92 Novel interpretation of the isotherms of multimodal ligands binding with DNA

The binding of ligands with DNA is a key moment in a whole range of cellular processes that provide not only the normal cell vital activity but also the development of some pathological processes. Depending on ligand type, structure of DNA adsorption centers, and physical–chemical conditions of the surrounding, the ligand may bind to DNA by several modes [1]. Particularly, adsorption isotherm of multimodal ligands binding to DNA in Scatchard’s coordinates has a concave shape with two brightly expressed linear areas in the region of small fillings. The analysis of such type of adsorption isotherm for determining of important binding parameters such as binding constant and number of adsorption centers (the part of DNA polymer with which one ligand molecule binds) presents difficulties. Practically in all cases, the analysis of such adsorption isotherm is carried out by linear parts of curves. Such analysis mode of experimental points is approximate method, since all registered of experimental points are roughly divided into two groups and they are treated by linear binding isotherm and therefore the binding parameters are determined. In the present work, the non-linear adsorption isotherm in Scatchard‘s coordinates is obtained which allowed, provided, the more precise treatment of all experimental points by unique curve which includes linear regions as well. Such mode of treatment of experimental points makes more precise the determination of not only binding constant and number of adsorption centers that correspond to the one ligand molecule binding, but also additional binding parameter – a proportion of adsorption centers of each binding to DNA type of multimodal ligand.     

Does CTCF mediate between nuclear organization and gene expression?

The multifunctional zinc‐finger protein CCCTC‐binding factor (CTCF) is a very strong candidate for the role of coordinating the expression level of coding sequences with their three‐dimensional position in the nucleus, apparently responding to a “code” in the DNA itself. Dynamic interactions between chromatin fibers in the context of nuclear architecture have been implicated in various aspects of genome functions. However, the molecular basis of these interactions still remains elusive and is a subject of intense debate. Here we discuss the nature of CTCF‐DNA interactions, the CTCF‐binding specificity to its binding sites and the relationship between CTCF and chromatin, and we examine data linking CTCF with gene regulation in the three‐dimensional nuclear space. We discuss why these features render CTCF a very strong candidate for the role and propose a unifying model, the “CTCF code,” explaining the mechanistic basis of how the information encrypted in DNA may be interpreted by CTCF into diverse nuclear functions.     

Structure and function of type II restriction endonucleases

More than 3000 type II restriction endonucleases have been discovered. They recognize short, usually palindromic, sequences of 4-8 bp and, in the presence of Mg(2+), cleave the DNA within or in close proximity to the recognition sequence. The orthodox type II enzymes are homodimers which recognize palindromic sites. Depending on particular features subtypes are classified. All structures of restriction enzymes show a common structural core comprising four beta-strands and one alpha-helix. Furthermore, two families of enzymes can be distinguished which are structurally very similar (EcoRI-like enzymes and EcoRV-like enzymes). Like other DNA binding proteins, restriction enzymes are capable of non-specific DNA binding, which is the prerequisite for efficient target site location by facilitated diffusion. Non-specific binding usually does not involve interactions with the bases but only with the DNA backbone. In contrast, specific binding is characterized by an intimate interplay between direct (interaction with the bases) and indirect (interaction with the backbone) readout. Typically approximately 15-20 hydrogen bonds are formed between a dimeric restriction enzyme and the bases of the recognition sequence, in addition to numerous van der Waals contacts to the bases and hydrogen bonds to the backbone, which may also be water mediated. The recognition process triggers large conformational changes of the enzyme and the DNA, which lead to the activation of the catalytic centers. In many restriction enzymes the catalytic centers, one in each subunit, are represented by the PD. D/EXK motif, in which the two carboxylates are responsible for Mg(2+) binding, the essential cofactor for the great majority of enzymes. The precise mechanism of cleavage has not yet been established for any enzyme, the main uncertainty concerns the number of Mg(2+) ions directly involved in cleavage. Cleavage in the two strands usually occurs in a concerted fashion and leads to inversion of configuration at the phosphorus. The products of the reaction are DNA fragments with a 3'-OH and a 5'-phosphate.     

