A combined structural and biochemical approach reveals translocation and stalling of UvrB on the DNA lesion as a mechanism of damage verification in bacterial nucleotide excision repair.

Nucleotide excision repair (NER) is a DNA repair pathway present in all domains of life. In bacteria, UvrA protein localizes the DNA lesion, followed by verification by UvrB helicase and excision by UvrC double nuclease. UvrA senses deformations and flexibility of the DNA duplex without precisely localizing the lesion in the damaged strand, an element essential for proper NER. Using a combination of techniques, we elucidate the mechanism of the damage verification step in bacterial NER. UvrA dimer recruits two UvrB molecules to its two sides. Each of the two UvrB molecules clamps a different DNA strand using its ß-hairpin element. Both UvrB molecules then translocate to the lesion, and UvrA dissociates. The UvrB molecule that clamps the damaged strand gets stalled at the lesion to recruit UvrC. This mechanism allows UvrB to verify the DNA damage and identify its precise location triggering subsequent steps in the NER pathway.

The OB-fold proteins of the Trypanosoma brucei editosome execute RNA-chaperone activity.

Sequence-deficient mitochondrial pre-mRNAs in African trypanosomes are substrates of a U-nucleotide-specific RNA editing reaction to generate translation-competent mRNAs. The reaction is catalyzed by a macromolecular protein complex termed the editosome. Editosomes execute RNA-chaperone activity to overcome the highly folded nature of pre-edited substrate mRNAs. The molecular basis for this activity is unknown. Here we test five of the OB-fold proteins of the Trypanosoma brucei editosome as candidates. We demonstrate that all proteins execute RNA-chaperone activity albeit to different degrees. We further show that the activities correlate to the surface areas of the proteins and we map the protein-induced RNA-structure changes using SHAPE-chemical probing. To provide a structural context for our findings we calculate a coarse-grained model of the editosome. The model has a shell-like structure: Structurally well-defined protein domains are separated from an outer shell of intrinsically disordered protein domains, which suggests a surface-driven mechanism for the chaperone activity.

RNA 3D Structure Modeling by Combination of Template-Based Method ModeRNA, Template-Free Folding with SimRNA, and Refinement with QRNAS.

RNA encompasses an essential part of all known forms of life. The functions of many RNA molecules are dependent on their ability to form complex three-dimensional (3D) structures. However, experimental determination of RNA 3D structures is laborious and challenging, and therefore, the majority of known RNAs remain structurally uncharacterized. To address this problem, computational structure prediction methods were developed that either utilize information derived from known structures of other RNA molecules (by way of template-based modeling) or attempt to simulate the physical process of RNA structure formation (by way of template-free modeling). All computational methods suffer from various limitations that make theoretical models less reliable than high-resolution experimentally determined structures. This chapter provides a protocol for computational modeling of RNA 3D structure that overcomes major limitations by combining two complementary approaches: template-based modeling that is capable of predicting global architectures based on similarity to other molecules but often fails to predict local unique features, and template-free modeling that can predict the local folding, but is limited to modeling the structure of relatively small molecules. Here, we combine the use of a template-based method ModeRNA with a template-free method SimRNA. ModeRNA requires a sequence alignment of the target RNA sequence to be modeled with a template of the known structure; it generates a model that predicts the structure of a conserved core and provides a starting point for modeling of variable regions. SimRNA can be used to fold small RNAs (<80 nt) without any additional structural information, and to refold parts of models for larger RNAs that have a correctly modeled core. ModeRNA can be either downloaded, compiled and run locally or run through a web interface at http://genesilico.pl/modernaserver/ . SimRNA is currently available to download for local use as a precompiled software package at http://genesilico.pl/software/stand-alone/simrna and as a web server at http://genesilico.pl/SimRNAweb . For model optimization we use QRNAS, available at http://genesilico.pl/qrnas .

Modeling of Protein-RNA Complex Structures Using Computational Docking Methods.

