Stephen R. Holbrook

Crystal structure of yeast phenylalanine transfer RNA. I. Crystallographic refinement.

We present the results of the Jimal stage of the X-ray crystallographic studies of yeast phenylalanine transfer RNA in an orthorhombic crystal form. The crystal structure of the transfer KNA has been refined by a least-squares procedure to minimize the difference between the observed (F,) and calculated (F’,) structure factors from X-ray diffraction patterns. The final crystallographic discrepancy index, R = IIF,, - Fcl/IF,, is 0.198, based upori 8426 structure factors with magnitudes over twice the estimated standard deviation, corresponding to 96.4% of the complete set of data with resolutions up t,o 2.7 A. During the refinement, bond lengths and angles within each phosphate group and each rmcleoside (base plus sugar) were constrained exactly to their appropriate standard values, while those for the linkages between the nucleosides and phosphates were elastically restrained close to their standard values. The details of the application of the constraint-restraint least-squares (CORELS) refinement method to the crystal structure of yeast phenylalanine tRNA are described in this paper. A complete list of atomic co-ordinates and the rigid group thermal factors are presented. The st,ereochemical details of this structrire and their frictional implications are described in the following paper.

Crystal structure of yeast phenylalanine transfer RNA: II. Structural features and functional implications☆

Abstract The structural features of yeast phenylalanine transfer RNA are analyzed and documented in detail, based on atomic co-ordinates obtained from an extensive crystallographic refinement of the crystal structure of the molecule at 2.7 A resolution (see preceding paper). We describe here: the relative orientation and the helicity of the base-paired stems; more definitive assignments of tertiary hydrogen bonds involving bases, riboses and phosphates; binding sites for magnesium hydrates, spermine and water; iriter-molecular contacts and base-stacking; flexibility of the molecule; conformational analysis of nucleotides in the structure. Among the more noteworthy features are a considerable irregularity in the helicity of the base-paired stems, a greater flexibility in the anticodon and aminoacyl acceptor arms, and a “coupling” among several conformational angles. The functional implications of these structural features are also discussed.

RNA structure: the long and the short of it.

The database of RNA structure has grown tremendously since the crystal structure analyses of ribosomal subunits in 2000–2001. During the past year, the trend toward determining the structure of large, complex biological RNAs has accelerated, with the analysis of three intact group I introns, A- and B-type ribonuclease P RNAs, a riboswitch–substrate complex and other structures. The growing database of RNA structures, coupled with efforts directed at the standardization of nomenclature and classification of motifs, has resulted in the identification and characterization of numerous RNA secondary and tertiary structure motifs. Because a large proportion of RNA structure can now be shown to be composed of these recurring structural motifs, a view of RNA as a modular structure built from a combination of these building blocks and tertiary linkers is beginning to emerge. At the same time, however, more detailed analysis of water, metal, ligand and protein binding to RNA is revealing the effect of these moieties on folding and structure formation. The balance between the views of RNA structure either as strictly a construct of preformed building blocks linked in a limited number of ways or as a flexible polymer assuming a global fold influenced by its environment will be the focus of current and future RNA structural biology.

SCOR: a structural classification of RNA database.

The Structural Classification of RNA (SCOR) database provides a survey of the three-dimensional motifs contained in 259 NMR and X-ray RNA structures. In one classification, the structures are grouped according to function. The RNA motifs, including internal and external loops, are also organized in a hierarchical classification. The 259 database entries contain 223 internal and 203 external loops; 52 entries consist of fully complementary duplexes. A classification of the well-characterized tertiary interactions found in the larger RNA structures is also included along with examples. The SCOR database is accessible at http://scor.lbl.gov.

High-resolution structure of RNase P protein from Thermotoga maritima

The structure of RNase P protein from the hyperthermophilic bacterium Thermotoga maritima was determined at 1.2-Å resolution by using x-ray crystallography. This protein structure is from an ancestral-type RNase P and bears remarkable similarity to the recently determined structures of RNase P proteins from bacteria that have the distinct, Bacillus type of RNase P. These two types of protein span the extent of bacterial RNase P diversity, so the results generalize the structure of the bacterial RNase P protein. The broad phylogenetic conservation of structure and distribution of potential RNA-binding elements in the RNase P proteins indicate that all of these homologous proteins bind to their cognate RNAs primarily by interaction with the phylogenetically conserved core of the RNA. The protein is found to dimerize through an extensive, well-ordered interface. This dimerization may reflect a mechanism of thermal stability of the protein before assembly with the RNA moiety of the holoenzyme.

