Stem Trace: an interactive visual tool for comparative RNA structure analysis.
MOTIVATION: Stem Trace is one of the latest tools available in STRUCTURELAB, an RNA structure analysis computer workbench. The paradigm used in STRUCTURELAB views RNA structure determination as a problem of dealing with a database of a large number of computationally generated structures. Stem Trace provides the capability to analyze this data set in a novel, visually driven, interactive and exploratory way. In addition to providing graphs at a high level of ion, it is also connected with complementary visualization tools which provide orthogonal views of the same data, as well as drawing of structures represented by a stem trace. Thus, on top of being an analysis tool, Stem Trace is a graphical user interface to an RNA structural information database. RESULTS: We illustrate Stem Trace's capabilities with several examples of the analysis of RNA folding data performed on 24 strains of HIV-1, HIV-2 and SIV sequences around the HIV dimerization region. This dimer linkage site has been found to play a role in encapsidation, reverse transcription, recombination, and inhibition of translation. Our examples show how Stem Trace elucidates preservation of structures in this region across the various strains of HIV. AVAILABILITY: The program can be made available upon request. It runs on SUN, SGI and DEC (Compaq) Unix workstations. (+info)
E-CELL: software environment for whole-cell simulation.
MOTIVATION: Genome sequencing projects and further systematic functional analyses of complete gene sets are producing an unprecedented mass of molecular information for a wide range of model organisms. This provides us with a detailed account of the cell with which we may begin to build models for simulating intracellular molecular processes to predict the dynamic behavior of living cells. Previous work in biochemical and genetic simulation has isolated well-characterized pathways for detailed analysis, but methods for building integrative models of the cell that incorporate gene regulation, metabolism and signaling have not been established. We, therefore, were motivated to develop a software environment for building such integrative models based on gene sets, and running simulations to conduct experiments in silico. RESULTS: E-CELL, a modeling and simulation environment for biochemical and genetic processes, has been developed. The E-CELL system allows a user to define functions of proteins, protein-protein interactions, protein-DNA interactions, regulation of gene expression and other features of cellular metabolism, as a set of reaction rules. E-CELL simulates cell behavior by numerically integrating the differential equations described implicitly in these reaction rules. The user can observe, through a computer display, dynamic changes in concentrations of proteins, protein complexes and other chemical compounds in the cell. Using this software, we constructed a model of a hypothetical cell with only 127 genes sufficient for transcription, translation, energy production and phospholipid synthesis. Most of the genes are taken from Mycoplasma genitalium, the organism having the smallest known chromosome, whose complete 580 kb genome sequence was determined at TIGR in 1995. We discuss future applications of the E-CELL system with special respect to genome engineering. AVAILABILITY: The E-CELL software is available upon request. SUPPLEMENTARY INFORMATION: The complete list of rules of the developed cell model with kinetic parameters can be obtained via our web site at: http://e-cell.org/. (+info)
The high-resolution crystal structure of the molybdate-dependent transcriptional regulator (ModE) from Escherichia coli: a novel combination of domain folds.
The molybdate-dependent transcriptional regulator (ModE) from Escherichia coli functions as a sensor of molybdate concentration and a regulator for transcription of operons involved in the uptake and utilization of the essential element, molybdenum. We have determined the structure of ModE using multi-wavelength anomalous dispersion. Selenomethionyl and native ModE models are refined to 1. 75 and 2.1 A, respectively and describe the architecture and structural detail of a complete transcriptional regulator. ModE is a homodimer and each subunit comprises N- and C-terminal domains. The N-terminal domain carries a winged helix-turn-helix motif for binding to DNA and is primarily responsible for ModE dimerization. The C-terminal domain contains the molybdate-binding site and residues implicated in binding the oxyanion are identified. This domain is divided into sub-domains a and b which have similar folds, although the organization of secondary structure elements varies. The sub-domain fold is related to the oligomer binding-fold and similar to that of the subunits of several toxins which are involved in extensive protein-protein interactions. This suggests a role for the C-terminal domain in the formation of the ModE-protein-DNA complexes necessary to regulate transcription. Modelling of ModE interacting with DNA suggests that a large distortion of DNA is not necessary for complex formation. (+info)
X-ray structure of T4 endonuclease VII: a DNA junction resolvase with a novel fold and unusual domain-swapped dimer architecture.
Phage T4 endonuclease VII (Endo VII), the first enzyme shown to resolve Holliday junctions, recognizes a broad spectrum of DNA substrates ranging from branched DNAs to single base mismatches. We have determined the crystal structures of the Ca2+-bound wild-type and the inactive N62D mutant enzymes at 2.4 and 2.1 A, respectively. The Endo VII monomers form an elongated, highly intertwined molecular dimer exhibiting extreme domain swapping. The major dimerization elements are two pairs of antiparallel helices forming a novel 'four-helix cross' motif. The unique monomer fold, almost completely lacking beta-sheet structure and containing a zinc ion tetrahedrally coordinated to four cysteines, does not resemble any of the known junction-resolving enzymes, including the Escherichia coli RuvC and lambda integrase-type recombinases. The S-shaped dimer has two 'binding bays' separated by approximately 25 A which are lined by positively charged residues and contain near their base residues known to be essential for activity. These include Asp40 and Asn62, which function as ligands for the bound calcium ions. A pronounced bipolar charge distribution suggests that branched DNA substrates bind to the positively charged face with the scissile phosphates located near the divalent cations. A model for the complex with a four-way DNA junction is presented. (+info)
The three-dimensional structure of the RNA-binding domain of ribosomal protein L2; a protein at the peptidyl transferase center of the ribosome.
