Biomedical subjects

Jane A Grasby

Publications and source records attributed to Jane A Grasby.

11 recordsLinked to original sources

PubMed2004

The role of phosphate groups in the VS ribozyme-substrate interaction.

The VS ribozyme trans-cleavage substrate interacts with the catalytic RNA via tertiary interactions. To study the role of phosphate groups in the ribozyme-substrate interaction, 18 modified substrates were synthesized, where an epimeric phosphorothioate replaces one of the phosphate diester linkages. Sites in the stem-loop substrate where phosphorothioate substitution impaired reaction cluster in two regions. The first site is the scissile phosphate diester linkage and nucleotides downstream of this and the second site is within the loop region. The addition of manganese ions caused recovery of the rate of reaction for phosphorothioate substitutions between A621 and A622 and U631 and C632, suggesting that these two phosphate groups may serve as ligands for two metal ions. In contrast, significant manganese rescue was not observed for the scissile phosphate diester linkage implying that electrophilic catalysis by metal ions is unlikely to contribute to VS ribozyme catalysis. In addition, an increase in the reaction rate of the unmodified VS ribozyme was observed when a mixture of magnesium and manganese ions acted as the cofactor. One possible explanation for this effect is that the cleavage reaction of the VS ribozyme is rate limited by a metal dependent docking of the substrate on the ribozyme.

Base Sequence↗

PubMed2004

The synthesis and properties of oligoribonucleotide-spermine conjugates.

Polyamines stabilise nucleic acids against chemical and enzymatic degradation, facilitate the formation of secondary and tertiary structures and enhance cellular uptake. Therefore methods for the syntheses of polyamine-nucleic acid conjugates are of interest. A route for the syntheses of RNA-spermine conjugates has been developed. The polyamine was introduced to the C-5 position of uridine via an ethyl tether and the molecule elaborated into a synthon suitable for oligoribonucleotide assembly. The resultant oligomers were components of the hairpin ribozyme. Characterisation of the spermine-conjugated catalytic RNA revealed that attachment of the polyamine was well tolerated in three of four positions, namely U41, U37 and U34, suggesting that conjugation to C-5 brings about minimal structural perturbation.

Base Sequence↗

PubMed2004

Sequence-specific cleavage of RNA in the absence of divalent metal ions by a DNAzyme incorporating imidazolyl and amino functionalities.

Two modified 2'-deoxynucleoside 5'-triphosphates have been used for the in vitro selection of a modified deoxyribozyme (DNAzyme) capable of the sequence-specific cleavage of a 12 nt RNA target in the absence of divalent metal ions. The modified nucleotides, a C5-imidazolyl-modified dUTP and 3-(aminopropynyl)-7-deaza-dATP were used in place of TTP and dATP during the selection and incorporate two extra protein-like functionalities, namely, imidazolyl (histidine analogue) and primary amino (lysine analogue) into the DNAzyme. The functional groups are analogous to the catalytic Lys and His residues employed during the metal-independent cleavage of RNA by the protein enzyme RNaseA. The DNAzyme requires no divalent metal ions or other cofactors for catalysis, remains active at physiological pH and ionic strength and can recognize and cleave a 12 nt RNA substrate with sequence specificity. This is the first example of a functionalized, metal-independent DNAzyme that recognizes and cleaves an all-RNA target in a sequence-specific manner. The selected DNAzyme is two orders of magnitude more efficient in its cleavage of RNA than an unmodified DNAzyme in the absence of metal ions and represents a rate enhancement of 10(5) compared with the uncatalysed hydrolysis of RNA.

Base Sequence↗

PubMed2003

Determination of the catalytic parameters of the N-terminal half of Escherichia coli ribonuclease E and the identification of critical functional groups in RNA substrates.

Ribonuclease E is required for the rapid decay and correct processing of RNA in Escherichia coli. A detailed understanding of the hydrolysis of RNA by this and related enzymes will require the integration of structural and molecular data with quantitative measurements of RNA hydrolysis. Therefore, an assay for RNaseE that can be set up to have relatively high throughput while being sensitive and quantitative will be advantageous. Here we describe such an assay, which is based on the automated high pressure liquid chromatography analysis of fluorescently labeled RNA samples. We have used this assay to optimize reaction conditions, to determine for the first time the catalytic parameters for a polypeptide of RNaseE, and to investigate the RNaseE-catalyzed reaction through the modification of functional groups within an RNA substrate. We find that catalysis is dependent on both protonated and unprotonated functional groups and that the recognition of a guanosine sequence determinant that is upstream of the scissile bond appears to consist of interactions with the exocyclic 2-amino group, the 7N of the nucleobase and the imino proton or 6-keto group. Additionally, we find that a ribose-like sugar conformation is preferred in the 5'-nucleotide of the scissile phosphodiester bond and that a 2'-hydroxyl group proton is not essential. Steric bulk at the 2' position in the 5'-nucleotide appears to be inhibitory to the reaction. Combined, these observations establish a foundation for the functional interpretation of a three-dimensional structure of the catalytic domain of RNaseE when solved.

