Kinetic oligonucleotide hybridization for monitoring kinetic folding of large RNAs.
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Base Sequence↗
Biomedical subjects
J R Williamson
Publications and source records attributed to J R Williamson.
A hierarchy of RNA subdomains in assembly of the central domain of the 30 S ribosomal subunit.
Beginning with the framework that has been developed for the assembly of the 30 S ribosomal subunit, we have identified a series of RNAs that are minimal binding sites for proteins S15, S6, S18, and S11 in the central domain from Thermus thermophilus. The minimal binding RNA for proteins S15, S6, and S18 consists of helix 22 and three-way junctions at both ends composed of portions of helices 20, 21, and 23. Addition of the remaining portion of helix 23 to this construct results in the minimal site for S11. Surprisingly, almost half of the central domain (helices 24, 25, and 26) is dispensable for binding the central domain proteins. Thus, at least two classes of RNA elements can be identified in ribosomal RNA. A protein-binding core element (such as helices 20, 21, 22, and 23) is required for the association of ribosomal proteins, whereas secondary binding elements (such as helices 24, 25, and 26) associate only with the preformed core RNP complex. Apparently, there may be a hierarchy of ribosomal RNA elements similar to the hierarchy of primary, secondary, and tertiary binding ribosomal proteins.
Base Sequence↗
Induced fit in RNA-protein recognition.
Two generalizations can be drawn from the recent rapid progress in understanding RNA-protein interactions. First, there is a great diversity of observed protein and RNA structural motifs. Second, formation of almost every RNA-protein complex that has been characterized involves conformational changes in the protein, the RNA, or both. The role of these conformational changes in the biological function of RNA-protein complexes is not at all clear. Whether or not conformational changes are a critical feature of ribonucleoprotein complex assembly or are an unimportant mechanistic detail, the ubiquity of these changes warrants careful consideration of their implications.
Molecular Structure↗
An optimal Mg(2+) concentration for kinetic folding of the tetrahymena ribozyme.
Divalent metal ions, such as Mg(2+), are generally required for tertiary structure formation in RNA. Although the role of Mg(2+) binding in RNA-folding equilibria has been studied extensively, little is known about the role of Mg(2+) in RNA-folding kinetics. In this paper, we explore the effect of Mg(2+) on the rate-limiting step in the kinetic folding pathway of the Tetrahymena ribozyme. Analysis of these data reveals the presence of a Mg(2+)-stabilized kinetic trap that slows folding at higher Mg(2+) concentrations. Thus, the Tetrahymena ribozyme folds with an optimal rate at 2 mM Mg(2+), just above the concentration required for stable structure formation. These results suggest that thermodynamic and kinetic folding of RNA are cooptimized at a Mg(2+) concentration that is sufficient to stabilize the folded form but low enough to avoid kinetic traps and misfolding.
Animals↗
Assignment of the L30-mRNA complex using selective isotopic labeling and RNA mutants.
The helix-loop-helix structure formed in the pre-mRNA and the mRNA of L30, a ribosomal protein from the yeast Saccharomyces cerevisiae, serves as an auto-regulatory binding site for the protein to suppress the L30 synthesis upon overproduction. Using a 33-nucleotide model RNA, the structures of the L30 binding site RNA in the presence and absence of the protein were investigated using nuclear magnetic resonance (NMR) spectroscopy. Homonuclear and(13)C/(15)N-based resonance assignments and spectral comparisons indicated that the purine-rich internal loop is dynamic in the free RNA but becomes ordered in the presence of L30 protein. Although the resonances in the loop region are sharper and more disperse in the bound RNA, their assignment was extremely challenging, due to spectral complexity and broadened resonances caused by local dynamics. Two strategies, namely selective(13)C/(15)N-labeling and NMR analyses of five complexes with RNA mutants, were used to overcome these difficulties. Only using these approaches could assignment of the internal loop resonances and identification of the unusual NOEs and nucleotide conformations within the internal loop be made. In the case of structural determination of the L30-mRNA complex, it was critical to be able to take advantage of the available biochemical information in order to complete the structure determination.
Base Sequence↗
Local folding coupled to RNA binding in the yeast ribosomal protein L30.
The ribosomal protein L30 from yeast Saccharomyces cerevisiae auto-regulates its own synthesis by binding to a structural element in both its pre-mRNA and its mRNA. The three-dimensional structures of L30 in the free (f L30) and the pre-mRNA bound (b L30) forms have been solved by nuclear magnetic resonance spectroscopy. Both protein structures contain four alternating alpha-helices and four beta-strands segments and adopt an overall topology that is an alphabetaalpha three-layer sandwich, representing a unique fold. Three loops on one end of the alphabetaalpha sandwich have been mapped as the RNA binding site on the basis of structural comparison, chemical shift perturbation and the inter-molecular nuclear Overhauser effects to the RNA. The structural and dynamic comparison of f L30 and b L30 reveals that local dynamics may play an important role in the RNA binding. The fourth helix in b L30 is longer than in f L30, and is stabilized by RNA binding. The exposed hydrophobic surface that is buried upon RNA binding may provide the energy necessary to drive secondary structure formation, and may account for the increased stability of b L30.
Amino Acid Sequence↗
Ligand-induced conformational changes observed in single RNA molecules.
