Browse
Submit Data
Databases
API
Help

Dataset Information

8 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Single-atom imino substitutions at A9 and A10 reveal distinct effects on the fold and function of the hairpin ribozyme catalytic core.

ABSTRACT: The hairpin ribozyme cleaves a phosphodiester bond within a cognate substrate. Structural and biochemical data indicate the conserved A9 and A10 bases reside close to the scissile bond but make distinct contributions to catalysis. To investigate these residues, we replaced the imino moiety of each base with N1-deazaadenosine. This single-atom change resulted in an 8-fold loss in k(obs) for A9 and displacement of the base from the active site; no effects were observed for A10. We propose that the imino moiety of A9 promotes a key water-mediated contact that favors transition-state formation, which suggests an enhanced chemical repertoire for RNA.

SUBMITTER: Spitale RC

PROVIDER: S-EPMC2754253 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Single-atom imino substitutions at A9 and A10 reveal distinct effects on the fold and function of the hairpin ribozyme catalytic core.

Spitale Robert C RC Volpini Rosaria R Mungillo Michael V MV Krucinska Jolanta J Cristalli Gloria G Wedekind Joseph E JE

Biochemistry 20090801 33

The hairpin ribozyme cleaves a phosphodiester bond within a cognate substrate. Structural and biochemical data indicate the conserved A9 and A10 bases reside close to the scissile bond but make distinct contributions to catalysis. To investigate these residues, we replaced the imino moiety of each base with N1-deazaadenosine. This single-atom change resulted in an 8-fold loss in k(obs) for A9 and displacement of the base from the active site; no effects were observed for A10. We propose that the ...[more]

PMID: 19634899

Similar Datasets

Mutational inhibition of ligation in the hairpin ribozyme: substitutions of conserved nucleobases A9 and A10 destabilize tertiary structure and selectively promote cleavage.

Project description:The hairpin ribozyme acts as a reversible, site-specific endoribonuclease that ligates much more rapidly than it cleaves cognate substrate. While the reaction pathway for ligation is the reversal of cleavage, little is known about the atomic and electrostatic details of the two processes. Here, we report the functional consequences of molecular substitutions of A9 and A10, two highly conserved nucleobases located adjacent to the hairpin ribozyme active site, using G, C, U, 2-aminopurine, 2,6-diaminopurine, purine, and inosine. Cleavage and ligation kinetics were analyzed, tertiary folding was monitored by hydroxyl radical footprinting, and interdomain docking was studied by native gel electrophoresis. We determined that nucleobase substitutions that exhibit significant levels of interference with tertiary folding and interdomain docking have relatively large inhibitory effects on ligation rates while showing little inhibition of cleavage. Indeed, one variant, A10G, showed a fivefold enhancement of cleavage rate and no detectable ligation, and we suggest that this property may be uniquely well suited to intracellular targeted RNA cleavage applications. Results support a model in which formation of a kinetically stable tertiary structure is essential for ligation of the hairpin ribozyme, but is not necessary for cleavage.

| S-EPMC2151026 | biostudies-literature

Molecular dynamics suggest multifunctionality of an adenine imino group in acid-base catalysis of the hairpin ribozyme.

Project description:Despite numerous structural and biochemical investigations, the catalytic mechanism of hairpin ribozyme self-cleavage remains elusive. To gain insight into the coupling of active site dynamics with activity of this small catalytic RNA, we analyzed a total of approximately 300 ns of molecular dynamics (MD) simulations. Our simulations predict improved global stability for an in vitro selected "gain of function" mutation, which is validated by native gel electrophoretic mobility shift assay. We observe that active site nucleobases and water molecules stabilize a geometry favorable to catalysis through a dynamic hydrogen bonding network. Simulations in which A38 is unprotonated show its N1 move into close proximity of the active site 2'-OH, indicating that A38 may act as a general base during cleavage, a role that has generally been discounted due to the longer distances observed in crystal structures involving inactivating substrate analogs. By contrast, simulations in which N1 of A38 is protonated place N1 in close proximity to the 5'-oxygen leaving group, which supports the proposal that A38 serves as a general acid. In analogy to protein enzymes, we discuss a plausible mechanism in which A38 acts bifunctionally and shuttles a proton directly from the 2'-OH to the 5'-oxygen. Furthermore, our simulations suggest an important role for protonation of N1 of A38 in promoting a favorable geometry similar to that observed in transition-state analog crystal structures, and support previously proposed roles of A38, G8, and long residency water molecules in transition-state stabilization.

| S-EPMC2661834 | biostudies-literature

Crystal structure of the catalytic core of an RNA-polymerase ribozyme.

