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Anna Marie Pyle

Publications and source records attributed to Anna Marie Pyle.

33 records · Page 2Linked to original sources
PubMed2004

Periodic cycles of RNA unwinding and pausing by hepatitis C virus NS3 helicase.

The NS3 helicase is essential for cytoplasmic RNA replication by the hepatitis C virus, and it is a representative member of helicase superfamily 2 (SF2). NS3 is an important model system for understanding unwinding activities of DExH/D proteins, and it has been the subject of extensive structural and mutational analyses. Despite intense interest in NS3, the molecular and kinetic mechanisms for RNA unwinding by this helicase have remained obscure. We have developed a combinatorial, time-resolved approach for monitoring the microscopic behaviour of a helicase at each nucleotide of a duplex substrate. By applying this analysis to NS3, we have independently established the 'physical' and 'kinetic' step size for unwinding of RNA (18 base pairs, in each case), which we relate to the stoichiometry of the functional, translocating species. Having obtained microscopic unwinding rate constants at each position along the duplex, we demonstrate that NS3 unwinds RNA through a highly coordinated cycle of fast ripping and local pausing that occurs with regular spacing along the duplex substrate, much like the stepping behaviour of cytoskeletal motor proteins.

Hepacivirus↗
PubMed2004

Backbone tracking by the SF2 helicase NPH-II.

Members of the DExH/D family of proteins, a subset of helicase superfamily 2 (SF2), are involved in virtually all aspects of RNA metabolism. NPH-II, a prototypical member of this protein family, exhibits robust RNA helicase activity in vitro. Using a series of modified substrates to explore the unwinding mechanism of NPH-II, we observed that the helicase tracks exclusively on the loading strand, where it requires covalent continuity and specifically recognizes the ribose-phosphate backbone. NPH-II unwinding was unaffected by lesions and nicks on the top strand, which has a minimal role in substrate recognition. NPH-II required physical continuity of phosphodiester linkages on the loading strand, although abasic regions were tolerated. These findings suggest that SF2 helicases are mechanistically distinct from other helicase families that can tolerate breaks in the loading strand and for which bases are the primary recognition determinant.

Binding Sites↗
PubMed2004

Solution structure of domain 5 of a group II intron ribozyme reveals a new RNA motif.

Domain 5 (D5) is the central core of group II intron ribozymes. Many base and backbone substituents of this highly conserved hairpin participate in catalysis and are crucial for binding to other intron domains. We report the solution structures of the 34-nucleotide D5 hairpin from the group II intron ai5 gamma in the absence and presence of divalent metal ions. The bulge region of D5 adopts a novel fold, where G26 adopts a syn conformation and flips down into the major groove of helix 1, close to the major groove face of the catalytic AGC triad. The backbone near G26 is kinked, exposing the base plane of the adjacent A-U pair to the solvent and causing bases of the bulge to stack intercalatively. Metal ion titrations reveal strong Mg(2+) binding to a minor groove shelf in the D5 bulge. Another distinct metal ion-binding site is observed along the minor groove side of the catalytic triad, in a manner consistent with metal ion binding in the ribozyme active site.

Base Sequence↗
PubMed2003

An alternative route for the folding of large RNAs: apparent two-state folding by a group II intron ribozyme.

