The dimeric form of the unphosphorylated response regulator BaeR

Bacterial response regulators (RRs) can regulate the expression of genes that confer antibiotic resistance; they contain a receiver and an effector domain and their ability to bind DNA is based on the dimerization state. This is triggered by phosphorylation of the receiver domain by a kinase. However, even in the absence of phosphorylation RRs can exist in equilibrium between monomers and dimers with phosphorylation shifting the equilibrium toward the dimer form. We have determined the crystal structure of the unphosphorylated dimeric BaeR from Escherichia coli. The dimer interface is formed by a domain swap at the receiver domain. In comparison with the unphosphorylated dimeric PhoP from Mycobacterium tuberculosis, BaeR displays an asymmetry of the effector domains.     

Molecular structure of the lampbrush loops nooses of the Y chromosome of Drosophila hydei

The molecular structure of the lampbrush loopforming fertility gene nooses from the short arm of the Y chromosome of Drosophila hydei is described on the basis of cloned DNA sequences which are characteristic for the sequence organization in the lampbrush loop. Y chromosomal lampbrush loops are organized into tandem repeat clusters of loop-specific repetitive DNA sequences and in interspersed repetitive DNA sequences with homologies elsewhere in the genome. In this paper, the basic properties of a repeat unit of the tandemly repeated sequence family ay1 are described. Moreover, it is shown that a loop contains several different domains carrying repeat clusters of the same repeated DNA family but with divergent sequence character. One of these clusters is characterized by an internal duplication of the basic repeat unit. We propose that the tandem repeat DNA family ay1 forms a frame of the lampbrush loop which is required for structural and functional reasons.     

A common switch in activation of the response regulators NtrC and PhoB: phosphorylation induces dimerization of the receiver modules.

During signal transduction, response regulators of two-component systems are phosphorylated in a conserved receiver module. Phosphorylation induces activation of the non-conserved output domain. We fused various domains of the response regulators NtrC, PhoB or CheB to the DNA binding domain of lambda repressor. Analysis of these hybrid proteins shows that the receiver modules of NtrC and PhoB are potential dimerization domains. In the unphosphorylated proteins, the ability of the receiver modules to dimerize is masked due to inhibition by their output domains. Inhibition can be relieved in two ways: phosphorylation of the receiver module or deletion of the output domain. In contrast, the receiver module of CheB lacks this ability for dimerization. We propose a model which groups response regulators into two classes. Common to both classes is the interaction between receiver and output domain in the unphosphorylated protein. In class I (e.g. NtrC and PhoB), this interaction leads to the inhibition of the receiver module. Phosphorylation relieves inhibition, thereby inducing activation via dimerization of the receiver modules. In class II (e.g. CheB), the interaction between receiver and output domain results in inhibition of the output domain. Phosphorylation relieves inhibition, thereby activating the output domain.     

Diversity of structure and function of DNA polymerase (gp43) of T4-related bacteriophages

The replication DNA polymerase (gp43) of the bacteriophage T4 is a member of the pol B family of DNA polymerases, which are found in all divisions of life in the biosphere. The enzyme is a modularly organized protein that has several activities in one polypeptide chain (900 amino acid residues). These include two catalytic functions, POL (polymerase) and EXO (3-exonuclease), and specific binding activities to DNA, the mRNA for gp43, deoxyribonucleotides (dNTPs), and other T4 replication proteins. The gene for this multifunctional enzyme (gene 43) has been preserved in evolution of the diverse group of T4-like phages in nature, but has diverged in sequence, organization, and specificity of the binding functions of the gene product. We describe here examples of T4-like phages where DNA rearrangements have created split forms of gene 43 consisting of two cistrons instead of one. These gene 43 variants specify separate gp43A (N-terminal) and gp43B (C-terminal) subunits of a split form of gp43. Compared to the monocistronic form, the interruption in contiguity of the gene 43 reading frame maps in a highly diverged sequence separating the code for essential components of two major modules of this pol B enzyme, the FINGERS and PALM domains, which contain the dNTP binding pocket and POL catalytic residues of the enzyme. We discuss the biological implications of these gp43 splits and compare them to other types of pol B splits in nature. Our studies suggest that DNA mobile elements may allow genetic information for pol B modules to be exchanged between organisms.Translated from Biokhimiya, Vol. 69, No. 11, 2004, pp. 1489–1496.Original Russian Text Copyright © 2004 by Petrov, Karam.     

