Concerted Formation of Macromolecular \u3cem\u3eSuppressor-mutator\u3c/em\u3e Transposition Complexes

Transposition of the maize Suppressor-mutator (Spm) transposon requires two element-encoded proteins, TnpA and TnpD. Although there are multiple TnpA binding sites near each element end, binding of TnpA to DNA is not cooperative, and the binding affinity is not markedly affected by the number of binding sites per DNA fragment. However, intermolecular complexes form cooperatively between DNA fragments with three or more TnpA binding sites. TnpD, itself not a sequence-specific DNA-binding protein, binds to TnpA and stabilizes the TnpA-DNA complex. The high redundancy of TnpA binding sites at both element ends and the protein-protein interactions between DNA-bound TnpA complexes and between these and TnpD imply a concerted transition of the element from a linear to a protein crosslinked transposition complex within a very narrow protein concentration range

Mdm2 binding to a conformationally sensitive domain on p53 can be modulated by RNA

AbstractBiochemical characterisation of the interaction of mdm2 protein with p53 protein has demonstrated that full-length mdm2 does not bind stably to p53–DNA complexes, contrasting with C-terminal truncations of mdm2 which do bind stably to p53–DNA complexes. In addition, tetrameric forms of the p53His175 mutant protein in the PAb1620+ conformation are reduced in binding to mdm2 protein. These data suggest that the mdm2 binding site in the BOX-I domain of p53 becomes concealed when either p53 binds to DNA or when the core domain of p53 is unfolded by missense mutation. This further suggests that the C-terminus of mdm2 protein contains a negative regulatory domain that affects mdm2 protein binding to a second, conformationally sensitive interaction site in the core domain of p53. We investigated whether there was a second docking site on p53 for mdm2 protein by examining the interaction of full-length mdm2 with p53 lacking the BOX-I domain. Although mdm2 protein did bind very weakly to p53 protein lacking the BOX-I domain, addition of RNA activated mdm2 protein binding to this truncated form of p53. These data provide evidence for three previously undefined regulatory stages in the p53–mdm2 binding reaction: (1) conformational changes in p53 protein due to DNA binding or point mutation conceals a secondary docking site of mdm2 protein; (2) the C-terminus of mdm2 is the primary determinant which confers this property upon mdm2 protein; and (3) mdm2 protein binding to this secondary interaction site within p53 can be stabilised by RNA

Protein-RNA interactions: a structural analysis

A detailed computational analysis of 32 protein-RNA complexes is presented. A number of physical and chemical properties of the intermolecular interfaces are calculated and compared with those observed in protein-double-stranded DNA and protein-single-stranded DNA complexes. The interface properties of the protein-RNA complexes reveal the diverse nature of the binding sites. van der Waals contacts played a more prevalent role than hydrogen bond contacts, and preferential binding to guanine and uracil was observed. The positively charged residue, arginine, and the single aromatic residues, phenylalanine and tyrosine, all played key roles in the RNA binding sites. A comparison between protein-RNA and protein-DNA complexes showed that whilst base and backbone contacts (both hydrogen bonding and van der Waals) were observed with equal frequency in the protein-RNA complexes, backbone contacts were more dominant in the protein-DNA complexes. Although similar modes of secondary structure interactions have been observed in RNA and DNA binding proteins, the current analysis emphasises the differences that exist between the two types of nucleic acid binding protein at the atomic contact level

From Nonspecific DNA–Protein Encounter Complexes to the Prediction of DNA–Protein Interactions

©2009 Gao, Skolnick. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.doi:10.1371/journal.pcbi.1000341DNA–protein interactions are involved in many essential biological activities. Because there is no simple mapping code between DNA base pairs and protein amino acids, the prediction of DNA–protein interactions is a challenging problem. Here, we present a novel computational approach for predicting DNA-binding protein residues and DNA–protein interaction modes without knowing its specific DNA target sequence. Given the structure of a DNA-binding protein, the method first generates an ensemble of complex structures obtained by rigid-body docking with a nonspecific canonical B-DNA. Representative models are subsequently selected through clustering and ranking by their DNA–protein interfacial energy. Analysis of these encounter complex models suggests that the recognition sites for specific DNA binding are usually favorable interaction sites for the nonspecific DNA probe and that nonspecific DNA–protein interaction modes exhibit some similarity to specific DNA–protein binding modes. Although the method requires as input the knowledge that the protein binds DNA, in benchmark tests, it achieves better performance in identifying DNA-binding sites than three previously established methods, which are based on sophisticated machine-learning techniques. We further apply our method to protein structures predicted through modeling and demonstrate that our method performs satisfactorily on protein models whose root-mean-square Ca deviation from native is up to 5 Å from their native structures. This study provides valuable structural insights into how a specific DNA-binding protein interacts with a nonspecific DNA sequence. The similarity between the specific DNA–protein interaction mode and nonspecific interaction modes may reflect an important sampling step in search of its specific DNA targets by a DNA-binding protein

Dynamics of \u3cem\u3eE. Coli\u3c/em\u3e Single Stranded DNA Binding (SSB) Protein-DNA Complexes

Single stranded DNA binding proteins (SSB) are essential to the cell as they stabilize transiently open single stranded DNA (ssDNA) intermediates, recruit appropriate DNA metabolism proteins, and coordinate fundamental processes such as replication, repair and recombination. Escherichia coli single stranded DNA binding protein (EcSSB) has long served as the prototype for the study of SSB function. The structure, functions, and DNA binding properties of EcSSB are well established: The protein is a stable homotetramer with each subunit possessing an N-terminal DNA binding core, a C-terminal protein-protein interaction tail, and an intervening intrinsically disordered linker (IDL). EcSSB wraps ssDNA in multiple DNA binding modes and can diffuse along DNA to remove secondary structures and remodel other protein-DNA complexes. This review provides an update on these features based on recent findings, with special emphasis on the functional and mechanistic relevance of the IDL and DNA binding modes

Nucleic acid - protein fingerprints. Novel protein classification based on nucleic acid - protein recognition

Protein chemistry uses protein description and classification based on molecular mass and isoelectric point as general features. Enzymes are also compared by enzymatic reaction constants, namely Km and kcat values. Proteins are also studied by binding to different oligonucleotides. Here we suggest a simple experimental method for such a comparison of DNA binding proteins, which we call "nucleic acid-protein fingerprints". The experimental design of the method is based on an use of short oligonucleotides immobilized inside microarray of hydrogel cells - biochip. As a first stage, we solved a simple experimental task: what is the shortest single strand oligonucleotide to be recognized by protein? We tested binding of oligonucleotides from 2 to 12 bases, and we have obtained unexpected result that tetranucleotide one is long enough for specific protein binding. This 4-mer can contain two universal bases - 5-nitroindole nucleoside analogue (Ni) and only two meaningful bases, like A, G, T and C. The result obtained opens a way for constructing the simplest protein binding microarray. This microarray consists of 16 meaningful dinucleotides, like AA, AG, CT, GG etc. Physical sequences of all the nucleotides were NiNiAA, etc, where Ni is bound to gel through the amino linker. We prepared such an array and tested it for specific binding of several DNA/RNA binding proteins, labeled with fluorescent dyes like Texas Red of Bodipy. We tested RNase A and Binase for binding on the simplest microarray. It contains only 16 units, and there is a significant difference in the binding patterns. The microarray based on 3-mers must contains 64 units and must have much more specificity. The new principle of protein classification based on nucleic acid-protein recognition has been proposed and experimentally proved. Such an experimental approach must lead to a universal classification of specific DNA/RNA binding proteins