Detection of oxygen-evolving Photosystem II centers inactive in plastoquinone reduction

Quite different estimates of the number of Photosystem II centers present in thylakoid membranes are obtained depending on the technique used in making the determination. By using brief saturating light flashes and measuring the electron transport per flash, we have obtained two values for the number of functional centers. When the electrons produced reduce the intersystem plastoquinone pool, there are about 1.7 mmol of active Photosystem II centers per mol chlorophyll, whereas there are at least 3 mmol of active centers per mol chlorophyll when certain halogenated benzoquinones are being reduced. There are also at least 3 mmol of terbutryn binding sites per mol of chlorophyll when this tightly binding herbicide is employed as a specific inhibitor of Photosystem II. Thus only about 60% of the membrane's total complement of Photosystem II centers are able to transfer electrons to Photosystem I at appreciable rates. Many functional assays requiring significant rates of turnover sample only this more active pool, whereas herbicide-binding studies and measurements of changes in the Photosystem II electron donor Z and electron acceptor Q_A performed by other investigators reveal, in addition, a large population of Photosystem II reaction centers that normally have negligible turnover numbers. However, these normally inactive centers readily transfer electrons to the halogenated benzoquinones and are then counted among the active centers. Therefore, it can be concluded that all of herbicide-binding sites represent centers with operative water-oxidizing reactions. It can also be concluded that there are few, if any, centers capable of binding more than a single herbicide molecule.     

Studies of the Escherichia coli Trp repressor binding to its five operators and to variant operator sequences.

The Escherichia coli Trp repressor binds to promoters of very different sequence and intrinsic activity. Its mode of binding to trp operator DNA has been studied extensively yet remains highly controversial. In order to examine the selectivity of the protein for DNA, we have used electromobility shift assays (EMSAs) to study its binding to synthetic DNA containing the core sequences of each of its five operators and of operator variants. Our results for DNA containing sequences of two of the operators, trpEDCBA and aroH are similar to those of previous studies. Up to three bands of lower mobility than the free DNA are obtained which are assigned to complexes of stoichiometry 1 : 1, 2 : 1 and 3 : 1 Trp repressor dimer to DNA. The mtr and aroL operators have not been studied previously in vitro. For DNA containing these sequences, we observe predominantly one retarded band in EMSA with mobility corresponding to 2 : 1 complexes. We have also obtained retardation of DNA containing the trpR operator sequence, which has only been previously obtained with super-repressor Trp mutants. This gives bands with mobilities corresponding to 1 : 1 and 2 : 1 complexes. In contrast, DNA containing containing a symmetrized trpR operator sequence, trpRs, gives a single retarded band with mobility corresponding solely to a 1 : 1 protein dimer-DNA complex. Using trpR operator variants, we show that a change in a single base pair in the core 20 base pairs can alter the number of retarded DNA bands in EMSA and the length of the DNase I footprint observed. This shows that the binding of the second dimer is sequence selective. We propose that the broad selectivity of Trp repressor coupled to tandem 2 : 1 binding, which we have observed with all five operator sequences, enables the Trp repressor to bind to a limited number of sites with diverse sequences. This allows it to co-ordinately control promoters of different intrinsic strength. This mechanism may be of importance in a number of promoters that bind multiple effector molecules.     

DNA binding properties of YB-1 and dbpA: binding to double-stranded, single-stranded, and abasic site containing DNAs.

A number of eukaryotic DNA binding proteins have been isolated by screening phage expression libraries with DNA probes containing the binding site of the DNA-binding protein. This methodology was employed here to isolate clones of the factor that interacts with the W box element of the human major histocompatibility complex HLA-DQB gene. Surprisingly, several cDNA clones of YB-1, a cDNA clone that was previously isolated with a CCAAT element-containing sequence were found. Independently, the screening of phage expression libraries with depurinated DNA resulted in the isolation of YB-1 and dbpA, a previously isolated cDNA that has homology to YB-1. Additional characterization of YB-1 showed that it bound a wide variety of DNA sequences and suggested that the binding of this protein is promiscuous. Furthermore, we show that both YB-1 and dbpA bind to depurinated DNA better than undamaged DNA and that the extent of specificity of binding is influenced by Mg2+. Due to the lack of sequence specificity and high degree of binding to depurinated DNA, we suggest that these proteins might be involved in chromosome functions such as maintenance of chromatin structure or DNA repair that do not require sequence-specific binding.     