BRCT domains: Easy as one,two, three

BRCA1 C-terminal (BRCT) domains are integral signaling modules in the DNA damage response (DDR). Aside from their established roles as phospho-peptide binding modules, BRCT domains have been implicated in phosphorylation-independent protein interactions, DNA binding and poly(ADP-ribose) (PAR) binding. These numerous functions can be attributed to the diversity in BRCT domain structure and architecture, where domains can exist as isolated single domains or assemble into higher order homo- or hetero-domain complexes. In this review, we incorporate recent structural and biochemical studies to demonstrate how structural features allow single and tandem BRCT domains to attain a high degree of functional diversity.Key words: BRCT domain, DNA repair, phosphorylation, phospho-peptide interaction, protein interaction, DNA binding, DNA damage response     

Members of the IclR family of bacterial transcriptional regulators function as activators and/or repressors

Members of the IclR family of regulators are proteins with around 250 residues. The IclR family is best defined by a profile covering the effector binding domain. This is supported by structural data and by a number of mutants showing that effector specificity lies within a pocket in the C-terminal domain. These regulators have a helix-turn-helix DNA binding motif in the N-terminal domain and bind target promoters as dimers or as a dimer of dimers. This family comprises regulators acting as repressors, activators and proteins with a dual role. Members of the IclR family control genes whose products are involved in the glyoxylate shunt in Enterobacteriaceae , multidrug resistance, degradation of aromatics, inactivation of quorum-sensing signals, determinants of plant pathogenicity and sporulation. No clear consensus exists on the architecture of DNA binding sites for IclR activators: the MhpR binding site is formed by a 15-bp palindrome, but the binding sites of PcaU and PobR are three perfect 10-bp sequence repetitions forming an inverted and a direct repeat. IclR-type positive regulators bind their promoter DNA in the absence of effector. The mechanism of repression differs among IclR-type regulators. In most of them the binding sites of RNA polymerase and the repressor overlap, so that the repressor occludes RNA polymerase binding. In other cases the repressor binding site is distal to the RNA polymerase, so that the repressor destabilizes the open complex.     

Purification and some properties of presumptive tof gene product of Coli phage 434.

Summary The presumptive tof gene product of Coli phage 434 has been purified from cells carrying imm–⁴³⁴ cIdv plasmid known to contain only some of the early genes of phage 434 and . It was detected and tentatively identified as tof protein primarily by its ability to specifically bind to phage 434 DNA. The protein has a molecular weight of about 11,000 and requires Mg²⁺ for specific DNA binding, unlike 434 cI-repressor.     

DNA binding domains in Tn3 transposase

Summary Various segments of Tn3 transposase were fused individually to -galactosidase, and the resulting fusion proteins were examined for their DNA binding ability by a nitrocellulose filter binding assay. Analyses of a series of the fusion proteins revealed that the N-terminal segment of the transposase (amino acid positions 1–242; the transposase gene encodes 1004 residues in all) had specific DNA binding ability for the 38 bp terminal inverted repeat (IR) sequence, and the central segment (amino acid positions 243–632) had non-specific DNA binding ability. Further analyses of each of the two regions revealed that the N-terminal segment could be divided into at least two subsegments (amino acid positions 1–86 and 87–242), neither of which had specific DNA binding ability, but which both possessed nonspecific DNA binding ability. The central segment included two subsegments (amino acid positions 243–289 and 439–505) with non-specific DNA binding ability. These results and other observations suggest that Tn3 transposase has several domains including those responsible for non-specific DNA binding, and a combination of two or more domains gives rise to specific DNA binding activity. The C-terminal segment of the transposase (amino acid positions 633-1004), which is very well conserved among transposases encoded by Tn3 family transposons, had no DNA binding ability. This segment may represent the main part of the catalytic domain responsible for the initiation step of transposition.     

Identifying Regulators for EAG1 Channels with a Novel Electrophysiology and Tryptophan Fluorescence Based Screen

Background

Ether-à-go-go (EAG) channels are expressed throughout the central nervous system and are also crucial regulators of cell cycle and tumor progression. The large intracellular amino- and carboxy- terminal domains of EAG1 each share similarity with known ligand binding motifs in other proteins, yet EAG1 channels have no known regulatory ligands.

Methodology/Principal Findings

Here we screened a library of small biologically relevant molecules against EAG1 channels with a novel two-pronged screen to identify channel regulators. In one arm of the screen we used electrophysiology to assess the functional effects of the library compounds on full-length EAG1 channels. In an orthogonal arm, we used tryptophan fluorescence to screen for binding of the library compounds to the isolated C-terminal region.

Conclusions/Significance

Several compounds from the flavonoid, indole and benzofuran chemical families emerged as binding partners and/or regulators of EAG1 channels. The two-prong screen can aid ligand and drug discovery for ligand-binding domains of other ion channels.     