Project description:Primordial organisms of the putative RNA world would have required polymerase ribozymes able to replicate RNA. Known ribozymes with polymerase activity best approximating that needed for RNA replication contain at their catalytic core the class I RNA ligase, an artificial ribozyme with a catalytic rate among the fastest of known ribozymes. Here we present the 3.0 angstrom crystal structure of this ligase. The architecture resembles a tripod, its three legs converging near the ligation junction. Interacting with this tripod scaffold through a series of 10 minor-groove interactions (including two A-minor triads) is the unpaired segment that contributes to and organizes the active site. A cytosine nucleobase and two backbone phosphates abut the ligation junction; their location suggests a model for catalysis resembling that of proteinaceous polymerases.

| S-EPMC3978776 | biostudies-literature

Wnt5a regulates ventral midbrain morphogenesis and the development of A9-A10 dopaminergic cells in vivo.

Project description:Wnt5a is a morphogen that activates the Wnt/planar cell polarity (PCP) pathway and serves multiple functions during development. PCP signaling controls the orientation of cells within an epithelial plane as well as convergent extension (CE) movements. Wnt5a was previously reported to promote differentiation of A9-10 dopaminergic (DA) precursors in vitro. However, the signaling mechanism in DA cells and the function of Wnt5a during midbrain development in vivo remains unclear. We hereby report that Wnt5a activated the GTPase Rac1 in DA cells and that Rac1 inhibitors blocked the Wnt5a-induced DA neuron differentiation of ventral midbrain (VM) precursor cultures, linking Wnt5a-induced differentiation with a known effector of Wnt/PCP signaling. In vivo, Wnt5a was expressed throughout the VM at embryonic day (E)9.5, and was restricted to the VM floor and basal plate by E11.5-E13.5. Analysis of Wnt5a-/- mice revealed a transient increase in progenitor proliferation at E11.5, and a precociously induced NR4A2+ (Nurr1) precursor pool at E12.5. The excess NR4A2+ precursors remained undifferentiated until E14.5, when a transient 25% increase in DA neurons was detected. Wnt5a-/- mice also displayed a defect in (mid)brain morphogenesis, including an impairment in midbrain elongation and a rounded ventricular cavity. Interestingly, these alterations affected mostly cells in the DA lineage. The ventral Sonic hedgehog-expressing domain was broadened and flattened, a typical CE phenotype, and the domains occupied by Ngn2+ DA progenitors, NR4A2+ DA precursors and TH+ DA neurons were rostrocaudally reduced and laterally expanded. In summary, we hereby describe a Wnt5a regulation of Wnt/PCP signaling in the DA lineage and provide evidence for multiple functions of Wnt5a in the VM in vivo, including the regulation of VM morphogenesis, DA progenitor cell division, and differentiation of NR4A2+ DA precursors.

| S-EPMC2568809 | biostudies-literature

RNA self-splicing by engineered hairpin ribozyme variants.

Project description:Small RNAs capable of self-cleavage and ligation might have been the precursors for the much more complex self-splicing group I and II introns in an early RNA world. Here, we demonstrate the activity of engineered hairpin ribozyme variants, which as self-splicing introns are removed from their parent RNA. In the process, two cleavage reactions are supported at the two intron-exon junctions, followed by ligation of the two generated exon fragments. As a result, the hairpin ribozyme, here acting as the self-splicing intron, is cut out. Two self-splicing hairpin ribozyme variants were investigated, one designed by hand, the other by a computer-aided approach. Both variants perform self-splicing, generating a cut-out intron and ligated exons.

| S-EPMC8754997 | biostudies-literature

Cobalt(III)hexaammine-dependent photocrosslinks in the hairpin ribozyme.

Project description:We have utilized the hairpin ribozyme, an RNA enzyme whose structure has been solved by high-resolution methods, to develop a new tool for mapping nucleobase-stacking interactions and potential metal-binding sites in RNA molecules. This tool involves the photoactivation of a specifically bound cobalt(III)hexaammine molecule at wavelengths corresponding to excitation of the metal ion complex only; no base excitation is involved. The photoexcitation initiates a process which strongly promotes the formation of a novel covalent bond or crosslink between one base (termed the "first base"), which is close in space to the excited cobalt(III)hexaammine complex, and another base upon which the first base is closely stacked. These crosslinked species can be isolated and sequenced; their activities can be analyzed to ensure that the crosslinked structures represent an active conformation of the molecule. We have shown that, as in electron transfer in DNA, several criteria must be met to result in the successful formation of these crosslinks. These include the appropriate oxidation potential of the first donor base, the stacking and close interaction of the two donor bases involved in the crosslink, and the binding of a specific cobalt(III)hexaammine molecule to the first donor base. Additionally, we have determined that this crosslinking is pH-sensitive, although the cause of this sensitivity remains unknown. This tool has proven useful in the past for the analysis of the hairpin ribozyme folded structure, and has been applied to identify potential metal-binding sites on the hairpin and extended hammerhead ribozymes.

| S-EPMC3897194 | biostudies-literature

Common fold in helix-hairpin-helix proteins.

