RNAMotif, an RNA secondary structure definition and search algorithm.

RNA molecules fold into characteristic secondary and tertiary structures that account for their diverse functional activities. Many of these RNA structures are assembled from a collection of RNA structural motifs. These basic building blocks are used repeatedly, and in various combinations, to form different RNA types and define their unique structural and functional properties. Identification of recurring RNA structural motifs will therefore enhance our understanding of RNA structure and help associate elements of RNA structure with functional and regulatory elements. Our goal was to develop a computer program that can describe an RNA structural element of any complexity and then search any nucleotide sequence database, including the complete prokaryotic and eukaryotic genomes, for these structural elements. Here we describe in detail a new computational motif search algorithm, RNAMotif, and demonstrate its utility with some motif search examples. RNAMotif differs from other motif search tools in two important aspects: first, the structure definition language is more flexible and can specify any type of base-base interaction; second, RNAMotif provides a user controlled scoring section that can be used to add capabilities that patterns alone cannot provide.

Transmembrane alpha-helices in the gap junction membrane channel: systematic search of packing models based on the pair potential function.

Recent progress in the field of electron cryo-microscopy and image analysis has shown that there is an overwhelming need to interpret medium resolution (5 to 10 A) three-dimensional maps. Traditional methods of fitting amino acid residues into electron density using molecular modeling programs must be supplemented with further analysis. We have used a potential of mean force (PMF) method, derived from Boltzmann statistics in protein structure, to generate models for the packing of alpha-helices, using pairwise potentials between amino acid residues. The approach was tested using the three-dimensional map of a recombinant cardiac gap junction membrane channel provided by electron cryo-crystallography (Unger et al., 1997; 1999a, 1999b) which had a resolution of 7.5 A in the membrane plane and 21 A in the vertical direction. The dodecameric channel was formed by the end-to-end docking of two hexamers, each of which displayed 24 rods of density in the membrane interior, which was consistent with an alpha-helical conformation for the four transmembrane domains of each connexin subunit. Based on the three-dimensional map and the amino acid sequence for the 4 transmembrane domains determined by hydropathy analysis, we used the modeling utility SymServ (Macke et al., 1998) to build hexameric connexons with 24 transmembrane alpha-helices. Canonical alpha-helices were aligned to the axes of the rods of density and translated along the density so that the center of masses coincided. The PMF function was used to evaluate 162,000 conformations for each of the 24 possible alpha-helical packing models. Since the different packing models yielded different energy distributions, the pair potential function appears to be a promising tool for evaluating the packing of alpha-helices in membrane proteins. The analysis will be refined by energy calculations based on the expectations that the outer boundary of the channel will be formed by hydrophobic residues in contact with the lipids.

Interactive modeling of supramolecular assemblies.

The modeling of supramolecular structure presents two major challenges: (1) managing the large amount of sequence, structural and biochemical data, and (2) presenting the data to the user in a flexible and comprehensible manner that addresses these problems. We describe a visualization environment for the creation and analysis of supramolecular models. A set of modular symmetry tools, collectively called SymGen, has been created, providing a flexible platform for the creation of complex assemblies, with interactive control of all symmetry elements and their parameters. A second tool, SymSearch, allows a range of parameters defined within SymGen to be sampled and the resulting conformations to be evaluated. The environment avoids information overload, caused by the large number of atoms in supramolecular complexes, by using a multiresolution spherical harmonic representation that allows the user to display only essential features. Spherical harmonics also enables control of the triangulation level, allowing the user to reduce the complexity of the geometric description to retain interactive speed. The visual fidelity of the surface data is retained by using texture maps that are independent of the resolution of the underlying triangulation. We describe the design and implementation of this environment, and three illustrative examples of its utility.

A phylogenetic definition of the major eubacterial taxa.

