Modeling active RNA structures using the intersection of conformational space: Application to the lead-activated ribozyme (original) (raw)
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Patterns of cleavages induced by lead ions in defined RNA secondary structure motifs
Journal of Molecular Biology, 1998
We have characterized the susceptibility of various RNA bulges, loops and other single-stranded sequences to hydrolysis promoted by Pb 2 . The reactivity of bulges depends primarily on the structural context of the¯anking base-pairs and the effect of nucleotide present at the 5 H side of the bulge is particularly strong. The ef®ciency of stacking interactions between the bulged residue and its neighbors seems to determine cleavage speci®city and ef®ciency. Hydrolysis of two-and three-nucleotide bulges depends only slightly on their nucleotide composition. In the case of terminal loops, the ef®ciency of their hydrolysis usually increases with the loop size and strongly depends on its nucleotide composition. Stable tetraloops UUCG, CUUG and GCAA are resistant to hydrolysis, while in some other loops of the GNRA family a single, weak cleavage occurs, suggesting the existence of structural subclasses within the family. A very ef®cient, speci®c hydrolysis of a phosphodiester bond in the singlestranded region adjacent to the stem in oligomer 12 resembles highly speci®c cleavages of some tRNA molecules. The reaction occurs in the presence of Pb 2 , but not in the presence of several other metal ions. The Pb 2 -cleavable RNA domain may be considered another example of leadzyme. The results of Pb 2 -induced hydrolysis in model RNA oligomers should be useful in interpretation of cleavage patterns of much larger, naturally occurring RNA molecules.
Functional Identification of Catalytic Metal Ion Binding Sites within RNA
PLoS Biology, 2005
The viability of living systems depends inextricably on enzymes that catalyze phosphoryl transfer reactions. For many enzymes in this class, including several ribozymes, divalent metal ions serve as obligate cofactors. Understanding how metal ions mediate catalysis requires elucidation of metal ion interactions with both the enzyme and the substrate(s). In the Tetrahymena group I intron, previous work using atomic mutagenesis and quantitative analysis of metal ion rescue behavior identified three metal ions (M A , M B , and M C ) that make five interactions with the ribozyme substrates in the reaction's transition state. Here, we combine substrate atomic mutagenesis with site-specific phosphorothioate substitutions in the ribozyme backbone to develop a powerful, general strategy for defining the ligands of catalytic metal ions within RNA. In applying this strategy to the Tetrahymena group I intron, we have identified the pro-S P phosphoryl oxygen at nucleotide C262 as a ribozyme ligand for M C . Our findings establish a direct connection between the ribozyme core and the functionally defined model of the chemical transition state, thereby extending the known set of transition-state interactions and providing information critical for the application of the recent group I intron crystallographic structures to the understanding of catalysis. Citation: Hougland JL, Kravchuk AV, Herschlag D, Piccirilli JA (2005) Functional identification of catalytic metal ion binding sites within RNA. PLoS Biol 3(9): e277. (JAP)
Order, dynamics and metal-binding in the lead-dependent ribozyme
Journal of Molecular Biology, 1998
The in vitro selected lead-dependent ribozyme is among the smallest and simplest of the known catalytic RNA motifs and has a unique metal ion speci®city for divalent lead. The conformation and dynamics of this ribozyme are analyzed here by NMR and chemical probing experiments. Complete assignments of the 1 H, 13 C, and 15 N resonances have been made, and the NMR chemical shift changes in the presence of Pb 2 , Mg 2 or high concentrations of Na show that there is no signi®cant structural change upon addition of either activating (Pb 2) or inhibitory (Mg 2) divalent ions. The 13 C NMR relaxation data indicate substantial dynamic uctuations on various timescales for active-site residues in this ribozyme. The combination of chemical probing and NMR experiments reveals a picture of the active site for the lead-dependent ribozyme that has both ordered and dynamic features.