Local mobility of nucleic acids as determined from crystallographic data. I: RNA and B form DNA

The local mobility of DNA and RNA can be described well by a segmented rigid body model in which the phosphates, riboses and bases are treated as independent groups. We have developed a computer program for extracting information about the translational and rotational mobility of these groups from X-ray diffraction data of single crystals of DNA and RNA fragments. We plan to extend our studies from the B DNA helix studied here to A and Z form helices for which diffraction data are available. The mobilities described here may be important in allowing the flexibility necessary for interaction of nucleic acids with proteins, ligands or other nucleic acids, and are crucial to our understanding of the factors governing both structural stability and motion of nucleic acids on a larger scale.

Identification of functional modules in protein complexes via hyperclique pattern discovery

Proteins usually do not act isolated in a cell but function within complicated cellular pathways, interacting with other proteins either in pairs or as components of larger complexes. While many protein complexes have been identified by large-scale experimental studies, due to a large number of false-positive interactions existing in current protein complexes 10, it is still difficult to obtain an accurate understanding of functional modules, which encompass groups of proteins involved in common elementary biological function. In this paper, we present a hyperclique pattern discovery approach for extracting functional modules (hyperclique patterns) from protein complexes. A hyperclique pattern is a type of association pattern containing proteins that are highly affiliated with each other. The analysis of hyperclique patterns shows that proteins within the same pattern tend to present in the protein complex together. Also, statistically significant annotations of proteins in a pattern using the Gene Ontology suggest that proteins within the same hyperclique pattern more likely perform the same function and participate in the same biological process. More interestingly, the 3-D structural view of proteins within a hyperclique pattern reveals that these proteins physically interactwith each other. In addition, we show that several hyperclique patterns corresponding to different functions can participate in the same protein complex as independent modules. Finally, we demonstrate that a hyperclique pattern can be involved in different complexes performing different higher-order biological functions, although the pattern corresponds to a specific elementary biological function.

PSoL: a positive sample only learning algorithm for finding non-coding RNA genes

MOTIVATION Small non-coding RNA (ncRNA) genes play important regulatory roles in a variety of cellular processes. However, detection of ncRNA genes is a great challenge to both experimental and computational approaches. In this study, we describe a new approach called positive sample only learning (PSoL) to predict ncRNA genes in the Escherichia coli genome. Although PSoL is a machine learning method for classification, it requires no negative training data, which, in general, is hard to define properly and affects the performance of machine learning dramatically. In addition, using the support vector machine (SVM) as the core learning algorithm, PSoL can integrate many different kinds of information to improve the accuracy of prediction. Besides the application of PSoL for predicting ncRNAs, PSoL is applicable to many other bioinformatics problems as well. RESULTS The PSoL method is assessed by 5-fold cross-validation experiments which show that PSoL can achieve about 80% accuracy in recovery of known ncRNAs. We compared PSoL predictions with five previously published results. The PSoL method has the highest percentage of predictions overlapping with those from other methods.

MeRNA: a database of metal ion binding sites in RNA structures

Metal ions are essential for the folding of RNA into stable tertiary structures and for the catalytic activity of some RNA enzymes. To aid in the study of the roles of metal ions in RNA structural biology, we have created MeRNA (Metals in RNA), a comprehensive compilation of all metal binding sites identified in RNA 3D structures available from the PDB and Nucleic Acid Database. Currently, our database contains information relating to binding of 9764 metal ions corresponding to 23 distinct elements, in 256 RNA structures. The metal ion locations were confirmed and ligands characterized using original literature references. MeRNA includes eight manually identified metal-ion binding motifs, which are described in the literature. MeRNA is searchable by PDB identifier, metal ion, method of structure determination, resolution and R-values for X-ray structure and distance from metal to any RNA atom or to water. New structures with their respective binding motifs will be added to the database as they become available. The MeRNA database will further our understanding of the roles of metal ions in RNA folding and catalysis and have applications in structural and functional analysis, RNA design and engineering. The MeRNA database is accessible at .

Anisotropic thermal-parameter refinement of the DNA dodecamer CGCGAATTCGCG by the segmented rigid-body method

A structure-factor least-squares refinement of the deoxyoligonucleotide (CGCGAATTCGCG)2 has been conducted using a model with constraints and restraints on the positional parameters and a segmented rigid-body representation for the anisotropic temperature factors. The macromolecule was divided into subgroups each of which was treated as a rigid body in terms of both positional and thermal parameters. For each subgroup, the thermal parameters determined were elements of translation, libration and correlation (TLS) tensors. This segmented rigidbody model of thermal motion has not previously been applied to the refinement of a macromolecular crystal structure. The anisotropic thermal-parameter refinement has significantly reduced the classical R factor as judged by the Hamilton test. The resulting difference Fourier map has a considerably lower noise level allowing fifteen additional low-occupancy water positions to be identified. In addition, analysis of the anisotropic thermal parameters has revealed new information about the local mobility of the groups in the oligonucleotide. Thus, the method of segmented rigid-body anisotropic temperature-factor refinement appears to be uniquely suited to macromolecules, especially nucleic acids, where high-resolution data are usually unavailable.