Through oligonucleotide signature analysis of 16S ribosomal RNAs, it is possible to define ten major groups of eubacteria. These are: (1) the Gram positive bacteria, (2) the purple photosynthetic bacteria and their relatives, (3) the spirochetes and their relatives, (4) the sulfur-dependent eubacteria and their relatives, (5) the bacteroides, flavobacteria and cytophagas and their relatives, (6) the cyanobacteria, (7) the green sulfur bacteria, (8) the green non-sulfur bacteria and their relatives, (9) the radio-resistant micrococci, and (10) the planctomyces and their relatives. Although no consensus exists as regards the taconomic terminology, these ten groupings are appropriately termed eubacterial Phyla or Divisions. The major subdivisions of those Phyla or Divisions that have been extensively characterized can also be defined by characteristic oligonucleotide signatures.

Biomolecular visualization using AVS.

Dataflow systems for scientific visualization are becoming increasingly sophisticated in their architecture and functionality. AVS, from Advanced Visual Systems Inc., is a powerful dataflow environment that has been applied to many computation and visualization tasks. An important, yet complex, application area is molecular modeling and biomolecular visualization. Problems in biomolecular visualization tax the capability of dataflow systems because of the diversity of operations that are required and because many operations do not fit neatly into the dataflow paradigm. Here we describe visualization strategies and auxiliary programs developed to enhance the applicability of AVS for molecular modelling. Our visualization strategy is to use general-purpose AVS modules and a small number of chemistry-specific modules. We have developed methods to control AVS using AVS-tool, a programmable interface to the AVS Command Line Interpreter (CLI), and have also developed NAB, a C-like language for writing AVS modules that has extensions for operating on proteins and nucleic acids. This strategy provides a flexible and extensible framework for a wide variety of molecular modeling tasks.

The coherence of synthetic telomeres.

The chromosomal telomeres of Oxytricha were synthesized and their ability to cohere examined on non-denaturing acrylamide gels containing the stabilizing cation K+. At least 5 different mobility species were observed, in addition to that of the monomeric telomere. By cohering synthetic telomeres containing different lengths of subtelomeric DNA, we showed that each of the different mobility species was a dimer of two telomeres. Since the different mobility species did not differ in numbers or sequences of nucleotides, they must correspond to different molecular shapes probably caused by different degrees of bending of the dimer. Paradoxically, telomeres with longer subtelomeric stems cohered more efficiently. In the presence of K+, solutions had to be heated to over 90 degrees before the telomeres separated. Various synthetic constructs, restriction endonuclease and dimethyl sulfate protection experiments showed that the only nucleotides involved in the cohered structures were the 16 base 'tails' of sequence 3'G4T4G4T4. Extension of this motif was actually inimical to coherence. Oligomers containing 2 G4T4 motifs protected their GN7 positions by forming dimers, those with 5 G4T4 could do so by internal folding, but the 3' terminal group of G4 was left unprotected. This suggests that only four groups of G4 are necessary for the cohered structure. Single-chain specific nuclease, S1, as well as osmium tetroxide, which oxidizes the thymine residues of single chains, reacted less efficiently with the cohered structures. Synthetic telomeres containing inosine replacing guanosine were not observed to cohere, indicating that the C2-NH2 is strongly stabilizing. The cohered structures appear to be unusually compact and sturdy units in which four G4 blocks form quadruplexes stabilized by K+. A new model for the cohered structure is presented.

The ribosomal database project.

The Ribosomal Database Project (RDP) is a curated database that offers ribosome data along with related programs and services. The offerings include phylogenetically ordered alignments of ribosomal RNA (rRNA) sequences, derived phylogenetic trees, rRNA secondary structure diagrams and various software packages for handling, analyzing and displaying alignments and trees. The data are available via ftp and electronic mail. Certain analytic services are also provided by the electronic mail server.

The structure of calcyclin reveals a novel homodimeric fold for S100 Ca(2+)-binding proteins.