Chemistry & Biology, 2007
Recent studies indicate that RNA function can be enhanced by the incorporation of conformationally restricted nucleotides. Herein, we use 8-bromoguanosine, a nucleotide analog with an enforced syn conformation, to elucidate the catalytic relevance of ribozyme structures. We chose to study the lead-dependent ribozyme (leadzyme) because structural models derived from NMR, crystal, and computational (MC-Sym) studies differ in which of the three active site guanosines (G7, G9, or G24) have a syn glycosidic torsion angle. Kinetic assays were carried out on 8BrG variants at these three guanosine positions. These data indicate that an 8BrG24 leadzyme is hyperactive, while 8BrG7 and 8BrG9 leadzymes have reduced activity. These findings support the computational model of the leadzyme, rather than the NMR and crystal structures, as being the most relevant to phosphodiester bond cleavage.
MetalionRNA: computational predictor of metal-binding sites in RNA structures
Bioinformatics, 2011
Motivation: Metal ions are essential for the folding of RNA molecules into stable tertiary structures and are often involved in the catalytic activity of ribozymes. However, the positions of metal ions in RNA 3D structures are difficult to determine experimentally. This motivated us to develop a computational predictor of metal ion sites for RNA structures. Results: We developed a statistical potential for predicting positions of metal ions (magnesium, sodium and potassium), based on the analysis of binding sites in experimentally solved RNA structures. The MetalionRNA program is available as a web server that predicts metal ions for RNA structures submitted by the user.
Cation-Specific Structural Accommodation within a Catalytic RNA
Biochemistry, 2006
Metal ions facilitate the folding of the hairpin ribozyme, but do not participate directly in catalysis. The metal complex cobalt (III) hexaammine supports folding and activity of the ribozyme and also mediates specific internucleotide photocrosslinks, several of which retain catalytic ability. These crosslinks imply that the active core structure organized by [Co(NH 3) 6 ] 3+ is different from that organized by Mg 2+ and that revealed in the crystal structure (1). Residues U+2 and C+3 of the substrate, in particular, adopt different conformations in [Co(NH 3) 6 ] 3+. U+2 is bulged out of loop A and stacked on residue G36, whereas the nucleotide at position +3 is stacked on G8, a nucleobase crucial for catalysis. Cleavage kinetics performed with +2 variants and a C+3 U variant correlate with the crosslinking observations. Variants that decreased cleavage rates in magnesium up to 70fold showed only subtle decreases or even increases in observed rates when assayed in [Co (NH 3) 6 ] 3+. Here, we propose a model of the [Co(NH 3) 6 ] 3+-mediated catalytic core generated by MC-SYM that is consistent with these data. Interactions between cations and RNA molecules are critical for the biological activity of RNA, in that metal ions promote RNA folding events and RNA-catalyzed reactions, including RNA processing reactions and peptide bond formation (2). In the hairpin and hammerhead ribozymes, cations function to facilitate folding into the active conformations, but play little or no direct role in catalysis (3-6). Folding and cleavage activity of the hairpin ribozyme can be supported by high concentrations (>1 M) of monovalent ions (4), moderate concentrations (2 to 20 mM) of magnesium and some other divalent ions (7), or by low concentrations (~1 mM) of the trivalent complex [Co(NH 3) 6 ] 3+.. This complex serves as an analogue of hexahydrated magnesium, in that it cannot make inner-sphere binding interactions with RNA (3). Catalysis by the hairpin ribozyme is preceded by a major conformational change, in which the two domains of the ribozyme-substrate complex come into close association with one another. This docking step is accompanied by changes in the orientation of the Watson-Crick helical elements within the complex, which can be monitored by biochemical and biophysical methods, including FRET, electrophoretic mobility, transient electric birefringence, and hydroxyl radical footprinting (8-10). Concomitantly, extensive interactions between the two major non-helical regions are formed, and result in the positioning of the likely catalytic bases, G8 and A38, at the scissile phosphodiester linkage. These latter conformational changes can be monitored by the photocrosslinking and fluorescence behavior of the affected nucleobases (11). The scope of overall conformational change can be visualized by comparing the NMR
