DOMPLOT: a program to generate schematic diagrams of the structural domain organization within proteins, annotated by ligand contacts.

A program is described for automatically generating schematic linear representations of protein chains in terms of their structural domains. The program requires the co-ordinates of the chain, the domain assignment, PROSITE information and a file listing all intermolecular interactions in the protein structure. The output is a PostScript file in which each protein is represented by a set of linked boxes, each box corresponding to all or part of a structural domain. PROSITE motifs and residues involved in ligand interactions are highlighted. The diagrams allow immediate visualization of the domain arrangement within a protein chain, and by providing information on sequence motifs, and metal ion, ligand and DNA binding at the domain level, the program facilitates detection of remote evolutionary relationships between proteins.     

HERA--a program to draw schematic diagrams of protein secondary structures

A program is described which generates hydrogen bonding diagrams of protein structures and optionally helical wheels and helical nets. The program can also beta-strands beta-strands and to automatically extract simple structural motifs such as hairpins or Greek keys. The program greatly reduces the effort required to produce these diagrams and offers considerable flexibility in the information which can be represented. The usefulness of the program is illustrated by several examples including comparing homologous families, correlating protein structure with attributes of individual residues, and extracting all examples of the psi-loop motif from the Brookhaven Data Bank.     

Methylphosphonates as probes of protein-nucleic acid interactions.

Deoxydinucleoside methylphosphonates were prepared by chemical synthesis and were introduced stereospecifically into the lac operator at two sites. These sites within d(ApApTpTpGpTpGpApGpCpGpGpApTpApApCpApApTpT), segment I, and d(ApApTpTpGpTpTpApTpCpCpGpCpTpCpApCpApApTpT), segment II, are indicated by p. Each segment containing a chiral methylphosphonate was annealed to the complementary unmodified segment. The interactions of these four modified lac operators with lac repressor were analyzed by the nitrocellulose filter binding assay. Introduction of either chiral phosphonate in segment II had little effect on the stability of the repressor-operator complex. When methylphosphonates were introduced into segment I, the affinity of lac repressor for the modified operators was shown to be dependent on the stereochemical configuration of the methylphosphonate.     

Recognition schemes for protein-nucleic acid interactions

The molecular forces involved in protein-nucleic acid interaction are electrostatic, stacking and hydrogen-bonding. These interactions have a certain amount of specificity due to the directional nature of such interactions and the spatial contributions of the steric effects of different substituent groups. Quantum chemical calculations on these interactions have been reported which clearly bring out such features. While the binding energies for electrostatic interactions are an order of magnitude higher, the differences in interaction energies for structures stabilised by hydrogen-bonding and stacking are relatively small. Thus, the molecular interactions alone cannot explain the highly specific nature of binding observed in certain segments of proteins and nucleic acids. It is therefore logical to assume that the sequence dependent three dimensional structures of these molecules help to place the functional groups in the correct geometry for a favourable interaction between the two molecules. We have carried out 2D-FT nuclear magnetic resonance studies on the oligonucleotide d-GGATCCGGATCC. This oligonucleotide sequence has two binding sites for the restriction enzyme Bam H1. Our studies indicate that the conformation of this DNA fragment is predominantly B-type except near the binding sites where the ribose ring prefers a³E conformation. This interesting finding raises the general question about the presence of specificity in the inherent backbone structures of proteins and nucleic acids as opposed to specific intermolecular interactions which may induce conformational changes to facilitate such binding.     

Quantitative approaches to monitor protein-nucleic acid interactions using fluorescent probes

Sequence-specific recognition of nucleic acids by proteins is required for nearly every aspect of gene expression. Quantitative binding experiments are a useful tool to measure the ability of a protein to distinguish between multiple sequences. Here, we describe the use of fluorophore-labeled oligonucleotide probes to quantitatively monitor protein/nucleic acid interactions. We review two complementary experimental methods, fluorescence polarization and fluorescence electrophoretic mobility shift assays, that enable the quantitative measurement of binding affinity. We also present two strategies for post-synthetic end-labeling of DNA or RNA oligonucleotides with fluorescent dyes. The approaches discussed here are efficient and sensitive, providing a safe and accessible alternative to the more commonly used radio-isotopic methods.     

A new electron microscopic method for studying protein-nucleic acid interactions.

