of these antibiotics within the DNA narrow groove].

It is shown that antibiotics actinomycin D (AM), netropsin (Nt), distamycin A (DM) and the propyl analogue of distamycin A (pDM) being complexed with DNA are located within the narrow groove of DNA. A comparative investigation of the 3H-dimethyl sulphate methylation extent of free calf thymus DNA and its complexes with AM, Nt, DM and pDM reveals that upon DNA saturation these antibiotics decrease the methylation level of the narrow groove (AM by 30%, pDM by 50%, DM by 65% and Nt by 70%). In the triple complex of DNA+AM+DM the methylation level of the narrow groove drops by 80%. The large groove is not shielded by these antibiotics at all. However, the methylation level of the large groove decreases by 50% for T6 phage DNA due to the presence of glucosyl residues linked to 5-hydroxymethylcytosine within the large groove. The binding of AM to DNA saturated with Nt or with the analogue of distamycin A (DM2) containing the 2 N-methylpyrrole residues has been investigated by spectrophotometry. The apparent number of binding sites for AM in these 2 complexes is about half as much as observed for free DNA while the saturation level of the binding decreased only by about 20%. This proves simultaneous presence of AM and Nt (DM2) within the narrow groove of DNA.

Animals↗

PubMedDate not supplied

[Specific DNA cleavage by an analog of netropsin containing a copper(II) chelating peptide Gly-Gly-His].

Experimental data are reported on DNA-cleaving activity of the synthetic netropsin analogs consisting of the two N-propylpyrrole carboxamide units linked covalently through two or three glycine residues to a copper-chelating tripeptide glycyl-glycyl-L-histidine. Incubation of DNA restriction fragment and netropsin analog in the presence of ascorbate, hydrogen peroxide and Cu2+ ions resulted in selective cleavage of the DNA at or near the preferred sites for binding of netropsin analog. A similar cleavage pattern is observed after X-ray irradiation of DNA complexes with netropsin analogs tethered with Cu2+ ions. The cleavage patterns are found to be dependent on the length of the connecting chain between the histidine-containing tripeptide and netropsin analog. The netropsin analog containing three glycine residues in the connecting chain, but not the analog with a shorter linker chain, can generate an intense cleavage of one of the two polynucleotide chains at a position corresponding to the presumed binding site for the dimeric ligand species. More than 50% of the total DNA can be cleaved at this position after X-ray irradiation. From analysis of the nucleotide sequences surrounding the preferred cleavage site on several DNA fragments we found that the consensus is 5'-TTTTNCA*AAA-3', where N is an arbitrary nucleotide. The Cu(2+)-mediated cleavage of DNA occurs at the second adenine (indicated by an asterisk) from the 5'-end of the sequence. The greatest cleavage activity is observed when the molar ratio of Cu2+ to the netropsin analog is equal to 0.5. Evidently, the Cu(2+)-ligated and unligated oligopeptide species interacts with each other to form a heterodimer bound to DNA at the cleavage site. To test the validity of this model we have studied the binding of unligated netropsin analog and netropsin analog complexed with Cu2+ ion to a self-complementary oligonucleotide 5'-GCGTTTTGCAAAACGC-3'. It is found that binding of Cu(2+)-ligated netropsin analog to the DNA oligomer preincubated with unligated form of the oligopeptide is a cooperative process for which interactions between the two bound ligands are responsible. The cooperativity parameter is estimated to be on the order of factor 6. Finally, a model is proposed in which a heterodimer stabilized by interligand beta-sheet binds in the minor DNA groove.

Amino Acid Sequence↗

PubMedDate not supplied

[Statistical fluctuations in processes of gene expression regulation: consideration of the problem from point of view of statistical mechanics].

The regulation of gene expression is a basic problem of biology. In some cases, the gene activity is regulated by specific binding of regulatory proteins to DNA. In terms of statistical mechanics, this binding is described as the process of adsorption of ligands on the one-dimensional lattice and has a probability nature. As a random physical process, the adsorption of regulatory proteins on DNA introduces a noise to the regulation of gene activity. We derived equations, which make it possible to estimate this noise in the case of the binding of the lac repressor to the operator and showed that these estimates correspond to experimental data. Many ligands are able to bind nonspecifically to DNA. Nonspecific binding is characterized by a lesser equilibrium constant but a greater number of binding sites on the DNA, as compared with specific binding. Relations are presented, which enable one to estimate the probability of the binding of a ligand on a specific site and on nonspecific sites on DNA. The competition between specific and nonspecific binding of regulatory proteins plays a great role in the regulation of gene activity. Similar to the one-dimensional "lattice gas" of particles, ligands adsorbed on DNA produce "one-dimensional" pressure on proteins located at the termini of free regions of DNA. This pressure, an analog of osmotic pressure, may be of importance in processes leading to changes in chromatin structure and activation of gene expression.

Adsorption↗

PubMedDate not supplied

[DNA-binding and antiviral activities of bis-netropsins].

The DNA-binding and antiviral activitus of bis-netropsins in which two monomers are attached covalently via three glycin residue were studied. These compounds have the same C-end groups but contain clusters with different numbers of lysine residues at the N-end of the molecule. In the homologous series of these compounds, bis-neropsins containing 15 and 31 branched lysine residues at the N-end of the molecule appear to be the most effective inhibitors of reproduction of the simplex herpes virus of type I in the Vero cell culture, including the virus versions resistant to aciclovir, ganciclovir, and other medicinal preparations. It was shown that the cytotoxicity of all the compounds studied is much lower than that of netropsin. The antiviral activity of the compounds correlates with their ability to selectively interact with the expanded clusters of the AT-pairs of DNA bases in the form of a monomer or a dimer, stabilized by interaction between the C-end halves of two bis-netropsin molecules bound at the neighboring overlapping binding sites on the DNA. The possible sites of their binding are the expanded clusters of AT-pairs at the origin of replication of OriS and OriL of the herpes virus.