A significant part of biology involves the formation of RNA-protein complexes. X-ray crystallography has added a few solved RNA-protein complexes to the repertoire; however, it remains challenging to capture these complexes and often only the unbound structures are available. This has inspired a growing interest in finding ways to predict these RNA-protein complexes. In this study, we show ways to approach this problem by computational docking methods, either with a fully automated NPDock server or with a workflow of methods for generation of many alternative structures followed by selection of the most likely solution. We show that by introducing experimental information, the structure of the bound complex is rendered far more likely to be within reach. This study is meant to help the user of docking software understand how to grapple with a typical realistic problem in RNA-protein docking, understand what to expect in the way of difficulties, and recognize the current limitations.

The architecture of the Schizosaccharomyces pombe CCR4-NOT complex.

CCR4-NOT is a large protein complex present both in cytoplasm and the nucleus of eukaryotic cells. Although it is involved in a variety of distinct processes related to expression of genetic information such as poly(A) tail shortening, transcription regulation, nuclear export and protein degradation, there is only fragmentary information available on some of its nine subunits. Here we show a comprehensive structural characterization of the native CCR4-NOT complex from Schizosaccharomyces pombe. Our cryo-EM 3D reconstruction of the complex, combined with techniques such as immunomicroscopy, RNA-nanogold labelling, docking of the available high-resolution structures and models of different subunits and domains, allow us to propose its full molecular architecture. We locate all functionally defined domains endowed with deadenylating and ubiquitinating activities, the nucleus-specific RNA-interacting subunit Mmi1, as well as surfaces responsible for protein-protein interactions. This information provides insight into cooperation of the different CCR4-NOT complex functions.

Structural and functional insights into tRNA binding and adenosine N1-methylation by an archaeal Trm10 homologue.

Purine nucleosides on position 9 of eukaryal and archaeal tRNAs are frequently modified in vivo by the post-transcriptional addition of a methyl group on their N1 atom. The methyltransferase Trm10 is responsible for this modification in both these domains of life. While certain Trm10 orthologues specifically methylate either guanosine or adenosine at position 9 of tRNA, others have a dual specificity. Until now structural information about this enzyme family was only available for the catalytic SPOUT domain of Trm10 proteins that show specificity toward guanosine. Here, we present the first crystal structure of a full length Trm10 orthologue specific for adenosine, revealing next to the catalytic SPOUT domain also N- and C-terminal domains. This structure hence provides crucial insights in the tRNA binding mechanism of this unique monomeric family of SPOUT methyltransferases. Moreover, structural comparison of this adenosine-specific Trm10 orthologue with guanosine-specific Trm10 orthologues suggests that the N1 methylation of adenosine relies on additional catalytic residues.

Structure and intrinsic disorder of the proteins of the Trypanosoma brucei editosome.

Mitochondrial pre-mRNAs in trypanosomatids undergo RNA editing to be converted into translatable mRNAs. The reaction is characterized by the insertion and deletion of uridine residues and is catalyzed by a macromolecular protein complex called the editosome. Despite intensive research, structural information for the majority of editosome proteins is still missing and no high resolution structure for the editosome exists. Here we present a comprehensive structural bioinformatics analysis of all proteins of the Trypanosoma brucei editosome. We specifically focus on the interplay between intrinsic order and disorder. According to computational predictions, editosome proteins involved in the basal reaction steps of the processing cycle are mostly ordered. By contrast, thirty percent of the amino acid content of the editosome is intrinsically disordered, which includes most prominently proteins with OB-fold domains. Based on the data we suggest a functional model, in which the structurally disordered domains of the complex are correlated with the RNA binding and RNA unfolding activity of the T. brucei editosome.

Structural and degradative aspects of ornithine decarboxylase antizyme inhibitor 2.

Ornithine decarboxylase (ODC) is the key enzyme in the polyamine biosynthetic pathway. ODC levels are controlled by polyamines through the induction of antizymes (AZs), small proteins that inhibit ODC and target it to proteasomal degradation without ubiquitination. Antizyme inhibitors (AZIN1 and AZIN2) are proteins homologous to ODC that bind to AZs and counteract their negative effect on ODC. Whereas ODC and AZIN1 are well-characterized proteins, little is known on the structure and stability of AZIN2, the lastly discovered member of this regulatory circuit. In this work we first analyzed structural aspects of AZIN2 by combining biochemical and computational approaches. We demonstrated that AZIN2, in contrast to ODC, does not form homodimers, although the predicted tertiary structure of the AZIN2 monomer was similar to that of ODC. Furthermore, we identified conserved residues in the antizyme-binding element, whose substitution drastically affected the capacity of AZIN2 to bind AZ1. On the other hand, we also found that AZIN2 is much more labile than ODC, but it is highly stabilized by its binding to AZs. Interestingly, the administration of the proteasome inhibitor MG132 caused differential effects on the three AZ-binding proteins, having no effect on ODC, preventing the degradation of AZIN1, but unexpectedly increasing the degradation of AZIN2. Inhibitors of the lysosomal function partially prevented the effect of MG132 on AZIN2. These results suggest that the degradation of AZIN2 could be also mediated by an alternative route to that of proteasome. These findings provide new relevant information on this unique regulatory mechanism of polyamine metabolism.