A new method for preparation of nucleic acid specimens for electron microscopy has been adapted to study the interaction of proteins with DNA. Both a detergent and a basic protein are added to the DNA-protein solution before spreading on a hypophase containing 0.2 m ammonium acetate. This method has been tested using T7 DNA and Escherichia coli RNA polymerase. Specifically bound enzyme molecules were clearly visible on the well extended DNA molecules; the binding sites were located at 0.59, 1.24, 1.57, and 1.86% of the total length of T7 DNA. Under carefully controlled conditions, 40–85% of the DNA molecules specifically bound at least one enzyme molecule.     

Roles of intrinsic disorder in protein-nucleic acid interactions

Interactions between proteins and nucleic acids typify the role of disordered segments, linkers, tails and other entities in the function of complexes that must form with high affinity and specificity but which must be capable of dissociating when no longer needed. While much of the emphasis in the literature has been on the interactions of disordered proteins with other proteins, disorder is also frequently observed in nucleic acids (particularly RNA) and in the proteins that interact with them. The interactions of disordered proteins with DNA most often manifest as molding of the protein onto the B-form DNA structure, although some well-known instances involve remodeling of the DNA structure that seems to require that the interacting proteins be disordered to various extents in the free state. By contrast, induced fit in RNA-protein interactions has been recognized for many years-the existence and prevalence of this phenomenon provides the clearest possible evidence that RNA and its interactions with proteins must be considered as highly dynamic, and the dynamic nature of RNA and its multiplicity of folded and unfolded states is an integral part of its nature and function.     

Characterization of bacteriophage T4 regA protein-nucleic acid interactions

The bacteriophage T4 regA protein is a translational repressor of a group of T4 early mRNAs. We have characterized the binding of regA protein to polynucleotides and to specific RNAs. Binding to nucleic acids was monitored by the quenching of the intrinsic tryptophan fluorescence of regA protein. regA protein exhibited differential affinities for the polynucleotides examined, with the order of affinity being poly(rU) greater than poly(dT) greater than poly(dU) = poly(rG) greater than poly(rC) = poly(rA). The binding site size calculated for regA protein binding to poly(rU) was n = 9 +/- 1 nucleotides. Cooperativity was observed in binding to multiple-site oligonucleotides, with a cooperativity parameter (omega) value of 10-22. To study the specific interaction between regA protein and T4 gene 44 mRNA, the affinity of regA protein for synthetic gene 44 RNA fragments was measured. The association constant (Ka) for regA protein binding to gene 44 RNA fragments was 100-fold higher than for binding to nontarget RNA. Study of variant gene 44 RNA fragments indicated that the nucleotides required for specific binding are contained within a 12-nucleotide sequence spanning -12 to -1, relative to the AUG codon. The bases of five nucleotides (indicated in upper case type) are critical for specific regA protein interaction with the gene 44 recognition element, 5'-aaUGAGgAaauu-3'. These studies further showed that formation of a regA protein-RNA complex involves a maximum of 2-3 ionic interactions and is primarily an enthalpy-driven process.     

Thermodynamic database for protein-nucleic acid interactions (ProNIT).

MOTIVATION: Protein-nucleic acid interactions are fundamental to the regulation of gene expression. In order to elucidate the molecular mechanism of protein-nucleic acid recognition and analyze the gene regulation network, not only structural data but also quantitative binding data are necessary. Although there are structural databases for proteins and nucleic acids, there exists no database for their experimental binding data. Thus, we have developed a Thermodynamic Database for Protein-Nucleic Acid Interactions (ProNIT). RESULTS: We have collected experimentally observed binding data from the literature. ProNIT contains several important thermodynamic data for protein-nucleic acid binding, such as dissociation constant (K(d)), association constant (K(a)), Gibbs free energy change (DeltaG), enthalpy change (DeltaH), heat capacity change (DeltaC(p)), experimental conditions, structural information of proteins, nucleic acids and the complex, and literature information. These data are integrated into a relational database system together with structural and functional information to provide flexible searching facilities by using combinations of various terms and parameters. A www interface allows users to search for data based on various conditions, with different display and sorting options, and to visualize molecular structures and their interactions. AVAILABILITY: ProNIT is freely accessible at the URL http://www.rtc.riken.go.jp/jouhou/pronit/pronit.html.     

