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A M Pyle

Publications and source records attributed to A M Pyle.

At least 19 recordsLinked to original sources
PubMed2001

Control of branch-site choice by a group II intron.

The branch site of group II introns is typically a bulged adenosine near the 3'-end of intron domain 6. The branch site is chosen with extraordinarily high fidelity, even when the adenosine is mutated to other bases or if the typically bulged adenosine is paired. Given these facts, it has been difficult to discern the mechanism by which the proper branch site is chosen. In order to dissect the determinants for branch-point recognition, new mutations were introduced in the vicinity of the branch site and surrounding domains. Single mutations did not alter the high fidelity for proper branch-site selection. However, several combinations of mutations moved the branch site systematically to new positions along the domain 6 stem. Analysis of those mutants, together with a new alignment of domain 5 and domain 6 sequences, reveals a set of structural determinants that appear to govern branch-site selection by group II introns.

Base Sequence↗
PubMed2001

Visualizing the solvent-inaccessible core of a group II intron ribozyme.

Group II introns are well recognized for their remarkable catalytic capabilities, but little is known about their three-dimensional structures. In order to obtain a global view of an active enzyme, hydroxyl radical cleavage was used to define the solvent accessibility along the backbone of a ribozyme derived from group II intron ai5gamma. These studies show that a highly homogeneous ribozyme population folds into a catalytically compact structure with an extensively internalized catalytic core. In parallel, a model of the intron core was built based on known tertiary contacts. Although constructed independently of the footprinting data, the model implicates the same elements for involvement in the catalytic core of the intron.

Base Sequence↗
PubMed2001

Guiding ribozyme cleavage through motif recognition: the mechanism of cleavage site selection by a group ii intron ribozyme.

The mechanism by which group II introns cleave the correct phosphodiester linkage was investigated by studying the reaction of mutant substrates with a ribozyme derived from intron ai5gamma. While fidelity was found to be quite high in most cases, a single mutation on the substrate (+1C) resulted in a dramatic loss of fidelity. When this mutation was combined with a second mutation that induces a bulge in the exon binding site 1/intron binding site 1 (EBS1/IBS1) duplex, the base-pairing register of the EBS1/IBS1 duplex was shifted and the cleavage site moved to a downstream position on the substrate. Conversely, when mismatches were incorporated at the EBS1/IBS1 terminus, the duplex was effectively truncated and cleavage occurred at an upstream site. Taken together, these data demonstrate that the cleavage site of a group II intron ribozyme can be tuned at will by manipulating the thermodynamic stability and structure of the EBS1/IBS1 pairing. The results are consistent with a model in which the cleavage site is not designated through recognition of specific nucleotides (such as the 5'-terminal residue of EBS1). Instead, the ribozyme detects a structure at the junction between single and double-stranded residues on the bound substrate. This finding explains the puzzling lack of phylogenetic conservation in ribozyme and substrate sequences near group II intron target sites.

Base Pair Mismatch↗
PubMed2001

Active disruption of an RNA-protein interaction by a DExH/D RNA helicase.

All aspects of cellular RNA metabolism and the replication of many viruses require DExH/D proteins that manipulate RNA in a manner that requires nucleoside triphosphates. Although DExH/D proteins have been shown to unwind purified RNA duplexes, most RNA molecules in the cellular environment are complexed with proteins. It has therefore been speculated that DExH/D proteins may also affect RNA-protein interactions. We demonstrate that the DExH protein NPH-II from vaccinia virus can displace the protein U1A from RNA in an active adenosine triphosphate-dependent fashion. NPH-II increases the rate of U1A dissociation by more than three orders of magnitude while retaining helicase processivity. This indicates that DExH/D proteins can effectively catalyze protein displacement from RNA and thereby participate in the structural reorganization of ribonucleoprotein assemblies.

3' Untranslated Regions↗
PubMed2001

Kinetic dissection of the multistep process in L1.ltrB intron mobility.

A kinetic characterization is presented for intron insertion into a target duplex DNA site during L1.ltrB intron mobility. This reaction is catalyzed by a ribonucleoprotein particle (RNP), consisting of a lariat form of group II intron RNA and the intron-encoded LtrA protein. In the first stage of intron mobility, the RNA component of the enzyme by itself inserts directly into the target ds DNA site, and thus, the RNP enzyme does not carry out turnover. Using single-turnover kinetics, we established an in vitro kinetic assay system and investigated mechanism of the intron RNA insertion process.