Genomic organization of IFI16, an interferon-inducible gene whose expression is associated with human myeloid cell differentiation: correlation of predicted protein domains with exon organization

The human IFI16 gene is a member of an interferon-inducible family of mouse and human genes closely linked on syntenic regions of chromosome 1. Expression of these genes is largely restricted to hemopoietic cells, and is associated with the differentiation of cells of the myeloid lineages. As a prelude to defining the mechanisms governing IFI16 expression, we have deduced its genomic organization using a combination of genomic cloning and polymerase chain reaction amplification of genomic DNA. IFI16 consists of ten exons and nine intervening introns spanning at least 28 kilobases (kb) of DNA. The reiterated domain structure of IFI16 protein is closely reflected in its intron/exon boundaries, and may represent the evolutionary fusion of several independent functional domains. Thus, exon 1 consists of 5' untranslated (UT) sequences and contains sequence motifs that may confer interferon-inducibility, and exon 2 encodes the lysine-rich amino-terminal (K) region, which possesses DNA-binding activity. Exon 3 codes for a domain which is poorly conserved between family members, except for a strongly retained basic motif likely to provide nuclear localization. The first of two 200 amino acid repeat domains that are the hallmark of this family (domain A) is represented jointly on exons 4 and 5, which are reiterated as exons 8 and 9, respectively, to encode the second 200 amino acid domain (B). Two intervening serine-threonine-rich domains (C and C'), unique to IFI16, are each encoded by single exons of identical length (exons 5 and 6). These domains are predicted to encode semi-rigid spacer domains between the 200 amino acid repeats. The reiterated nature of exons 4 to 6 and the insertion of introns into a single reading frame strongly suggest that IFI16 and related genes arose by a series of exon duplications, some of which antedated speciation into mouse and humans. Several alternative mRNA cap sites downstream of a TATA consensus sequence were defined, using primer extension analysis of mRNA. Sequencing of ~1.7 kb of DNA upstream of this region revealed no recognizable consensus elements for induction by interferon- (interferon-/ß-stimulated response elements), but two motifs resembling interferon- activation sites were located. IFNs and both induce IFI16 mRNA expression in myeloid cells. Interferon- inducibility of IFI16 may be regulated by an interferon-/ß-stimulated response consensus element in the 5' UT exon, as a similar motif is conserved in the corresponding position in the related myeloid cell nuclear differentiation antigen gene. An interferon--activation site consensus was also located in this region of IFI16.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M63838     

Modeling of Nucleotide Binding Domains of ABC Transporter Proteins Based on a F1-ATPase/recA Topology: Structural Model of the Nucleotide Binding Domains of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

Members of the ABC transporter superfamily contain two nucleotide binding domains. To date, the three dimensional structure of no member of this super-family has been elucidated. To gain structural insight, the known structures of several other nucleotides binding proteins can be used as a framework for modeling these domains. We have modeled both nucleotide binding domains of the protein CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) using the two similar domains of mitochondrial F₁-ATPase. The models obtained, provide useful insights into the putative functions of these domains and their possible interaction as well as a rationale for the basis of Cystic Fibrosis causing mutations. First, the two nucleotide binding domains (folds) of CFTR are each predicted to span a 240–250 amino acid sequence rather than the 150–160 amino acid sequence originally proposed. Second, the first nucleotide binding fold, is predicted to catalyze significant rates of ATP hydrolysis as a catalytic base (E504) resides near the phosphate of ATP. This prediction has been verified experimentally [Ko, Y.H., and Pedersen, P.L. (1995) J. Biol. Chem. 268, 24330-24338], providing support for the model. In contrast, the second nucleotide binding fold is predicted at best to be a weak ATPase as the glutamic acid residue is replaced with a glutamine. Third, F508, which when deleted causes 70% of all cases of cystic fibrosis, is predicted to lie in a cleft near the nucleotide binding pocket. All other disease causing mutations within the two nucleotide binding domains of CFTR either reside near the Walker A and Walker B consensus motifs in the heart of the nucleotide binding pocket, or in the C motif which lies outside but near the nucleotide binding pocket. Finally, the two nucleotide binding domains of CFTR are predicted to interact, and in one of the two predicted orientations, F508 resides near the interface.This is the first report where both nucleotide binding domains of an ABC transporter and their putative domain-domain interactions have been modeled in three dimensions. The methods and the template used in this work can be used to analyze the structures and function of the nucleotide binding domains of all other members of the ABC transporter super-family.     