Despite a growing literature on the folding of RNA, our understanding of tertiary folding in large RNAs derives from studies on a small set of molecular examples, with primary focus on group I introns and RNase P RNA. To broaden the scope of RNA folding models and to better understand group II intron function, we have examined the tertiary folding of a ribozyme (D135) that is derived from the self-splicing ai5gamma intron from yeast mitochondria. The D135 ribozyme folds homogeneously and cooperatively into a compact, well-defined tertiary structure that includes all regions critical for active-site organization and substrate recognition. When D135 was treated with increasing concentrations of Mg(2+) and then subjected to hydroxyl radical footprinting, similar Mg(2+) dependencies were seen for internalization of all regions of the molecule, suggesting a highly cooperative folding behavior. In this work, we show that global folding and compaction of the molecule have the same magnesium dependence as the local folding previously observed. Furthermore, urea denaturation studies indicate highly cooperative unfolding of the ribozyme that is governed by thermodynamic parameters similar to those for forward folding. In fact, D135 folds homogeneously and cooperatively from the unfolded state to its native, active structure, thereby demonstrating functional reversibility in RNA folding. Taken together, the data are consistent with two-state folding of the D135 ribozyme, which is surprising given the size and multi-domain structure of the RNA. The findings establish that the accumulation of stable intermediates prior to formation of the native state is not a universal feature of RNA folding and that there is an alternative paradigm in which the folding landscape is relatively smooth, lacking rugged features that obstruct folding to the native state.

Binding Sites↗
PubMed2003

RNA structure comparison, motif search and discovery using a reduced representation of RNA conformational space.

Given the wealth of new RNA structures and the growing list of RNA functions in biology, it is of great interest to understand the repertoire of RNA folding motifs. The ability to identify new and known motifs within novel RNA structures, to compare tertiary structures with one another and to quantify the characteristics of a given RNA motif are major goals in the field of RNA research; however, there are few systematic ways to address these issues. Using a novel approach for visualizing and mathematically describing macromolecular structures, we have developed a means to quantitatively describe RNA molecules in order to rapidly analyze, compare and explore their features. This approach builds on the alternative eta,theta convention for describing RNA torsion angles and is executed using a new program called PRIMOS. Applying this methodology, we have successfully identified major regions of conformational change in the 50S and 30S ribosomal subunits, we have developed a means to search the database of RNA structures for the prevalence of known motifs and we have classified and identified new motifs. These applications illustrate the powerful capabilities of our new RNA structural convention, and they suggest future adaptations with important implications for bioinformatics and structural genomics.

Algorithms↗
PubMed2003

Domains 2 and 3 interact to form critical elements of the group II intron active site.

Group II introns are self-splicing RNA molecules that also behave as mobile genetic elements. The secondary structure of group II intron RNAs is typically described as a series of six domains that project from a central wheel. Most structural and mechanistic analyses of the intron have focused on domains 1 and 5, which contain the residues essential for catalysis, and on domain 6, which contains the branch-point adenosine. Domains 2 and 3 (D2, D3) have been shown to make important contributions to intronic activity; however, information about their function is quite limited. To elucidate the role of D2 and D3 in group II ribozyme catalysis, we built a series of multi-piece ribozyme constructs based on the ai5gamma group II intron. These constructs are designed to shed light on the roles of D2 and D3 in some of the major reactions catalyzed by the intron: 5'-exon cleavage, branching, and substrate hydrolysis. Reactions with these constructs demonstrate that D3 stimulates the chemical rate constant of group II intron reactions, and that it behaves as a form of catalytic effector. However, D3 is unable to associate independently with the ribozyme core. Docking of D3 is mediated by a short duplex that is found at the base of D2. In addition to recruiting D3 into the core, the D2 stem directs the folding of the adjacent j(2/3) linker, which is among the most conserved elements in the group II intron active site. In turn, the D2 stem contributes to 5'-splice site docking and ribozyme conformational change. Nucleotide analog interference mapping suggests an interaction between the D2 stem and D3 that builds on the known theta-theta' interaction and extends it into D3. These results establish that D3 and the base of D2 are key elements of the group II intron core and they suggest a hierarchy for active-site assembly.

Base Sequence↗
PubMed2003

A group II intron inserted into a bacterial heat-shock operon shows autocatalytic activity and unusual thermostability.