DNA↗
PubMed2000

A tertiary interaction that links active-site domains to the 5' splice site of a group II intron.

Group II introns are self-splicing RNAs that are commonly found in the genes of plants, fungi, yeast and bacteria. Little is known about the tertiary structure of group II introns, which are among the largest natural ribozymes. The most conserved region of the intron is domain 5 (D5), which, together with domain 1 (D1), is required for all reactions catalysed by the intron. Despite the importance of D5, its spatial relationship and tertiary contacts to other active-site constituents have remained obscure. Furthermore, D5 has never been placed directly at a site of catalysis by the intron. Here we show that a set of tertiary interactions (lambda-lambda') links catalytically essential regions of D5 and D1, creating the framework for an active-site and anchoring it at the 5' splice site. Highly conserved elements similar to components of the lambda-lambda' interaction are found in the eukaryotic spliceosome.

Base Pairing↗
PubMed2000

The DExH protein NPH-II is a processive and directional motor for unwinding RNA.

All aspects of cellular RNA metabolism and processing involve DExH/D proteins, which are a family of enzymes that unwind or manipulate RNA in an ATP-dependent fashion. DExH/D proteins are also essential for the replication of many viruses, and therefore provide targets for the development of therapeutics. All DExH/D proteins characterized to date hydrolyse nucleoside triphosphates and, in most cases, this activity is stimulated by the addition of RNA or DNA. Several members of the family unwind RNA duplexes in an NTP-dependent fashion in vitro; therefore it has been proposed that DExH/D proteins couple NTP hydrolysis to RNA conformational change in complex macromolecular assemblies. Despite the central role of DExH/D proteins, their mechanism of RNA helicase activity remains unknown. Here we show that the DExH protein NPH-II unwinds RNA duplexes in a processive, unidirectional fashion with a step size of roughly one-half helix turn. We show that there is a quantitative connection between ATP utilization and helicase processivity, thereby providing direct evidence that DExH/D proteins can function as molecular motors on RNA.

Kinetics↗
PubMed2000

Metal ion binding sites in a group II intron core.

Group II introns are catalytic RNA molecules that require divalent metal ions for folding, substrate binding, and chemical catalysis. Metal ion binding sites in the group II core have now been elucidated by monitoring the site-specific RNA hydrolysis patterns of bound ions such as Tb(3+) and Mg(2+). Major sites are localized near active site elements such as domain 5 and its surrounding tertiary interaction partners. Numerous sites are also observed at intron substructures that are involved in binding and potentially activating the splice sites. These results highlight the locations of specific metal ions that are likely to play a role in ribozyme catalysis.

Base Sequence↗
PubMed1999

Antagonistic substrate binding by a group II intron ribozyme.

In this study, the thermodynamic properties of substrate-ribozyme recognition were explored using a system derived from group II intron ai5gamma. Substrate recognition by group II intron ribozymes is of interest because any nucleic ac?id sequence can be targeted, the recognition sequence can be quite long (>/=13 bp), and reaction can proceed with a very high degree of sequence specificity. Group II introns target their substrates throug?h the formation of base-pairing interactions with two regions of the intron (EBS1 and EBS2), which are usually located far apart in the secondary structure. These structures pair with adjacent, corresponding sites (IBS1 and IBS2) on the substrate. In order to understand the relative energetic contribution of each base-pairing interaction (EBS1-IBS1 or EBS2-IBS2) to substrate binding energy, the free energy of each helix was measured. The individual helices were found to have base-pairing free energies similar to those calculated for regular RNA duplexes of the same sequence, suggesting that each recognition helix derives its binding energy from base-pairing interactions alone and that each helix can form independently. Most interestingly, it was found that the sum of the measured individual free energies (approximately 20 kcal/mol) was much higher than the known free energy for substrate binding (approximately 12 kcal/mol). This indicates that certain group II intron ribozymes can bind their substrates in an antagonistic fashion, paying a net energetic penalty upon binding the full-length substrate. This loss of binding energy is not due to weakening of individual helices, but appears to be linked to ribozyme conformational changes induced by substrate binding. This coupling between substrate binding and ribozyme conformational rearrangement may provide a mechanism for lowering overall substrate binding energy while retaining the full information content of 13 bp, thus resulting in a mechanism for ensuring sequence specificity.