The S100 calcium-binding proteins are implicated as effectors in calcium-mediated signal transduction pathways. The three-dimensional structure of the S100 protein calcyclin has been determined in solution in the apo state by NMR spectroscopy and a computational strategy that incorporates a systematic docking protocol. This structure reveals a symmetric homodimeric fold that is unique among calcium-binding proteins. Dimerization is mediated by hydrophobic contacts from several highly conserved residues, which suggests that the dimer fold identified for calcyclin will serve as a structural paradigm for the S100 subfamily of calcium-binding proteins.

The Ribosomal Database Project.

The Ribosomal Database Project (RDP) complies ribosomal sequences and related data, and redistributes them in aligned and phylogenetically ordered form to its user community. It also offers various software packages for handling, analyzing and displaying sequences. In addition, the RDP offers (or will offer) certain analytic services. At present the project is in an intermediate stage of development.

Active site structure of Rieske-type proteins: electron nuclear double resonance studies of isotopically labeled phthalate dioxygenase from Pseudomonas cepacia and Rieske protein from Rhodobacter capsulatus and molecular modeling studies of a Rieske center.

Continuous wave electron nuclear double resonance (CW ENDOR) spectra of [delta-15N,epsilon(-14)N]histidine-labeled phthalate dioxygenase (PDO) from Pseudomonas cepacia were recorded and found to be virtually identical to those previously recorded from [delta,epsilon-15N2]histidine-labeled protein [Gurbiel, R. J., Batie, C. J., Sivaraja, M., True, A. E., Fee, J. A., Hoffman, B. M., & Ballou, D. P. (1989) Biochemistry 28, 4861-4871]. Thus, the two histidine residues, previously shown to ligate one of the irons in the cluster [cf. Gurbiel et al. 1989)], both coordinate the metal at the N(delta) position of their imidazole rings. Pulsed ENDOR studies showed that the "remote", noncoordinating nitrogen of the histidine imidazole ring could be observed from the Rieske protein in a sample of Rhodobacter capsulatus cytochrome bc1 complex uniformly labeled with 15N but not in a sample of PDO labeled with [delta-15N,epsilon-14N]histidine, but this atom was easily observed with a sample of Rh. capsulatus cytochrome bc1 complex that had been uniformly labeled with 15N; this confirmed the conclusion from the CW ENDOR studies that ligation is exclusively via N(delta) for both ligands in the PDO center. Modifications in the algorithms previously used to simulate 14N ENDOR spectra permitted us to compute spectra without any constraints on the relative orientation of hyperfine and quadrupole tensors. This new algorithm was used to analyze current and previously published spectra, and slightly different values for the N-Fe-N angle and imidazole ring rotation angles are presented [cf. Gurbiel et al. (1989) Gurbiel, R. J., Ohnishi, T., Robertson, D. E., Daldal, F., and Hoffman, B. M. (1991) Biochemistry 30, 11579-11584]. This analysis has permitted us to refine the proposed structure of the [2Fe-2S] Rieske-type cluster and rationalize some of the properties of these novel centers. Although the spectra of cytochrome bc1 complex from Rh. capsulatus are of somewhat lower resolution than those obtained with samples of PDO, our analysis nevertheless permits the conclusion that the geometry of the cluster is essentially the same for all Rieske and Rieske-type proteins. Structural constraints inferred from the spectroscopic results permitted us to apply the principles of distance geometry to arrive at possible three-dimensional models of the active site structure of Rieske protein from Rh. capsulatus. Results from this test case indicate that similar procedures should be generally useful in metalloprotein systems. We also recorded the pulsed and CW ENDOR spectra of 57Fe-labeled PDO, and the resulting data were used to derive the full hyperfine tensors for both Fe(III) and Fe(II) ions, including their orientations relative to the g tensor. The A tensor of the ferric ion is nominally isotropic, while the A tensor of the ferrous ion is axial, having A(parallel) > A(perpendicular); both tensors are coincident with the observed g tensor, with A(parallel) of the ferrous ion lying along the maximum g-value, g1. These results were examined using refinements of existing theories of spin-coupling in [2Fe-2S]+ clusters, and it is concluded that current theories are not adequate to fully describe the experimental results.