Computational modeling of protein-RNA complex structures.

Protein-RNA interactions play fundamental roles in many biological processes, such as regulation of gene expression, RNA splicing, and protein synthesis. The understanding of these processes improves as new structures of protein-RNA complexes are solved and the molecular details of interactions analyzed. However, experimental determination of protein-RNA complex structures by high-resolution methods is tedious and difficult. Therefore, studies on protein-RNA recognition and complex formation present major technical challenges for macromolecular structural biology. Alternatively, protein-RNA interactions can be predicted by computational methods. Although less accurate than experimental measurements, theoretical models of macromolecular structures can be sufficiently accurate to prompt functional hypotheses and guide e.g. identification of important amino acid or nucleotide residues. In this article we present an overview of strategies and methods for computational modeling of protein-RNA complexes, including software developed in our laboratory, and illustrate it with practical examples of structural predictions.

Molecular evolution of dihydrouridine synthases.

BACKGROUND: Dihydrouridine (D) is a modified base found in conserved positions in the D-loop of tRNA in Bacteria, Eukaryota, and some Archaea. Despite the abundant occurrence of D, little is known about its biochemical roles in mediating tRNA function. It is assumed that D may destabilize the structure of tRNA and thus enhance its conformational flexibility. D is generated post-transcriptionally by the reduction of the 5,6-double bond of a uridine residue in RNA transcripts. The reaction is carried out by dihydrouridine synthases (DUS). DUS constitute a conserved family of enzymes encoded by the orthologous gene family COG0042. In protein sequence databases, members of COG0042 are typically annotated as "predicted TIM-barrel enzymes, possibly dehydrogenases, nifR3 family". RESULTS: To elucidate sequence-structure-function relationships in the DUS family, a comprehensive bioinformatic analysis was carried out. We performed extensive database searches to identify all members of the currently known DUS family, followed by clustering analysis to subdivide it into subfamilies of closely related sequences. We analyzed phylogenetic distributions of all members of the DUS family and inferred the evolutionary tree, which suggested a scenario for the evolutionary origin of dihydrouridine-forming enzymes. For a human representative of the DUS family, the hDus2 protein suggested as a potential drug target in cancer, we generated a homology model. While this article was under review, a crystal structure of a DUS representative has been published, giving us an opportunity to validate the model. CONCLUSIONS: We compared sequences and phylogenetic distributions of all members of the DUS family and inferred the phylogenetic tree, which provides a framework to study the functional differences among these proteins and suggests a scenario for the evolutionary origin of dihydrouridine formation. Our evolutionary and structural classification of the DUS family provides a background to study functional differences among these proteins that will guide experimental analyses.

Crystal structure of a fructokinase homolog from Halothermothrix orenii.

Fructokinase (FRK; EC 2.7.1.4) catalyzes the phosphorylation of d-fructose to d-fructose 6-phosphate (F6P). This irreversible and near rate-limiting step is a central and regulatory process in plants and bacteria, which channels fructose into a metabolically active state for glycolysis. Towards understanding the mechanism of FRK, here we report the crystal structure of a FRK homolog from a thermohalophilic bacterium Halothermothrixorenii (Hore_18220 in sequence databases). The structure of the Hore_18220 protein reveals a catalytic domain with a Rossmann-like fold and a beta-sheet "lid" for dimerization. Based on comparison of Hore_18220 to structures of related proteins, we propose its mechanism of action, in which the lid serves to regulate access to the substrate binding sites. Close relationship of Hore_18220 and plant FRK enzymes allows us to propose a model for the structure and function of FRKs.