Binding Sites↗
PubMed1999

Site-specific labeling of RNA with fluorophores and other structural probes.

Site-specific probes provide a powerful tool for structure and function studies of nucleic acids, especially in elucidating tertiary structures of large ribozymes and other folded RNA molecules. Among many types of extrinsic labels, fluorophores are most attractive because they can provide structural information at millisecond time resolution, thus allowing real-time observation of structural transition during biological function. Methods for introducing fluorophores in RNA molecules are summarized here. These methods are robust and readily applicable to the labeling of other types of probes. However, as each case of RNA modification is unique, fine tuning of the general methodology is beneficial.

Amines↗
PubMed1999

Calculating the electrostatic properties of RNA provides new insights into molecular interactions and function.

Solutions to the nonlinear Poisson-Boltzmann equation were used to obtain the electrostatic potentials of RNA molecules that have known three-dimensional structures. The results are described in terms of isopotential contours and surface electrostatic potential maps. Both representations have unexpected features: 'cavities' within isopotential contours and areas of enhanced negative potential on molecular surfaces. Intriguingly, the sites of unusual electrostatic features correspond to functionally important regions, suggesting that electrostatic properties play a key role in RNA recognition and stabilization. These calculations reveal that the electrostatic potentials generated by RNA molecules have a variety of functionally important characteristics that cannot be discerned by simple visual inspection of the molecular structure.

Anticodon↗
PubMed1998

Stepping through an RNA structure: A novel approach to conformational analysis.

Drawing from the growing database of complex three-dimensional RNA structures, a systematic method has been developed for classifying and analyzing the variety of conformations adopted by nucleic acids. This method is based on the development of a reduced representation for nucleic acid backbone conformation, simplifying the formidable eight-dimensional problem that has long complicated nucleic acid conformational analysis. Two pseudotorsion angles (eta and theta) have been defined, based on the selection of two appropriate pivot points along the RNA backbone, P and C4'. These pseudotorsions, together with a complete library of conventional torsion angles, can be calculated for any RNA structure or all-atom model using a new program called AMIGOS. Having computed eta and theta pseudotorsions for each position on an RNA molecule, they can be represented on a two-dimensional plot similar to the phi-phi plots that have traditionally been used for protein conformational analysis. Like a Ramachandran plot, clusters of residues appear at discrete regions on an eta-theta plot. Nucleotides within these clusters share conformational properties, often belonging to the same type of structural motif such as A-platforms, sheared tandem purine-purine pairs and GNRA tetraloops. An eta-theta plot provides a two-dimensional representation of the conformational properties of an entire RNA molecule, facilitating rapid analysis of structural features. In addition to the utility of eta-theta plots for intuitive visualization of conformational space, the pseudotorsional convention described here should significantly simplify approaches to macromolecular modeling of RNA structure.

Algorithms↗
PubMed1998

Defining functional groups, core structural features and inter-domain tertiary contacts essential for group II intron self-splicing: a NAIM analysis.

Group II introns are self-splicing RNA molecules that are of considerable interest as ribozymes, mobile genetic elements and examples of folded RNA. Although these introns are among the most common ribozymes, little is known about the chemical and structural determinants for their reactivity. By using nucleotide analog interference mapping (NAIM), it has been possible to identify the nucleotide functional groups (Rp phosphoryls, 2'-hydroxyls, guanosine exocyclic amines, adenosine N7 and N6) that are most important for composing the catalytic core of the intron. The majority of interference effects occur in clusters located within the two catalytically essential Domains 1 and 5 (D1 and D5). Collectively, the NAIM results indicate that key tetraloop-receptor interactions display a specific chemical signature, that the epsilon-epsilon' interaction includes an elaborate array of additional features and that one of the most important core structures is an uncharacterized three-way junction in D1. By combining NAIM with site-directed mutagenesis, a new tertiary interaction, kappa-kappa', was identified between this region and the most catalytically important section of D5, adjacent to the AGC triad in stem 1. Together with the known zeta-zeta' interaction, kappa-kappa' anchors D5 firmly into the D1 scaffold, thereby presenting chemically essential D5 functionalities for participation in catalysis.