The complete nucleotide sequence of the Pseudomonas gene coding for carboxypeptidase G2

The complete nucleotide sequence of the Pseudomonas chromosomal gene coding for the enzyme carboxypeptidase G2 (CPG2) has been determined. The nucleotide sequence obtained has been confirmed by comparing the predicted amino acid sequence with that of randomly derived peptide fragments and by N-terminal sequencing of the purified protein. The gene has been shown to code for a 22 amino acid signal peptide at its N-terminus which closely resembles the signal peptides of other secreted proteins. An alternative 36 amino acid signal peptide which may function in Pseudomonas has also been identified. The codon utilisation of the gene is influenced by the high G + C (67.2%) content of the DNA and exhibits a 92.8% preference for codons ending in G or C. This unusual codon preference may contribute to the generally observed weak expression of Pseudomonas genes in Escherichia coli. A region of DNA upstream of the structural gene has also been sequenced and a ribosome binding site and two putative promoter sequences identified.     

High Resolution Structural Studies of Cro Repressor Protein and Implications for DNA Recognition

Abstract

Cro repressor is a small dimeric protein that binds to specific sites on the DNA of bacteriophage λ. The structure of Cro has been determined and suggests that the protein binds to its sequence-specific sites with a pair of two-fold related α-helices of the protein located within successive major grooves of the DNA.

From the known three-dimensional structure of the repressor, model building and energy refinement have been used to develop a detailed model for the presumed complex between Cro and DNA. Recognition of specific DNA binding sites appears to occur via multiple hydrogen bonds between amino acid side chains of the protein and base pair atoms exposed within the major groove of DNA. The Cro:DNA model is consistent with the calculated electrostatic potential energy surface of the protein.

From a series of amino acid sequence and gene sequence comparisons, it appears that a number of other DNA-binding proteins have an α-helical DNA-binding region similar to that seen in Cro. The apparent sequence homology includes not only DNA-binding proteins from different bacteriophages, but also gene-regulatory proteins from bacteria and yeast. It has also been found that the conformations of part of the presumed DNA-binding regions of Cro repressor, λ repressor and CAP gene activator proteins are strikingly similar. Taken together, these results strongly suggest that a two-helical structural unit occurs in the DNA-binding region of many proteins that regulate gene expression. However, the results to date do not suggest that there is a simple one-to-one recognition code between amino acids and bases.

Crystals have been obtained of complexes of Cro with six-base-pair and nine-basepair DNA oligomers, and X-ray analysis of these co-crystals is in progress.     

Identification of a novel HIV-1 TAR RNA bulge binding protein.

The Tat protein binds to TAR RNA to stimulate the expression of the human immunodeficiency virus type 1 (HIV-1) genome. Tat is an 86 amino acid protein that contains a short region of basic residues (aa49-aa57) that are required for RNA binding and TAR is a 59 nucleotide stem-loop with a tripyrimidine bulge in the upper stem. TAR is located at the 5' end of all viral RNAs. In vitro, Tat specifically interacts with TAR by recognising the sequence of the bulge and upper stem, with no requirement for the loop. However, in vivo the loop sequence is critical for activation, implying a requirement for accessory cellular TAR RNA binding factors. A number of TAR binding cellular factors have been identified in cell extracts and various models for the function of these factors have been suggested, including roles as coactivators and inhibitors. We have now identified a novel 38 kD cellular factor that has little general, single-stranded or double-stranded RNA binding activity, but that specifically recognises the bulge and upper stem region of TAR. The protein, referred to as BBP (bulge binding protein), is conserved in mammalian and amphibian cells and in Schizosaccharomyces pombe but is not found in Saccharomyces cerevisiae. BBP is an effective competitive inhibitor of Tat binding to TAR in vitro. Our data suggest that the bulge-stem recognition motif in TAR is used to mediate cellular factor/RNA interactions and indicates that Tat action might be inhibited by such competing reactions in vivo.