Project description:Helix-hairpin-helix (HhH) is a widespread motif involved in non-sequence-specific DNA binding. The majority of HhH motifs function as DNA-binding modules, however, some of them are used to mediate protein-protein interactions or have acquired enzymatic activity by incorporating catalytic residues (DNA glycosylases). From sequence and structural analysis of HhH-containing proteins we conclude that most HhH motifs are integrated as a part of a five-helical domain, termed (HhH)(2) domain here. It typically consists of two consecutive HhH motifs that are linked by a connector helix and displays pseudo-2-fold symmetry. (HhH)(2) domains show clear structural integrity and a conserved hydrophobic core composed of seven residues, one residue from each alpha-helix and each hairpin, and deserves recognition as a distinct protein fold. In addition to known HhH in the structures of RuvA, RadA, MutY and DNA-polymerases, we have detected new HhH motifs in sterile alpha motif and barrier-to-autointegration factor domains, the alpha-subunit of Escherichia coli RNA-polymerase, DNA-helicase PcrA and DNA glycosylases. Statistically significant sequence similarity of HhH motifs and pronounced structural conservation argue for homology between (HhH)(2) domains in different protein families. Our analysis helps to clarify how non-symmetric protein motifs bind to the double helix of DNA through the formation of a pseudo-2-fold symmetric (HhH)(2) functional unit.

| S-EPMC102670 | biostudies-literature

Antigenomic delta ribozyme variants with mutations in the catalytic core obtained by the in vitro selection method.

Project description:We have used the in vitro selection method to search for catalytically active variants of the antigenomic delta ribozyme with mutations in the regions that constitute the ribozyme active site: L3, J1/4 and J4/2. In the initial combinatorial library 16 nt positions were randomized and the library contained a full representation of all possible sequences. Following ten cycles of selection-amplification several catalytically active ribozyme variants were identified. It turned out that one-third of the variants contained only single mutation G80U and their activity was similar to that of the wild-type ribozyme. Unexpectedly, in the next one-third of the variants the C76 residue, which was proposed to play a crucial role in the ribozyme cleavage mechanism, was mutated. In these variants, however, a cytosine residue was present in a neighboring position to the polynucleotide chain. It shows that the ribozyme catalytic core possesses substantial 'structural plasticity' and the capacity of functional adaptation. Four selected ribozyme variants were subjected to more detailed analysis. It turned out that the variants differed in their relative preferences towards Mg2+, Ca2+ and Mn2+ ions. Thus, the functional properties of the variants were dependent on both the structure of their catalytic sites and divalent metal ions performing catalysis.

| S-EPMC1388270 | biostudies-literature

Hairpin ribozyme-antisense RNA constructs can act as molecular Lassos.

Project description:We have developed a novel class of antisense agents, RNA Lassos, which are capable of binding to and circularizing around complementary target RNAs. The RNA Lasso consists of a fixed sequence derived from the hairpin ribozyme and an antisense segment whose size and sequence can be varied to base pair with accessible sites in the target RNA. The ribozyme catalyzes self-processing of the 5'- and 3'-ends of a transcribed Lasso precursor and ligates the processed ends to produce a circular RNA. The circular and linear forms of the self-processed Lasso coexist in an equilibrium that is dependent on both the Lasso sequence and the solution conditions. Lassos form strong, noncovalent complexes with linear target RNAs and form true topological linkages with circular targets. Lasso complexes with linear RNA targets were detected by denaturing gel electrophoresis and were found to be more stable than ordinary RNA duplexes. We show that expression of a fusion mRNA consisting of a sequence from the murine tumor necrosis factor-alpha (TNF-alpha) gene linked to luciferase reporter can be specifically and efficiently blocked by an anti-TNF Lasso. We also show in cell culture experiments that Lassos directed against Fas pre-mRNA were able to induce a change in alternative splicing patterns.

| S-EPMC2588507 | biostudies-literature

Pressure modulates the self-cleavage step of the hairpin ribozyme.

Project description:The ability of certain RNAs, denoted as ribozymes, to not only store genetic information but also catalyse chemical reactions gave support to the RNA world hypothesis as a putative step in the development of early life on Earth. This, however, might have evolved under extreme environmental conditions, including the deep sea with pressures in the kbar regime. Here we study pressure-induced effects on the self-cleavage of hairpin ribozyme by following structural changes in real-time. Our results suggest that compression of the ribozyme leads to an accelerated transesterification reaction, being the self-cleavage step, although the overall process is retarded in the high-pressure regime. The results reveal that favourable interactions between the reaction site and neighbouring nucleobases are strengthened under pressure, resulting therefore in an accelerated self-cleavage step upon compression. These results suggest that properly engineered ribozymes may also act as piezophilic biocatalysts in addition to their hitherto known properties.

| S-EPMC5379106 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data