Group II intron RNAs fold into catalytically active structures that catalyze their own self-splicing and subsequent transposition into DNA. Because of their remarkable enzymatic properties, it has been of interest to find new group II introns with novel properties. Here we report the cloning, sequencing, and mechanistic characterization of a new group II intron from the bacterium Azotobacter vinelandii (the AV intron). Although it bears the characteristics of the group IIB1 class, the AV intron is unusually G-C rich, and it has unusual insertion sequences and a minimal dependence on the EBS2-IBS2 tertiary interaction. The AV intron is the first bacterial intron that has been found to reside in a housekeeping gene which, in this case, encodes a heat-shock protein (hsp60). Consistent with a potential role in heat-shock regulation, kinetic analysis reveals that AV intron self-splicing is activated only at elevated temperatures. This suggests a novel pathway for the regulation of heat shock in prokaryotes and provides a first example of a thermally tolerant group II intron RNA.

Azotobacter vinelandii↗
PubMed2003

The pathway for DNA recognition and RNA integration by a group II intron retrotransposon.

Group II intron RNPs are mobile genetic elements that attack and invade duplex DNA. In this work, we monitor the invasion reaction in vitro and establish a quantitative kinetic framework for the steps of this complex cascade. We find that target site specificity is achieved after DNA binding, which occurs nonspecifically. RNP searches the bound DNA before undergoing a conformational change that is associated with identification of its specific binding site. The study reveals a facile equilibrium between intron invasion and splicing, indicating that RNP invasion of top strand DNA is a relatively unfavorable event. Group II mobility must therefore depend on the trapping of invasion products, potentially through interaction of the intron-encoded protein with the DNA target and/or initiation of reverse transcription.

Bacterial Proteins↗
PubMed2002

Metal ions in the structure and function of RNA.

It is becoming increasingly clear that RNA is more than a passive carrier of genetic information. Folded RNA molecules play key roles in almost every aspect of cellular metabolism, including protein transport, RNA splicing, peptide bond formation, and translational regulation. This is facilitated by the multifunctional nature of RNA biopolymers which can serve as rigid structural scaffolds, conformational switches, and catalysts for chemical reactions. In all cases, metal ions play a crucial role in RNA function. For folded RNA molecules, the pathway for adopting proper tertiary structure, and the stabilization of that structure, depends on specific and nonspecific interactions with certain classes of metal ions. There is a rapidly expanding repertoire of RNA structural motifs that typically sequester metal ions, and these are being studied using new spectroscopic and chemical methodologies. Many ribozymes (catalytic RNA molecules) depend on metal ions as cofactors that are explicitly involved in the chemical mechanism of catalysis. All of these functions are exemplified by recent studies of group II introns, which are among the largest ribozymes found in Nature. In this case, there are specific roles for metal ions in the folding pathway, the tertiary structure and the chemical mechanism.

Magnetic Resonance Spectroscopy↗
PubMed2002

The hepatitis C viral NS3 protein is a processive DNA helicase with cofactor enhanced RNA unwinding.

The RNA helicase/protease NS3 plays a central role in the RNA replication of hepatitis C virus (HCV), a cytoplasmic RNA virus that represents a major worldwide health problem. NS3 is, therefore, an important drug target in the effort to combat HCV. Most work has focused on the protease, rather than the helicase, activities of the enzyme. In order to further characterize NS3 helicase activity, we evaluated individual stages of duplex unwinding by NS3 alone and in complex with cofactor NS4A. Despite a putative replicative role in RNA unwinding, we found that NS3 alone is a surprisingly poor helicase on RNA, but that RNA activity is promoted by cofactor NS4A. In contrast, NS3 alone is a highly processive helicase on DNA. Phylogenetic analysis suggests that this robust DNA helicase activity is not vestigial and may have specifically evolved in HCV. Given that HCV has no replicative DNA intermediate, these findings suggest that NS3 may have the capacity to affect host DNA.

Base Sequence↗
PubMed2002

mda-5: An interferon-inducible putative RNA helicase with double-stranded RNA-dependent ATPase activity and melanoma growth-suppressive properties.