Thermodynamic databases for proteins and protein-nucleic acid interactions

Thermodynamic data regarding proteins and their interactions are important for understanding the mechanisms of protein folding, protein stability, and molecular recognition. Although there are several structural databases available for proteins and their complexes with other molecules, databases for experimental thermodynamic data on protein stability and interactions are rather scarce. Thus, we have developed two electronically accessible thermodynamic databases. ProTherm, Thermodynamic Database for Proteins and Mutants, contains numerical data of several thermodynamic parameters of protein stability, experimental methods and conditions, along with structural, functional, and literature information. ProNIT, Thermodynamic Database for Protein-Nucleic Acid Interactions, contains thermodynamic data for protein-nucleic acid binding, experimental conditions, structural information of proteins, nucleic acids and the complex, and literature information. These data have been incorporated into 3DinSight, an integrated database for structure, function, and properties of biomolecules. A WWW interface allows users to search for data based on various conditions, with different display and sorting options, and to visualize molecular structures and their interactions. These thermodynamic databases, together with structural databases, help researchers gain insight into the relationship among structure, function, and thermodynamics of proteins and their interactions, and will become useful resources for studying proteins in the postgenomic era.     

Analysis of ion concentration effects on the kinetics of protein-nucleic acid interactions: Application to lac repressor-operator interactions

The effects of monovalent and divalent cations on the bimolecular rate constant of the reaction of a positively charged ligand with a nucleic acid polyanion are analyzed for two possible reaction mechanisms. One mechanism postulates that the association reaction occurs without intermediates, and that ion effects on the rate constant result entirely from the screening of the charged reactants by ionic atmospheres of low molecular weight ions (a screening-controlled mechanism). This mechanism is analyzed by analogy with the Bronsted-Bjerrum theory for the kinetics of interaction of low molecular weight ions. The second mechanism to be considered here postulates the existence of a ligand-DNA intermediate which is in rapid equilibrium with the reactants (pre-equilibrium mechanism). Ion concentration effects on the association rate constants for the pre-equBibrium mechanism result mainly from the release of counterions from the DNA upon formation of the intermediate. Both of the above mechanisms predict that the logarithm of the association rate constant,_a, will be a linear function of the logarithm of the monovalent cation concentration, [M+] (in the absence of competition by divalent cations or anions). Knowledge of the salt dependences of K_a and of the observed equilibrium constant k_obs of the ligand-nucleic acid interaction should usually be sufficient to determine whether a screening-controlled mechanism or a pre-equilibrium mechanism is suitable to describe the process. If the association reaction can be described by a pre-equilibrium mechanism, the number of ionic interactions involved in the ligand-nucleic acid intermediate can be estimated. This analysis, extended to include the effects of divalent cations on screening or on the pre-equilibrium step, is applied to literature data on the salt dependence of the kinetics of the interaction of lac represser with lac operator DNA. When the operator is present on bacteriophage λ DNA, the observed reaction kinetics are consistent with the formation of an intermediate repressor-DNA complex in a pre-equilibrium step. On the other hand, the kinetics of association of toe represser with synthetic foe operator fragments may be an example of a screening-controlled reaction.     

Alpha-S-cysteinylthymine: a model for protein-nucleic acid cross-linking.

Crystals of alpha-S-cysteinylthymine, C8H12CIN3O4S, formula weight 281.72, are orthorhombic, space group P212121, with a=9.499 (1), b=24.072 (4), and c=5.012 (1) A, V=1146.1 (2) A3, and Z=4. The structure was determined by the direct method and refined by a full-matrix least-squares procedure to a final residual, R=0.043, using 1277 diffractometer data. From the structure, a three-dimensional model for the radiation-induced interaction of thymine residues and cysteine residues could be postulated.     

Kinetics of protein-nucleic acid interactions: use of salt effects to probe mechanisms of interaction

The kinetics of protein-nucleic acid interactions are discussed with particular emphasis on the effects of salt concentration and valence on the observed rate constants. A general review is given of the use of experimentally determined salt dependences of observed kinetic parameters as a tool to probe the mechanism of interaction. Quantitative analysis of these salt dependences, through the application of polyelectrolyte theory, can be used to distinguish reactions which occur in a single step from those reactions which involve distinct intermediates. For those rate constants which display a large salt dependence, in either the association or dissociation reaction, this is due to the high concentration of counterions (e.g., Na+) in the vicinity of the nucleic acid which are subsequently released (or bound in the case of dissociation) at some point before the rate limiting step of the reaction. A general discussion of other features which affect protein-nucleic acid kinetics, such as nucleic acid length and the ratio of nonspecific to specific DNA binding sites (in the case of sequence specific binding proteins), is also given. The available data on the nucleic acid binding kinetics of small ligands (ions, dyes, oligopeptides), nonspecific binding proteins (T4 gene 32 protein, fd gene 5 and Escherichia coli SSB), and sequence specific binding proteins (lac repressor, RNA polymerase, Eco RI restriction endonuclease) are discussed with emphasis on the interpretation of the experimentally determined salt dependences.     