MODOMICS: a database of RNA modification pathways. 2008 update.

MODOMICS, a database devoted to the systems biology of RNA modification, has been subjected to substantial improvements. It provides comprehensive information on the chemical structure of modified nucleosides, pathways of their biosynthesis, sequences of RNAs containing these modifications and RNA-modifying enzymes. MODOMICS also provides cross-references to other databases and to literature. In addition to the previously available manually curated tRNA sequences from a few model organisms, we have now included additional tRNAs and rRNAs, and all RNAs with 3D structures in the Nucleic Acid Database, in which modified nucleosides are present. In total, 3460 modified bases in RNA sequences of different organisms have been annotated. New RNA-modifying enzymes have been also added. The current collection of enzymes includes mainly proteins for the model organisms Escherichia coli and Saccharomyces cerevisiae, and is currently being expanded to include proteins from other organisms, in particular Archaea and Homo sapiens. For enzymes with known structures, links are provided to the corresponding Protein Data Bank entries, while for many others homology models have been created. Many new options for database searching and querying have been included. MODOMICS can be accessed at http://genesilico.pl/modomics.

Crystal structure of the Escherichia coli 23S rRNA:m5C methyltransferase RlmI (YccW) reveals evolutionary links between RNA modification enzymes.

Methylation is the most common RNA modification in the three domains of life. Transfer of the methyl group from S-adenosyl-l-methionine (AdoMet) to specific atoms of RNA nucleotides is catalyzed by methyltransferase (MTase) enzymes. The rRNA MTase RlmI (rRNA large subunit methyltransferase gene I; previously known as YccW) specifically modifies Escherichia coli 23S rRNA at nucleotide C1962 to form 5-methylcytosine. Here, we report the crystal structure of RlmI refined at 2 A to a final R-factor of 0.194 (R(free)=0.242). The RlmI molecule comprises three domains: the N-terminal PUA domain; the central domain, which resembles a domain previously found in RNA:5-methyluridine MTases; and the C-terminal catalytic domain, which contains the AdoMet-binding site. The central and C-terminal domains are linked by a beta-hairpin structure that has previously been observed in several MTases acting on nucleic acids or proteins. Based on bioinformatics analyses, we propose a model for the RlmI-AdoMet-RNA complex. Comparative structural analyses of RlmI and its homologs provide insight into the potential function of several structures that have been solved by structural genomics groups and furthermore indicate that the evolutionary paths of RNA and DNA 5-methyluridine and 5-methylcytosine MTases have been closely intertwined.

YbeA is the m3Psi methyltransferase RlmH that targets nucleotide 1915 in 23S rRNA.

Pseudouridines in the stable RNAs of Bacteria are seldom subjected to further modification. There are 11 pseudouridine (Psi) sites in Escherichia coli rRNA, and further modification is found only at Psi1915 in 23S rRNA, where the N-3 position of the base becomes methylated. Here, we report the identity of the E. coli methyltransferase that specifically catalyzes methyl group addition to form m(3)Psi1915. Analyses of E. coli rRNAs using MALDI mass spectrometry showed that inactivation of the ybeA gene leads to loss of methylation at nucleotide Psi1915. Methylation is restored by complementing the knockout strain with a plasmid-encoded copy of ybeA. Homologs of the ybeA gene, and thus presumably the ensuing methylation at nucleotide m(3)Psi1915, are present in most bacterial lineages but are essentially absent in the Archaea and Eukaryota. Loss of ybeA function in E. coli causes a slight slowing of the growth rate. Phylogenetically, ybeA and its homologs are grouped with other putative S-adenosylmethionine-dependent, SPOUT methyltransferase genes in the Cluster of Orthologous Genes COG1576; ybeA is the first member to be functionally characterized. The YbeA methyltransferase is active as a homodimer and docks comfortably into the ribosomal A site without encroaching into the P site. YbeA makes extensive interface contacts with both the 30S and 50S subunits to align its active site cofactor adjacent to nucleotide Psi1915. Methylation by YbeA (redesignated RlmH for rRNA large subunit methyltransferase H) possibly functions as a stamp of approval signifying that the 50S subunit has engaged in translational initiation.