Human melanoma cells can be reprogrammed to terminally differentiate and irreversibly lose proliferative capacity by appropriate pharmacological manipulation. Subtraction hybridization identified melanoma differentiation-associated gene-5 (mda-5) as a gene induced during differentiation, cancer reversion, and programmed cell death (apoptosis). This gene contains both a caspase recruitment domain and putative DExH group RNA helicase domains. Atypical helicase motifs of MDA-5 deviate from consensus sequences but are well conserved in a potentially new group of cloned and hypothetical proteins. mda-5 is an early response gene inducible by IFN and tumor necrosis factor-alpha, responding predominantly to IFN-beta. Protein kinase C activation by mezerein further augments mda-5 expression induced by IFN-beta. Expression of mda-5 is controlled transcriptionally by IFN-beta, and the MDA-5 protein localizes in the cytoplasm. mda-5 displays RNA-dependent ATPase activity, and ectopic expression of mda-5 in human melanoma cells inhibits colony formation. In these contexts, mda-5 may function as a mediator of IFN-induced growth inhibition and/or apoptosis. MDA-5 is a double-stranded RNA-dependent ATPase that contains both a caspase recruitment domain and RNA helicase motifs, with a confirmed association with growth and differentiation in human melanoma cells.

Adenosine Triphosphatases↗
PubMed2002

Productive folding to the native state by a group II intron ribozyme.

Group II introns are large catalytic RNA molecules that fold into compact structures essential for the catalysis of splicing and intron mobility reactions. Despite a growing body of information on the folded state of group II introns at equilibrium, there is currently no information on the folding pathway and little information on the ionic requirements for folding. Folding isotherms were determined by hydroxyl radical footprinting for the 32 individual protections that are distributed throughout a group II intron ribozyme derived from intron ai5gamma. The isotherms span a similar range of Mg(2+) concentrations and share a similar index of cooperativity. Time-resolved hydroxyl radical footprinting studies show that all regions of the ribozyme fold slowly and with remarkable synchrony into a single catalytically active structure at a rate comparable to those of other ribozymes studied thus far. The rate constants for the formation of tertiary contacts and recovery of catalytic activity are identical within experimental error. Catalytic activity analyses in the presence of urea provide no evidence that the slow folding of the ai5gamma intron is attributable to the presence of unproductive kinetic traps along the folding pathway. Taken together, the data suggest that the rate-limiting step for folding of group II intron ai5gamma occurs early along the reaction pathway. We propose that this behavior resembles protein folding that is limited in rate by high contact order, or the need to form key tertiary interactions from partners that are located far apart in the primary or secondary structure.

Base Pairing↗
PubMed2002

Group II introns: highly specific endonucleases with modular structures and diverse catalytic functions.

Group II introns are large catalytic RNAs with a remarkable repertoire of reactions. Here we present construct designs and protocols that were used to develop a set of kinetic frameworks for studying the structure and reaction mechanisms of group II introns and ribozymes derived from them. In addition, we discuss application of these systems to structure/function analysis of the ai5gamma group II intron.

Catalysis↗
PubMed1998

Analysis of putative RNase sensitivity and protease insensitivity of demethylation activity in extracts from rat myoblasts.

The mechanism for demethylation of DNA in rat myoblasts has recently been studied using a new in vitro system that monitors demethylation in whole cell extracts. Previous investigations using this system had indicated that demethylation is resistant to conditions that are normally assumed to denature or digest proteins. Remarkably, it was reported that the activity appeared to be sensitive to the action of ribonuclease, suggesting a role for RNA in the demethylation of DNA. This manuscript reports that, upon further purification of the extract, demethylation activity has properties that are different. When subjected to more rigorous procedures for digestion of proteins, the demethylase activity disappears. Furthermore, RNase sensitivity of the extract disappears when a quantity of unmethylated competitor DNA is added to the reaction mix or when extracts treated with RNase are subsequently treated with protease. Although a role for RNA cannot be completely discounted, it is unlikely that this demethyl-ation reaction involves RNA cofactors or ribozyme components. These results have important implications for the mechanism of DNA demethylation and they exemplify the potential pitfalls of experiments in which new biological roles for RNA are evaluated using RNase sensitivity experiments.

Animals↗