Nonrandom chromosomal distribution of spontaneous breakpoints and satellite DNA clusters in two geographically distant populations of Chironomus riparius (Diptera: Chironomidae)

Two geographically distant populations of Chironomus riparius (syn. C. thummi) from two environmentally polluted sites (Santena, Italy and Varna, Bulgaria) show numerous somatic and inherited chromosomal aberrations (inversions, deletions and deficiencies). Fifty-five percent of the observed breakpoints occurred in at least two larvae from both populations. Breakpoints occurring twice or more were considered as common structural chromosomal breakpoints. We tested whether such common breakpoints in larvae of the two polluted populations had a random chromosomal distribution or occurred preferentially in specific heterochromatic regions. Distribution of common breakpoints was not random, and proximal regions of first and third chromosome had significantly more common breakpoints than distal ones. By FISH we identified and mapped 56 chromosomal sections containing clusters of two tandem-repetitive satellite DNA families called Hinf and Alu elements. Like the common breakpoints, these repetitive DNA clusters appeared to be significantly more abundant in regions of constitutive heterochromatin such as the pericentromeric regions, while in distal sections of chromosomal arms they were rare or absent. Twenty-four out of 45 common breakpoints (i.e., 53.3%) occurred in cytogenetic sections where Alu and Hinf satellite DNA probes hybridized. The frequency of co-localization between common breakpoints and repetitive DNA hybridization signals was significantly higher than expected by chance. We hypothesize that spontaneous or induced breaks occur more frequently in sections containing blocks of repetitive DNA.     

Multidomain and Multifunctional Glycosyl Hydrolases from the Extreme Thermophile Caldicellulosiruptor Isolate Tok7B.1

DNA sequencing techniques have revealed widespread molecular diversity of the genomic organization of apparently closely related bacteria (as judged from SSU rDNA sequence similarity). We have previously described the extreme thermophile Caldicellulosiruptor saccharolyticus, which is unusual in possessing multi-catalytic, multidomain arrangements for the majority of its glycosyl hydrolases. We report here the sequencing of three gene clusters of glycosyl hydrolases from Caldicellulosiruptor sp. strain Tok7B.1. These clusters are not closely linked, and each is different in its organization from any described for Cs. saccharolyticus. The catalytic domains of the enzymes belong to glycosyl hydrolase families 5, 9, 10, 43, 44, and 48. The cellulose binding domains (CBDs) of these enzymes from Caldicellulosiruptor sp. Tok7B.1 are types IIIb, IIIc, or VI. A number of individual catalytic and binding domains have been expressed in Escherichia coli, and biochemical data are reported on the purified enzymes for cellulose degradation encoded by engineered derivatives of celB and celE. Received: 12 November 1999 / Accepted: 30 November 1999     

Genetic linkage mapping in peach using morphological,RFLP and RAPD markers

We have constructed a genetic linkage map of peach [Prunus persica (L.) Batsch] consisting of RFLP, RAPD and morphological markers, based on 71 F₂ individuals derived from the self-fertilization of four F₁ individuals of a cross between New Jersey Pillar and KV 77119. This progeny, designated as the West Virginia (WV) family, segregates for genes controlling canopy shape, fruit flesh color, and flower petal color, size and number. The segregation of 65 markers, comprising 46 RFLP loci, 12 RAPD loci and seven morphological loci, was analyzed. Low-copy genomic and cDNA probes were used in the RFLP analysis. The current genetic map for the WV family contains 47 markers assigned to eight linkage groups covering 332 centi Morgans (cM) of the peach nuclear genome. The average distance between two adjacent markers is 8 cM. Linkage was detected between Pillar (Pi) and double flowers (Dl) RFLP markers linked to Pi and flesh color () loci were also found. Eighteen markers remain unassigned. The individuals analyzed for linkage were not a random sample of all F₂ trees, as an excess of pillar trees were chosen for analysis. Because of this, Pi and eight other markers that deviated significantly from the expected Mendelian ratios (e.g., 121 or 31) were not eliminated from the linkage analysis. Genomic clones that detect RFLPs in the WV family also detect significant levels of polymorphism among the 34 peach cultivars examined. Unique fingerprint patterns were created for all the cultivars using only six clones detecting nine RFLP fragments. This suggests that RFLP markers from the WV family have a high probability of being polymorphic in crosses generated with other peach cultivars, making them ideal for anchor loci. This possibility was examined by testing RFLP markers developed with the WV family in three other unrelated peach families. In each of these three peach families respectively 43%, 54% and 36% of RFLP loci detected in the WV family were also polymorphic. This finding supports the possibility that these RFLP markers may serve as anchor loci in many other peach crosses.