Base Sequence↗

PubMed2003

Dynamic evidence for metal ion catalysis in the reaction mediated by a flap endonuclease.

On the basis of structural work, metal ions are proposed to play a catalytic role in reactions mediated by many phosphoryl transfer enzymes. To gain dynamic support for such mechanisms, the role of metal ion cofactors in phosphate diester hydrolysis catalysed by a flap endonuclease has been studied. The pH maximal rate profiles were measured in the presence of various metal ion cofactors; in each case, a single ionic form of the enzyme/cofactor accounts for the pH dependence. The kinetic pK(a)s display good correlation with the acidity of the corresponding hexahydrated metal ions, which strongly suggests a role for metal-bound hydroxide, or its equivalent ionic species, in the reaction. Comparing rates of reaction in the pH-independent regions, a small negative beta(nuc) value is observed. This suggests that expected trends in the nucleophilicity of the various metal-bound hydroxides are balanced by a second form of metal ion catalysis that is related to the acidity of the hexahydrated metal ion. This is likely to be either electrophilic catalysis or leaving group activation.

Catalysis↗

PubMed2002

The role of magnesium ions and 2'-hydroxyl groups in the VS ribozyme-substrate interaction.

The minimal substrate of the trans-cleaving Neurospora VS ribozyme has a stem-loop structure and interacts with the ribozyme by RNA tertiary interactions that remain only partially defined. The magnesium ion dependence of the catalytic parameters of a trans-cleaving VS-derived ribozyme were studied. The turnover number of the catalytic RNA was found to depend on the binding of at least three magnesium ions, with an apparent magnesium ion dissociation constant of 16mM, but K(M) was observed to be metal ion independent in the millimolar range. To address the role of 2'-hydroxyl groups of the VS substrate RNA in interactions with the ribozyme, 23 altered substrates, each with a single 2'-deoxyribonucleoside substitution, were synthesised and their kinetic properties in the VS ribozyme reaction were analysed. The removal of five 2'-hydroxyl groups, at positions G620, A621, U628, C629 and G630 inhibited the reaction, whereas at two sites, G623 and A639, reaction was stimulated by the modification. Substitution of G620 with a 2'-deoxynucleoside was expected to inhibit the reaction, in line with the critical role of this 2'-hydroxyl group in the transesterification reaction. Altered substrates in which a 2'-O-methyl nucleoside replaced A621, U628, C629 and G630 were prepared and characterised. Although removal of the hydroxyl group of A621 inhibited the turnover number of the ribozyme significantly, this activity was recovered upon 2'-O-methyl adenosine substitution, suggesting that the 2'-oxygen atom of this nucleoside forms an important contact within the ribozyme active site. A cluster of residues within the loop region of the substrate, were more modestly affected by 2'-deoxynucleoside substitution. In two cases, magnesium binding was impaired, suggesting that stem-loop I is a possible magnesium ion binding site.

Base Sequence↗

PubMed2002

A conserved tyrosine residue aids ternary complex formation, but not catalysis, in phage T5 flap endonuclease.

The flap endonucleases, or 5' nucleases, are involved in DNA replication and repair. They possess both 5'-3' exonucleolytic activity and the ability to cleave bifurcated, or branched DNA, in an endonucleolytic, structure-specific manner. These enzymes share a great degree of structural and sequence similarity. Conserved acidic amino acids, whose primary role appears to be chelation of essential divalent cation cofactors, lie at the base of the active site. A loop, or helical archway, is located above the active site. A conserved tyrosine residue lies at the base of the archway in phage T5 flap endonuclease. This residue is conserved in the structures of all flap endonucleases analysed to date. We mutated the tyrosine 82 codon in the cloned T5 5' nuclease to one encoding phenylalanine. Detailed analysis of the purified Y82F protein revealed only a modest (3.5-fold) decrease in binding affinity for DNA compared with wild-type in the absence of cofactor. The modified nuclease retains both structure-specific endonuclease and exonuclease activities. Kinetic analysis was performed using a newly developed single-cleavage assay based on hydrolysis of a fluorescently labelled oligonucleotide substrate. Substrate and products were resolved by denaturing HPLC. Steady-state kinetic analysis revealed that loss of the tyrosine hydroxyl function did not significantly impair k(cat). Pre-steady state analysis under single-turnover conditions also demonstrated little change in the rate of reaction compared to the wild-type protein. The pH dependence of the kinetic parameters for the Y82F enzyme-catalysed reaction was bell-shaped as for the wild-type protein. Thus, Y82 does not play a role in catalysis. However, steady-state analysis did detect a large (approximately 300-fold) defect in K(M). These results imply that this conserved tyrosine plays a key role in ternary complex formation (protein-DNA-metal ion), a prerequisite for catalysis.