Base Sequence↗
PubMed1998

A map of the binding site for catalytic domain 5 in the core of a group II intron ribozyme.

Group II introns are ribozymes with a complex tertiary architecture that is of great interest as a model for RNA folding. Domain 5 (D5) is a highly conserved region of the intron that is considered one of the most critical structures in the catalytic core. Despite its central importance, the means by which D5 interacts with other core elements is unclear. To obtain a map of potential interaction sites, dimethyl sulfate was used to footprint regions of the intron that are involved in D5 binding. These studies were complemented by measurements of D5 binding to a series of truncated intron derivatives. In this way, the minimal region of the intron required for strong D5 association was defined and the sites most likely to represent thermodynamically significant positions of tertiary contact were identified. These studies show that ground-state D5 binding is mediated by tertiary contacts to specific regions of D1, including a tetraloop receptor and an adjacent three-way junction. In contrast, D2 and D3 are not found to stabilize D5 association. These data highlight the significance of D1-D5 interactions and will facilitate the identification of specific tertiary contacts between them.

Base Sequence↗
PubMed1998

The DEAH-box protein PRP22 is an ATPase that mediates ATP-dependent mRNA release from the spliceosome and unwinds RNA duplexes.

Of the proteins required for pre-mRNA splicing, at least four, the DEAH-box proteins, are closely related due to the presence of a central 'RNA helicase-like' region, and extended homology through a large portion of the protein. A major unresolved question is the function of these proteins. Indirect evidence suggests that several of these proteins are catalysts for important structural rearrangements in the spliceosome. However, the mechanism for the proposed alterations is presently unknown. We present evidence that PRP22, a DEAH-box protein required for mRNA release from the spliceosome, unwinds RNA duplexes in a concentration- and ATP-dependent manner. This demonstrates that PRP22 can modify RNA structure directly. We also show that the PRP22-dependent release of mRNA from the spliceosome is an ATP-dependent process and that recombinant PRP22 is an ATPase. Non-hydrolyzable ATP analogs did not substitute for ATP in the RNA-unwinding reaction, suggesting that ATP hydrolysis is required for this reaction. Specific mutation of a putative ATP phosphate-binding motif in the recombinant protein eliminated the ATPase and RNA-unwinding capacity. Significantly, these data suggest that the DEAH-box proteins act directly on RNA substrates within the spliceosome.

Adenosine Triphosphatases↗
PubMed1998

Sequence specificity of a group II intron ribozyme: multiple mechanisms for promoting unusually high discrimination against mismatched targets.

Group II intron ai5 gamma was reconstructed into a multiple-turnover ribozyme that efficiently cleaves small oligonucleotide substrates in-trans. This construct makes it possible to investigate sequence specificity, since second-order rate constants (kcat/K(m), or the specificity constant) can be obtained and compared with values for mutant substrates and with other ribozymes. The ribozyme used in this study consists of intron domains 1 and 3 connected in-cis, together with domain 5 as a separate catalytic cofactor. This ribozyme has mechanistic features similar to the first step of reverse-splicing, in which a lariat intron attacks exogenous RNA and DNA substrates, and it therefore serves as a model for the sequence specificity of group II intron mobility. To quantitatively evaluate the sequence specificity of this ribozyme, the WT kcat/Km value was compared to individual kcat/Km values for a series of mutant substrates and ribozymes containing single base changes, which were designed to create mismatches at varying positions along the two ribozyme-substrate recognition helices. These mismatches had remarkably large effects on the discrimination index (1/relative kcat/K(m)), resulting in values > 10,000 in several cases. The delta delta G++ for mismatches ranged from 2 to 6 kcal/mol depending on the mismatch and its position. The high specificity of the ribozyme is attributable to effects on duplex stabilization (1-3 kcal/mol) and unexpectedly large effects on the chemical step of reaction (0.5-2.5 kcal/mol). In addition, substrate association is accompanied by an energetic penalty that lowers the overall binding energy between ribozyme and substrate, thereby causing the off-rate to be faster than the rate of catalysis and resulting in high specificity for the cleavage of long target sequences (> or = 13 nucleotides).

Base Composition↗