Nucleic Acids Research, 2009
RNA triphosphatases (RTPases) are involved in the addition of the distinctive cap structure found at the 5' ends of eukaryotic mRNAs. Fungi, protozoa and some DNA viruses possess an RTPase that belongs to the triphosphate tunnel metalloenzyme family of enzymes that can also hydrolyze nucleoside triphosphates. Previous crystallization studies revealed that the phosphohydrolase catalytic core is located in a hydrophilic tunnel composed of antiparallel b-strands. However, all past efforts to obtain structural information on the interaction between RTPases and their substrates were unsuccessful. In the present study, we used computational molecular docking to model the binding of a nucleotide substrate into the yeast RTPase active site. In order to confirm the docking model and to gain additional insights into the molecular determinants involved in substrate recognition, we also evaluated both the phosphohydrolysis and the inhibitory potential of an important number of nucleotide analogs. Our study highlights the importance of specific amino acids for the binding of the sugar, base and triphosphate moieties of the nucleotide substrate, and reveals both the structural flexibility and complexity of the active site. These data illustrate the functional features required for the interaction of an RTPase with a ligand and pave the way to the use of nucleotide analogs as potential inhibitors of RTPases of pathogenic importance.
Proceedings of the National Academy of Sciences, 1994
A three-dimensional model of Ml RNA, the catalytic RNA subunit of RNase P from Eschenchia col, was constructed with the aid of a computer. The modeling process took Into account data fom chemical and enzymatic protection experiments, phylogenetic analysis, studies of the activities of mutants, and the kinetics of reactions catalyzed by the binding of substrate to Ml RNA. The model provides a plausible picture of the binding to Ml RNA of the tRNA domain of a precursor tRNA substrate. The scIssile bond and adjacent segments of the amoal acceptor stem of a precursor tRNA substrate can fit Into a deft that leads to the phylogenetically conserved, central part of the structure.
Computational chemistry with RNA secondary structures
2004
The secondary structure for nucleic acids provides a level of description that is both abstract enough to allow for efficient algorithms and realistic enough to provide a good approximate to the thermodynamic and kinetics properties of RNA structure formation. The secondary structure model has furthermore been successful in explaining salient features of RNA evolution in nature and in the test tube. In this contribution we review the computational chemistry of RNA secondary structures using a simplified algorithmic approach for explanation.
Biopolymers, 2006
The RNA recognition motif (RRM) is one of the most common RNA binding domains. We have investigated the contribution of three highly conserved aromatic amino acids to RNA binding by the N-terminal RRM of the U1A protein. Recently, we synthesized a modified base (A-4CPh) in which a phenyl group is tethered to adenine using a linker of 4 methylene groups. The substitution of this base for adenine in the target RNA selectively stabilizes the complex formed with a U1A protein in which one of the conserved aromatic amino acids is replaced with Ala (Phe56Ala). In this article, we report molecular dynamics (MD) simulations that probe the structural consequences of the substitution of A-4CPh for adenine in the wild type and Phe56Ala U1A-RNA complexes and in the free RNA. The simulations suggest that A-4CPh stabilizes the complex formed with Phe56Ala by adopting a folded conformation in which the tethered phenyl group fills the site occupied by the phenyl group of Phe56 in the wild-type complex. In contrast, an extended conformation of A-4CPh is predicted to be most stable in the complex formed with the wild-type protein. The calculations indicate A-4CPh is in an extended conformation in the free RNA. Therefore, preorganizing the structure of the phenyl-tethered base for binding may improve both the affinity and specificity of the RNA containing A-4CPh for the Phe56Ala U1A protein. Taken together, the previous experimental work and the calculations reported here suggest a general design strategy for altering RRM-RNA complex stability.