Bacteriophages↗

PubMed2002

Interactions of mutant and wild-type flap endonucleases with oligonucleotide substrates suggest an alternative model of DNA binding.

Previous structural studies on native T5 5' nuclease, a member of the flap endonuclease family of structure-specific nucleases, demonstrated that this enzyme possesses an unusual helical arch mounted on the enzyme's active site. Based on this structure, the protein's surface charge distribution, and biochemical analyses, a model of DNA binding was proposed in which single-stranded DNA threads through the archway. We investigated the kinetic and substrate-binding characteristics of wild-type and mutant nucleases in relation to the proposed model. Five basic residues R33, K215, K241, R172, and R216, are all implicated in binding branched DNA substrates. All these residues except R172 are involved in binding to duplex DNA carrying a 5' overhang. Replacement of either K215 or R216 with a neutral amino acid did not alter kcat appreciably. However, these mutant nucleases displayed significantly increased values for Kd and Km. A comparison of flap endonuclease binding to pseudoY substrates and duplexes with a single-stranded 5' overhang suggests a better model for 5' nuclease-DNA binding. We propose a major revision to the binding model consistent with these biophysical data.

Base Sequence↗

PubMed2002

RNA footprinting analysis using ion pair reverse phase liquid chromatography.

Hydroxyl radical footprinting is a powerful technique often employed in characterization of the tertiary interactions between proteins and nucleic acids. Following the generation of a nucleic acid "ladder" either by chemical or enzymatic reactions, the radiolabeled products are traditionally separated by denaturing gel electrophoresis and further quantified by phosphorimaging techniques. Here we report the use of ion pair reverse phase liquid chromatography to analyze the products of an RNA footprinting reaction using fluorescently labeled RNA molecules. This technique offers several advantages over existing procedures, including rapid analysis, automation, and direct quantification of the cleavage products without the need to employ radiolabeling. To illustrate the resolving power of this technique, we have analyzed the products of base hydrolysis, generated from a fluorescently labeled RNA molecule and have subsequently used this method to define the solvent accessibility of the substrate strand as it docks with the hairpin ribozyme.

Base Sequence↗

PubMed2002

Holliday junction binding and processing by the RuvA protein of Mycoplasma pneumoniae.

The RuvA, RuvB and RuvC proteins of Escherichia coli act together to process Holliday junctions formed during recombination and DNA repair. RuvA has a well-defined DNA binding surface that is sculptured specifically to accommodate a Holliday junction and allow subsequent loading of RuvB and RuvC. A negatively charged pin projecting from the centre limits binding of linear duplex DNA. The amino-acid sequences forming the pin are highly conserved. However, in certain Mycoplasma and Ureaplasma species the structure is extended by four amino acids and two acidic residues forming a crucial charge barrier are missing. We investigated the significance of these differences by analysing RuvA from Mycoplasma pneumoniae. Gel retardation and surface plasmon resonance assays revealed that this protein binds Holliday junctions and other branched DNA structures in a manner similar to E. coli RuvA. Significantly, it binds duplex DNA more readily. However it does not support branch migration mediated by E. coli RuvB and when bound to junction DNA is unable to provide a platform for stable binding of E. coli RuvC. It also fails to restore radiation resistance to an E. coli ruvA mutant. The data presented suggest that the modified pin region retains the ability to promote junction-specific DNA binding, but acts as a physical obstacle to linear duplex DNA rather than as a charge barrier. They also indicate that such an obstacle may interfere with the binding of a resolvase. Mycoplasma species may therefore process Holliday junctions via uncoupled branch migration and resolution reactions.

Amino Acid Sequence↗

PubMed2002

The RuvABC resolvasome.

The RuvABC resolvasome of Escherichia coli catalyses the resolution of Holliday junctions that arise during genetic recombination and DNA repair. This process involves two key steps: branch migration, catalysed by the RuvB protein that is targeted to the Holliday junction by the structure specific RuvA protein, and resolution, which is catalysed by the RuvC endonuclease. We have quantified the interaction of the RuvA protein with synthetic Holliday junctions and have shown that the binding of the protein is highly structure-specific, and leads to the formation of a complex containing two tetramers of RuvA per Holliday junction. Our data are consistent with two tetramers of RuvA binding to the DNA recombination intermediate in a co-operative manner. Once formed this complex prevents the binding of RuvC to the Holliday junction. However, the formation of a RuvAC complex can be observed following sequential addition of the RuvC and RuvA proteins. Moreover, by examining the DNA recognition properties of a mutant RuvA protein (E55R, D56K) we show that the charge on the central pin is critical for directing the structure-specific binding by RuvA.

Bacterial Proteins↗