Animals↗

PubMedDate not supplied

[The interaction of lamda-Cro repressor and its mutant covalent S-S-dimer lamda-CroV55C with symmetric and asymmetric DNA].

Binding of lamda-Cro protein and mutant CroV55C disulfide bonded dimer to synthetic olygonucleotide duplexes were studied using a competition with distamycin A. The equilibrium binding constants for lamda operator OR3 and duplexes contained symmetry left or right halves of OR3 with one base pair deletion or insertion in center of duplex were calculated. The higher binding constant for Cro was detected with 17 bp symmetry duplex consist two left halves of OR3, for the mutant CroV55C higher binding constant was detected with 16 bp derivate of this duplex with the central GC base pair deletion.

Bacteriophage lambda↗

PubMedDate not supplied

[Interaction of trivaline with single-stranded polyribonucleotides].

Binding of tripeptide H-Val3-(NH)2-Dns (TVP) to polyribonucleotides was studied by fluorescence methods, circular and flow linear dichroism, equilibrium dialysis and electron microscopy. It was found that TVP binds to poly(U) in monomer, dimer and tetramer forms with binding constants of about 10(3), 40, 18.10(4) M, respectively. The cooperativity parameter for peptide dimer binding is 2000. The peptide forms tetramer complexes with poly(A), poly(C), poly(G) also. The formation of a complex between the peptide tetramer and nucleic acid is accompanied by a significant increase in the fluorescence intensity. The cooperative binding of TVP dimers to poly(U), poly(A), poly(C) is accompanied by a dramatic decrease in the flexibility of polynucleotide chains. However, it has a small effect (if any) on the flexibility of the poly(G) chain. The observed similarity of thermodynamic, optical and hydrodynamic++ properties of TVP complexes with single-stranded and double-stranded nucleic acids may reflect a similarity in the geometries of peptide complexes with nucleic acids. Electron microscopy studies show that peptide binding to poly(U) and dsDNA leads to compactization of the nucleic acids caused by interaction between the peptide tetramers bound to a nucleic acid. At the first stage of the compactization process the well-organized rod-like particles are formed, each consisting of one or more single-stranded polynucleotide fibers. Increasing the peptide concentration stimulates a side-by-side association and folding of the rods with the formation of macromolecular "leech-like" structures with the thickness of 20-50 nm.

Circular Dichroism↗

PubMedDate not supplied

[Interaction of a cysteine-containing peptide with DNA].

Cystine peptide dimer (Lys-Gly-Val-Cys-Val-N2H2Dns)2 with S-S bridge was synthesized and its interactions with DNA and synthetic polynucleotides have been studied by optical spectroscopy methods. By recording fluorescent titration curves we have shown that the affinity of the peptide to different synthetic polynucleotides decreases in the order: poly(dG).poly(dC) greater than poly(dA).poly(dT) greater than poly(dGC).poly(dGC). The stability of complexes to increasing concentrations of NaCl diminishes in the same order. The association constant is about 20-fold greater for peptide binding to poly(dG).poly(dC) than to poly(dA).poly(dT). By using circular dichroism and fluorescence measurements we have shown that the peptide competes for the binding sites on DNA with two minor-groove binding antibiotics--distamycin A and sybiromycin. These results have suggested that the peptide also binds in the DNA minor groove. Investigation of the interactions between such peptides and DNA may be useful for constructing ligands with combined specificity to DNA.

Amino Acid Sequence↗

PubMedDate not supplied

[Synthetic DNA-bindings ligands containing reaction centers specific for AT- and GC-pairs in DNA].

In the present communication design, synthesis and DNA binding activities of three bis-netropsins and two netropsin analogs containing two N-propylpyrrolecarboxamide fragments linked covalently to peptides Gly-Gly-(analog I) and Val-Val-Val-Gly-Gly-(analog II) are reported. Each bis-netropsin consists of two netropsin-like fragments attached to peptides -Gly-Cys-Gly-NH2 (compound IIIa), H-Gly-Cys-Gly-Gly-Gly-(compound IV) or Gly-Cys-Sar-NH2 (compound IIIb) which are linked symmetrically via S-S bonds. Physico-chemical studies show that each bis-netropsin carries 6 AT-specific reaction centers and covers approximately 10 base pairs upon binding to poly(dA).poly(dT). This indicates that two netropsin-like fragments of the bis-netropsin molecule are implicated in specific interaction with DNA base pairs. The peptide fragments of bis-netropsins IIIa and IV form small beta-sheets containing two-GC-specific reaction centers. The DNase I cleavage patterns of bis-netropsin-DNA complexes visualized by high resolution gel electrophoresis show that the preferred binding sites for bis-netropsins IIIa and IV are identical and contain two runs of three or more AT pairs separated by two GC pairs. Specificity determinants of netropsin analog II binding in the beta-associated dimeric form are identical to those of bis-netropsin IIIa thereby indicating that there is a similarity in the structure of complexes formed by these ligands with DNA. In the monomeric form analog II exhibits binding specificity identical to that of analog I. Replacement of C-terminal glycine residues by sarcosines in the peptide fragments of bis-netropsin IIIa leads to a decrease in the affinity of ligand for DNA.

Base Composition↗