Use of difference boundary sedimentation velocity to investigate nonspecific protein-nucleic acid interactions

The difference boundary sedimentation velocity technique of Schachman and co-workers is demonstrated to be applicalbe to the measurement of binding constants (Kobsd) in the range 10(2)-10(5) M(-1) for the nonspecific interactions of proteins with DNA. The difference technique can reproducibly detect a 2% change in the sedimentation coefficient of the DNA upon binding ligands, corresponding to average extents of association as low as 10 molecules of protein (in the cases of Escherichia coli lac repressor and E. coli RNA polymerase) per molecule of bacteriophage T7 DNA. At these low binding densities, it is plausible to assume that the primary effect of ligand binding is on the buoyant mass of the complex and not on the frictional coefficient of the flexible DNA coil. Binding constants calculated by using this assumption agree well with literature values for the nonspecific interactions of RNase and lac repressor proteins with double-stranded DNA. Advantages of the method are that it is relatively rapid, requires the optical detection of the DNA only, and can be performed on small amounts of sample. The method appears useful for surveying (to an accuracy of +/-50% in Kobsd or +/-10% in log Kobsd) the effects of solution variables on Kobsd of protein-DNA interactions. Applications of the method to the nonspecific interactions of RNA polymerase core and holoenzymes with T7 DNA are discussed.     

Theoretical studies on protein-nucleic acid interactions. I. Interaction of positively charged amino acids with nucleic acid fragments

Coulombic interactions between the side chains of charged amino acids (Arg⁺, Lys⁺, and His⁺) and negatively charged phosphate groups of nucleic acid fragments have been studied theoretically. Diribose monophosphate and dideoxyribose monophosphate are chosen as model systems for single-stranded RNA and DNA, respectively. The interaction energies have been calculated by second-order perturbation theory using simplified formulas for individual terms. The interaction energy in this formalism is a sum of electrostatic, polarization, dispersion, and repulsive energies. Our results show that about 90% of the total interaction energy is contributed by the electrostatic term alone. Contribution from the repulsive term exceeds that from the dispersion term. Calculated interaction energies suggest that Lys⁺ and His⁺ form more stable complexes with RNA than with single-stranded DNA. On the other hand, Arg⁺ has a higher affinity for DNA than for RNA. The affinity of nucleic acids for the three amino acids is in the order Lys⁺ > His⁺ > Arg⁺. Further, the basic amino acid residues form more stable complexes with A-DNA than with B-DNA. The role of the Coulombic interactions in the specific recognition of nucleic acids by proteins is discussed.     

Use of an ALFexpress DNA sequencer to analyze protein-nucleic acid interactions by band shift assay

Gel retardation analysis, or band shift assay, is technically the simplest method to investigate protein-nucleic acid interactions. In this report, we describe a nonradioactive band shift assay using a fluorescent DNA target and an ALFexpress automatic DNA sequencer in place of the current method that utilizes radioactively end-labeled DNA target and a standard electrophoresis unit. In our study, the dsDNA targets were obtained by annealing two synthetic oligonucleotides or by PCR. In both cases, a molecule of indodicarbocyanine (CY5) was attached at the 5' OH end of one of the two synthetic oligonucleotides, with a ratio of one molecule of fluorescent dye per molecule of dsDNA. To demonstrate the feasibility of this new band shift assay method, the DNA-binding proteins selected as models were the BlaI and AmpR repressors, which are involved in the induction of the Bacillus licheniformis 749/I and Citrobacter freundii beta-lactamases, respectively. The results show that the use of an automatic DNA sequencer allows easy gel retardation analysis and provides a fast, sensitive, and quantitative method. The ALFexpress DNA sequencer has the same limit of detection as a laser fluorescence scanner and can be used instead of a FluorImager or a Molecular Imager.     

LINKER: a program to generate linker sequences for fusion proteins

The construction of functional fusion proteins often requires a linker sequence that adopts an extended conformation to allow for maximal flexibility. Linker sequences are generally selected based on intuition. Without a reliable selection criterion, the design of such linkers is often difficult, particularly in situations where longer linker sequences are required. Here we describe a program called LINKER which can automatically generate a set of linker sequences that are known to adopt extended conformations as determined by X-ray crystallography and NMR. The only required input to the program is the desired linker sequence length. The program is specifically designed to assist in fusion protein construction. A number of optional input parameters have been incorporated so that users are able to enhance sequence selection based on specific applications. The program output simply contains a set of sequences with a specified length. This program should be a useful tool in both the biotechnology industry and biomedical research. It can be accessed through the Web page http://www.fccc. edu/research/labs/feng/linker.html.     

Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions

The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this protocol, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided.