Biomedical subjects

James R Williamson

Publications and source records attributed to James R Williamson.

At least 19 recordsLinked to original sources

PubMed2006

New RNA labeling methods offer dramatic sensitivity enhancements in 2H NMR relaxation spectra.

A new labeling strategy is presented that greatly facilitates the measurement of 2H spin relaxation rates in RNA molecules as a probe of pico- to nanosecond time scale dynamics. In this labeling scheme the sugar positions are uniformly 13C-labeled, with position 2' protonated and all other sites on the sugar deuterated. Pulse sequences are presented for measurement of 2H R1 and R2 relaxation rates at positions 1', 3', and 4' with sensitivity gains that are on the order of 5-fold relative to previous methods that employed random fractional deuteration. The improved sensitivity is transformative and facilitates the study of motion in moderately sized RNA molecules with good sensitivity. The utility of the approach is demonstrated with an application to HIV-2 TAR, where the site-specific measures of molecular dynamics at sugar positions obtained here complement previous studies of dynamics at aromatic sites in the molecule.

Carbon Isotopes↗

PubMed2006

Measurement of long-range 1H-19F scalar coupling constants and their glycosidic torsion dependence in 5-fluoropyrimidine-substituted RNA.

Long-range scalar 5J(H1',F) couplings were observed in 5-fluoropyrimidine-substituted RNA. We developed a novel S3E-19F-alpha,beta-edited NOESY experiment for quantitation of these long-range scalar 5J(H1',F) couplings, where the J-couplings can be extracted from inspection of intraresidual (H1',H6) NOE cross-peaks. Quantum chemical calculations were exploited to investigate the relation between scalar couplings and conformations around the glycosidic bond in oligonucleotides. The theoretical dependence of the observed 5J(H1',F) couplings on the torsion angle chi can be described by a generalized Karplus relationship. The corresponding density functional theory (DFT) analysis is outlined. Additional NMR experiments facilitating the resonance assignments of 5-fluoropyrimidine-substituted RNAs are described, and chemical shift changes due to altered shielding in the presence of fluorine-19 (19F) are presented.

Glycosides↗

PubMed2006

The RNA Ontology Consortium: an open invitation to the RNA community.

The aim of the RNA Ontology Consortium (ROC) is to create an integrated conceptual framework-an RNA Ontology (RO)-with a common, dynamic, controlled, and structured vocabulary to describe and characterize RNA sequences, secondary structures, three-dimensional structures, and dynamics pertaining to RNA function. The RO should produce tools for clear communication about RNA structure and function for multiple uses, including the integration of RNA electronic resources into the Semantic Web. These tools should allow the accurate description in computer-interpretable form of the coupling between RNA architecture, function, and evolution. The purposes for creating the RO are, therefore, (1) to integrate sequence and structural databases; (2) to allow different computational tools to interoperate; (3) to create powerful software tools that bring advanced computational methods to the bench scientist; and (4) to facilitate precise searches for all relevant information pertaining to RNA. For example, one initial objective of the ROC is to define, identify, and classify RNA structural motifs described in the literature or appearing in databases and to agree on a computer-interpretable definition for each of these motifs. To achieve these aims, the ROC will foster communication and promote collaboration among RNA scientists by coordinating frequent face-to-face workshops to discuss, debate, and resolve difficult conceptual issues. These meeting opportunities will create new directions at various levels of RNA research. The ROC will work closely with the PDB/NDB structural databases and the Gene, Sequence, and Open Biomedical Ontology Consortia to integrate the RO with existing biological ontologies to extend existing content while maintaining interoperability.

Databases, Genetic↗

PubMed2005

An assembly landscape for the 30S ribosomal subunit.

Self-assembling macromolecular machines drive fundamental cellular processes, including transcription, messenger RNA processing, translation, DNA replication and cellular transport. The ribosome, which carries out protein synthesis, is one such machine, and the 30S subunit of the bacterial ribosome is the preeminent model system for biophysical analysis of large RNA-protein complexes. Our understanding of 30S assembly is incomplete, owing to the challenges of monitoring the association of many components simultaneously. Here we have developed a method involving pulse-chase monitored by quantitative mass spectrometry (PC/QMS) to follow the assembly of the 20 ribosomal proteins with 16S ribosomal RNA during formation of the functional particle. These data represent a detailed and quantitative kinetic characterization of the assembly of a large multicomponent macromolecular complex. By measuring the protein binding rates at a range of temperatures, we find that local transformations throughout the assembling subunit have similar but distinct activation energies. Thus, the prevailing view of 30S assembly as a pathway proceeding through a global rate-limiting conformational change must give way to one in which the assembly of the complex traverses a landscape dotted with various local conformational transitions.

Kinetics↗

PubMed2005

RNA helical packing in solution: NMR structure of a 30 kDa GAAA tetraloop-receptor complex.

Tertiary interactions are critical for proper RNA folding and ribozyme catalysis. RNA tertiary structure is often condensed through long-range helical packing interactions mediated by loop-receptor motifs. RNA structures displaying helical packing by loop-receptor interactions have been solved by X-ray crystallography, but not by NMR. Here, we report the NMR structure of a 30 kDa GAAA tetraloop-receptor RNA complex. In order to stabilize the complex, we used a modular design in which the RNA was engineered to form a homodimer, with each subunit containing a GAAA tetraloop phased one helical turn apart from its cognate 11-nucleotide receptor domain. The structure determination utilized specific isotopic labeling patterns (2H, 13C and 15N) and refinement against residual dipolar couplings. We observe a unique and highly unusual chemical shift pattern for an adenosine platform interaction that reveals a spectroscopic fingerprint for this motif. The structure of the GAAA tetraloop-receptor interaction is well defined solely from experimental NMR data, shows minor deviations from previously solved crystal structures, and verifies the previously inferred hydrogen bonding patterns within this motif. This work demonstrates the feasibility of using engineered homodimers as modular systems for the determination of RNA tertiary interactions by NMR.

Base Sequence↗

PubMed2005

The binding interface between Bacillus stearothermophilus ribosomal protein S15 and its 5'-translational operator mRNA.

The Bacillus stearothermophilus ribosomal protein S15 (BS15) binds a purine-rich three-helix junction motif in the central domain of 16S ribosomal RNA (rRNA) as well as a translational operator located in the 5'-untranslated region (5'-UTR) of its cognate messenger RNA (mRNA). An in-frame fusion between the 5'-UTR of the BS15 gene and beta-galactosidase (lacZ) was prepared, and tested for BS15-dependent translational repression of lacZ activity in Escherichia coli. The presence of BS15 in trans represses lacZ activity 24-fold. A series of detailed point mutations in BS15 were tested for their effects upon translational repression of lacZ activity. These point mutations demonstrated that the 5'-UTR-BS15 binding interface utilizes many of the same conserved amino acid residues implicated in the binding of BS15 to 16S rRNA. The data demonstrate that the S15 protein can bind to an RNA target motif based primarily upon appropriate minor groove and sugar-phosphate backbone contacts, irrespective of the specific RNA sequence.

5' Untranslated Regions↗

PubMed2005

The catalytic diversity of RNAs.

The natural RNA enzymes catalyse phosphate-group transfer and peptide-bond formation. Initially, metal ions were proposed to supply the chemical versatility that nucleotides lack. In the ensuing decades, structural and mechanistic studies have substantially altered this initial viewpoint. Whereas self-splicing ribozymes clearly rely on essential metal-ion cofactors, self-cleaving ribozymes seem to use nucleotide bases for their catalytic chemistry. Despite the overall differences in chemical features, both RNA and protein enzymes use similar catalytic strategies.

Animals↗

PubMed2005

Dual modes of RNA-silencing suppression by Flock House virus protein B2.

As a counter-defense against antiviral RNA silencing during infection, the insect Flock House virus (FHV) expresses the silencing suppressor protein B2. Biochemical experiments show that B2 binds to double-stranded RNA (dsRNA) without regard to length and inhibits cleavage of dsRNA by Dicer in vitro. A cocrystal structure reveals that a B2 dimer forms a four-helix bundle that binds to one face of an A-form RNA duplex independently of sequence. These results suggest that B2 blocks both cleavage of the FHV genome by Dicer and incorporation of FHV small interfering RNAs into the RNA-induced silencing complex.

Crystallography↗

PubMed2004

Shape-specific nucleotide binding of single-stranded RNA by the GLD-1 STAR domain.

Proteins containing the STAR RNA-binding domain fulfill vital roles in RNA biogenesis, yet a detailed understanding of STAR domain RNA binding specificity is lacking. In Caenorhabditis elegans, the STAR protein GLD-1 directly binds the 28 nucleotide recognition element TGE within the 3' untranslated region of tra-2 mRNA. The GLD-1:TGE interaction promotes translational silencing of tra-2 mRNA, marking a pivotal event in the spermatogenesis to oogenesis switch in C.elegans hermaphrodites. By measuring the binding affinities of both GLD-1 and TGE mutants, we have explored the molecular determinants of STAR domain specificity. Site-directed GLD-1 mutants were guided by sequence homology with human splicing factor 1 (SF1), for which an RNA:protein complex structure is available in the work done by Liu et al. The RNA binding affinity of 11 mutant GLD-1 proteins was measured, and their binding specificity was assessed with a series of TGE RNAs containing natural or modified nucleotides. This combinatorial analysis of both RNA and protein mutants revealed a diverse array of specificities of individual nucleotide-binding pockets along the interface. At nucleotide position 18, adenosine appears to be specified by the overall shape of a pocket lined with aliphatic side-chains. At position 19, the high preference for cytidine is dependent on both the length of an amino acid side-chain and the identity of terminal functional groups. The nucleotide 21 binding pocket exhibits low discrimination for cytidine, and accommodates most nucleobases. The highly hydrophobic binding interface and apparent small number of hydrogen bonding read-out interactions at these positions is consistent with our finding that few amino acids seem to function individually in establishing binding specificity. Rather, specificity is conferred by the shape of the nucleotide-binding pocket. Our data provide the first detailed, quantitative analysis of the STAR domain, and highlight features of STAR:RNA recognition that are distinct among single-stranded RNA-binding proteins.

Alanine↗

PubMed2004

RNA tertiary structure and cooperative assembly of a large ribonucleoprotein complex.

The mechanisms that govern the ordered assembly of multiprotein ribonucleoprotein complexes are not well understood. The in vitro reconstitution of the small subunit of the bacterial ribosome provides a tractable system for the detailed study of ordered assembly. We present a quantitative thermodynamic description of the hierarchical binding of ribosomal proteins to 16S rRNA during assembly of the platform of the 30S ribosomal subunit. The binding of S8, S11, S15, and the S6:S18 heterodimer to the central domain of 16S rRNA has been measured both individually and in combination using isothermal titration calorimetry and gel mobility shift assays. Both enthalpy and free energy measurements demonstrate the cooperative binding of S15 and the S6:S18 heterodimer, but no cooperativity is observed for either S8 or S11. The results define a thermodynamic framework that describes cooperative platform assembly.

Bacteria↗

PubMed2004

Enzymatic synthesis and 19F NMR studies of 2-fluoroadenine-substituted RNA.

The production of isotopically labeled RNA remains critical to current NMR structural studies. One approach to obtain simple NMR spectra is to label with a nucleus that is not naturally occurring in RNA. Fluorine-19 can serve as a sensitive site-specific probe upon incorporation into RNA. Here we report the efficient in vitro enzymatic synthesis of 2-fluoroadenosine-5'-triphosphate and its incorporation into the HIV-2 transactivation region (TAR) of RNA by DNA template-directed transcription using phage T7 RNA polymerase. We provide unequivocal evidence for this 19F-substituted base analogue capability to selectively interact with uracil, forming 2F-A-U base pairs in RNA. The introduction of a 2-fluoroadenyl substitution is relatively nonperturbing and provides us with uniquely positioned, sensitive NMR reporter groups to monitor structural changes in the local RNA environment.

Adenine↗

PubMed2004

Specificity of the STAR/GSG domain protein Qk1: implications for the regulation of myelination.

Inadequate formation and maintenance of myelin is the basis for several neurodegenerative disorders, including leukodystrophy and multiple sclerosis. In mice, oligodendrocyte differentiation and subsequent formation of myelin requires the Quaking gene. Mutation of this gene leads to embryonic lethality or to a trembling phenotype characteristic of dysmyelination. Quaking encodes Qk1, a member of the highly conserved STAR/GSG family of RNA-binding proteins that function as master developmental regulators in higher eukaryotes. Qk1 has been implicated in the regulation of alternative splicing, stability, and translation control of mRNAs that code for myelin structural components in glial cells. We have used quantitative gel mobility shift and fluorescence polarization assays to define the nucleotide sequence specificity of the Qk1 STAR/GSG domain, and to probe the interaction between Qk1 and the 3'-untranslated region (UTR) of myelin basic protein (MBP) mRNA. The results show that Qk1 recognizes a hexanucleotide consensus element that is similar although not identical to the specificity determinant recognized by the Caenorhabditis elegans STAR/GSG protein GLD-1. Several consensus sites are present in the 3'-UTR of MBP mRNA. The highest affinity site is located within the RNA localization region, suggesting a possible role for Qk1 in restricting MBP mRNA to the myelin compartment.

3' Untranslated Regions↗

PubMed2004

Enthalpy arrays.

We report the fabrication of enthalpy arrays and their use to detect molecular interactions, including protein-ligand binding, enzymatic turnover, and mitochondrial respiration. Enthalpy arrays provide a universal assay methodology with no need for specific assay development such as fluorescent labeling or immobilization of reagents, which can adversely affect the interaction. Microscale technology enables the fabrication of 96-detector enthalpy arrays on large substrates. The reduction in scale results in large decreases in both the sample quantity and the measurement time compared with conventional microcalorimetry. We demonstrate the utility of the enthalpy arrays by showing measurements for two protein-ligand binding interactions (RNase A + cytidine 2'-monophosphate and streptavidin + biotin), phosphorylation of glucose by hexokinase, and respiration of mitochondria in the presence of 2,4-dinitrophenol uncoupler.

2,4-Dinitrophenol↗

PubMed2004

A three-fluorophore FRET assay for high-throughput screening of small-molecule inhibitors of ribosome assembly.

In one of the first steps of prokaryotic ribosome assembly, the ribosomal protein S15 binds to a three-way junction in the central domain of the 16S rRNA. Binding causes a conformational change that is required for subsequent binding events. Using a novel fluorescence resonance energy transfer assay with three fluorophores, two on the RNA and one on the S15 protein, small-molecule libraries can be screened for potential inhibitors of this initial step in ribosome assembly. The employment of three fluorophores allows both the conformational change of the RNA and the binding of S15 to be monitored in a single assay.

Aminoglycosides↗

PubMed2004

Joint X-ray and NMR refinement of the yeast L30e-mRNA complex.

L30e, a Saccharomyces cervisiae ribosomal protein, regulates its own expression by binding to a purine-rich asymmetric internal loop located in both its pre-mRNA and mature mRNA. A crystal structure of an MBP-L30e fusion protein in complex with an RNA containing the pre-mRNA regulatory site was solved at 3.24 A. Interestingly, the structure of the RNA differed from that observed in a previously determined NMR structure of the complex. Analysis of the NMR data led to the identification of a single imino proton resonance in the internal loop that had been incorrectly assigned and was principally responsible for the erroneous RNA structure. A structure refinement was performed using both the X-ray diffraction data and the NMR-derived distance and angle restraints. The joint NMR and X-ray refinement resulted in improved stereochemistry and lower crystallographic R factors. The RNA internal loop of the MBP-L30e-mRNA complex adopts the canonical K-turn fold.

Crystallization↗

PubMed2003

RNA target specificity of the STAR/GSG domain post-transcriptional regulatory protein GLD-1.

The post-transcriptional regulation of gene expression underlies several critical developmental phenomena. In metazoa, gene products that are expressed, silenced and packaged during oogenesis govern early developmental processes prior to nascent transcription activation. Furthermore, tissue-specific alternative splicing of several transcription factors controls pattern formation and organ development. A highly conserved family of proteins containing a STAR/GSG RNA-binding domain is essential to both processes. Here, we identify the consensus STAR-binding element (SBE) required for specific mRNA recognition by GLD-1, a key regulator of Caenorhabditis elegans germline development. We have identified and verified new GLD-1 repression targets containing this sequence. The results suggest additional functions of GLD-1 in X-chromosome silencing and early embryogenesis. The SBE is present in Quaking and How mRNA targets, suggesting that STAR protein specificity is highly conserved. Similarities between the SBE and the branch-site signal indicate a possible competition mechanism for STAR/GSG regulation of splicing variants.

Alternative Splicing↗

PubMed2003

Application of NMR SHAPES screening to an RNA target.

Several NMR screening techniques have been developed in recent years to aid in the identification of lead drug compounds. These NMR methods have traditionally been used for protein targets, and here we examine their applicability for an RNA target. We used the SHAPES compound library to test three different NMR screening methodologies: the saturation transfer difference (STD), the 2D trNOESY, and the WaterLOGSY experiments. We found that the WaterLOGSY experiment was the most sensitive method for our RNA target, the P4P6 domain of the Tetrahymena thermophila Group I intron. Using the WaterLOGSY experiment, we found that 23 of the 112 SHAPES compounds interact with P4P6. To identify which of these 23 hits bind through nonspecific interactions, we counterscreened with a linear duplex RNA control and identified one of the SHAPES compounds as interacting with P4P6 specifically. We thus demonstrated that the WaterLOGSY experiment in combination with the SHAPES compound library can be used to efficiently find RNA binding lead compounds.

Animals↗

PubMed2003

Inherent protein structural flexibility at the RNA-binding interface of L30e.

The Saccharomyces cerevisiae ribosomal protein L30 autoregulates its own expression by binding to a purine-rich internal loop in its pre-mRNA and mRNA. NMR studies of L30 and its RNA complex showed that both the internal loop of the RNA as well as a region of the protein become substantially more ordered upon binding. A crystal structure of a maltose binding protein (MBP)-L30 fusion protein with two copies in the asymmetric unit has been determined. The flexible RNA-binding region in the L30 copies has two distinct conformations, one resembles the RNA bound form solved by NMR and the other is unique. Structure prediction algorithms also had difficulty accurately predicting this region, which is consistent with conformational flexibility seen in the NMR and X-ray crystallography studies. Inherent conformational flexibility may be a hallmark of regions involved in intermolecular interactions.

Amino Acid Sequence↗