Ribosomal protein L2 is the largest protein component in the ribosome. It is located at or near the peptidyl transferase center and has been a prime candidate for the peptidyl transferase activity. It binds directly to 23S rRNA and plays a crucial role in its assembly. The three-dimensional structure of the RNA-binding domain of L2 from Bacillus stearothermophilus has been determined at 2.3 A resolution by X-ray crystallography using the selenomethionyl MAD method. The RNA-binding domain of L2 consists of two recurring motifs of approximately 70 residues each. The N-terminal domain (positions 60-130) is homologous to the OB-fold, and the C-terminal domain (positions 131-201) is homologous to the SH3-like barrel. Residues Arg86 and Arg155, which have been identified by mutation experiments to be involved in the 23S rRNA binding, are located at the gate of the interface region between the two domains. The molecular architecture suggests how this important protein has evolved from the ancient nucleic acid-binding proteins to create a 23S rRNA-binding domain in the very remote past. (+info)
Crystal structure of a heparin- and integrin-binding segment of human fibronectin.
The crystal structure of human fibronectin (FN) type III repeats 12-14 reveals the primary heparin-binding site, a clump of positively charged residues in FN13, and a putative minor site approximately 60 A away in FN14. The IDAPS motif implicated in integrin alpha4beta1 binding is at the FN13-14 junction, rendering the critical Asp184 inaccessible to integrin. Asp184 clamps the BC loop of FN14, whose sequence (PRARI) is reminiscent of the synergy sequence (PHSRN) of FN9. Mutagenesis studies prompted by this observation reveal that both arginines of the PRARI sequence are important for alpha4beta1 binding to FN12-14. The PRARI motif may represent a new class of integrin-binding sites. The spatial organization of the binding sites suggests that heparin and integrin may bind in concert. (+info)
A trans-acting peptide activates the yeast a1 repressor by raising its DNA-binding affinity.
The cooperative binding of gene regulatory proteins to DNA is a common feature of transcriptional control in both prokaryotes and eukaryotes. It is generally viewed as a simple energy coupling, through protein-protein interactions, of two or more DNA-binding proteins. In this paper, we show that the simple view does not account for the cooperative DNA binding of a1 and alpha2, two homeodomain proteins from budding yeast. Rather, we show through the use of chimeric proteins and synthetic peptides that, upon heterodimerization, alpha2 instructs a1 to bind DNA. This change is induced by contact with a peptide contributed by alpha2, and this contact converts a1 from a weak to a strong DNA-binding protein. This explains, in part, how high DNA-binding specificity is achieved only when the two gene regulatory proteins conjoin. We also provide evidence that features of the a1-alpha2 interaction can serve as a model for other examples of protein-protein interactions, including that between the herpes virus transcriptional activator VP16 and the mammalian homeodomain-containing protein Oct-l. (+info)
Golgi structure in three dimensions: functional insights from the normal rat kidney cell.
Three-dimensional reconstructions of portions of the Golgi complex from cryofixed, freeze-substituted normal rat kidney cells have been made by dual-axis, high-voltage EM tomography at approximately 7-nm resolution. The reconstruction shown here ( approximately 1 x 1 x 4 microm3) contains two stacks of seven cisternae separated by a noncompact region across which bridges connect some cisternae at equivalent levels, but none at nonequivalent levels. The rest of the noncompact region is filled with both vesicles and polymorphic membranous elements. All cisternae are fenestrated and display coated buds. They all have about the same surface area, but they differ in volume by as much as 50%. The trans-most cisterna produces exclusively clathrin-coated buds, whereas the others display only nonclathrin coated buds. This finding challenges traditional views of where sorting occurs within the Golgi complex. Tubules with budding profiles extend from the margins of both cis and trans cisternae. They pass beyond neighboring cisternae, suggesting that these tubules contribute to traffic to and/or from the Golgi. Vesicle-filled "wells" open to both the cis and lateral sides of the stacks. The stacks of cisternae are positioned between two types of ER, cis and trans. The cis ER lies adjacent to the ER-Golgi intermediate compartment, which consists of discrete polymorphic membranous elements layered in front of the cis-most Golgi cisterna. The extensive trans ER forms close contacts with the two trans-most cisternae; this apposition may permit direct transfer of lipids between ER and Golgi membranes. Within 0.2 microm of the cisternae studied, there are 394 vesicles (8 clathrin coated, 190 nonclathrin coated, and 196 noncoated), indicating considerable vesicular traffic in this Golgi region. Our data place structural constraints on models of trafficking to, through, and from the Golgi complex. (+info)