We present the first demonstration that fluorescence resonance energy transfer can be used to track the motion of a single molecule undergoing conformational changes. As a model system, the conformational changes of individual three-helix junction RNA molecules induced by the binding of ribosomal protein S15 or Mg(2+) ions were studied by changes in single-molecule fluorescence. The transition from an open to a folded configuration was monitored by the change of fluorescence resonance energy transfer between two different dye molecules attached to the ends of two helices in the RNA junction. Averaged behavior of RNA molecules closely resembles that of unlabeled molecules in solution determined by other bulk assays, proving that this approach is viable and suggesting new opportunities for studying protein-nucleic acids interactions. Surprisingly, we observed an anomalously broad distribution of RNA conformations at intermediate ion concentrations that may be attributed to foldability differences among RNA molecules. In addition, an experimental scheme was developed where the real-time response of single molecules can be followed under changing environments. As a demonstration, we repeatedly changed Mg(2+) concentration in the buffer while monitoring single RNA molecules and showed that individual RNA molecules can measure the instantaneous Mg(2+) concentration with 20-ms time resolution, making it the world's smallest Mg(2+) meter.
Base Sequence↗
Epidermal growth factor and angiotensin II regulation of extracellular signal-regulated protein kinase in rat liver epithelial WB cells.
Activation of extracellular signal-regulated protein kinase (ERK) is considered essential for mitogenesis. In the present study, rat liver epithelial WB cells were used to investigate the relative roles of Ca2+, protein kinase C (PKC), and protein tyrosine phosphorylation in mitogenesis and activation of the ERK pathway stimulated by epidermal growth factor (EGF) and angiotensin II (Ang II). The sensitivity of the ERK pathway to Ca2+ was studied by using 1,2-bis (O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) to chelate intracellular Ca2+ and a low extracellular Ca2+ concentration to prevent Ca2+ influx. Agonist-induced PKC activation was diminished by inhibition of PKC by GF-109203X (bisindolylmaleimide) or by down-regulation of PKC by long-term treatment of the cells with phorbol myristate acetate (PMA). Our results show that although activation of PKC was critical for mitogenesis induced by Ang II or EGF, the initial activation of ERK by both agonists in these cells was essentially independent of PKC activation and was insensitive to Ca2+ mobilization. This is in contrast to the findings in some cell types that exhibit a marked dependency on mobilization of Ca2+ and/or PKC activation. On the other hand, an obligatory tyrosine phosphorylation step for activation of ERK was indicated by the use of protein tyrosine kinase inhibitors, which profoundly inhibited the activation of ERK by EGF, Ang II, and PMA. Additional experiments indicated that tyrosine phosphorylation by a cytosolic tyrosine kinase may represent a general mechanism for G-protein coupled receptor mediated ERK activation.
Angiotensin II↗
The PUMILIO-RNA interaction: a single RNA-binding domain monomer recognizes a bipartite target sequence.
Translational repression of hunchback (hb) mRNA in the posterior of the Drosophila embryo requires two copies of a bipartite sequence, the Nanos Response Element (NRE), located in the 3' untranslated region of the mRNA. The PUMILIO (PUM) protein is thought to bind the NREs and thereby repress hb translation. The RNA-binding domain of PUM defines an evolutionarily conserved family of RNA-binding proteins, the PUM-Homology Domain (PUM-HD) proteins, which have been identified in yeast, plants, and animals. The PUM RNA-binding domain, the Drosophila PUM-HD (DmPUM-HD), has been shown previously to recognize nucleotides in both the 5' and 3' halves of the NRE, suggesting that a dimer of PUM might recognize one NRE. Here, we analyze the RNA-binding affinity and stoichiometry of the DmPUM-HD and find that one DmPUM-HD monomer binds independently and with equal affinity to each NRE (KD approximately 0.5 nM). We detect no cooperative interactions between DmPUM-HD monomers bound at adjacent sites. Our results imply that a single DmPUM-HD protein recognizes nucleotides in both the 5' and 3' NRE half-sites. Based on our estimate of the intraembryonic concentration of PUM (>40 nM), we propose that in vivo nearly all NREs are occupied by a PUM monomer.
Animals↗
A self-organizing neural system for learning to recognize textured scenes.
A self-organizing ARTEX model is developed to categorize and classify textured image regions. ARTEX specializes the FACADE model of how the visual cortex sees, and the ART model of how temporal and prefrontal cortices interact with the hippocampal system to learn visual recognition categories and their names. FACADE processing generates a vector of boundary and surface properties, notably texture and brightness properties, by utilizing multi-scale filtering, competition, and diffusive filling-in. Its context-sensitive local measures of textured scenes can be used to recognize scenic properties that gradually change across space, as well as abrupt texture boundaries. ART incrementally learns recognition categories that classify FACADE output vectors, class names of these categories, and their probabilities. Top-down expectations within ART encode learned prototypes that pay attention to expected visual features. When novel visual information creates a poor match with the best existing category prototype, a memory search selects a new category with which classify the novel data. ARTEX is compared with psychophysical data, and is bench marked on classification of natural textures and synthetic aperture radar images. It outperforms state-of-the-art systems that use rule-based, backpropagation, and K-nearest neighbor classifiers.
Discrimination, Psychological↗
The role of cytosolic reductive stress in oxidant formation and diabetic complications.
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Cytosol↗
Exposing the kinetic traps in RNA folding.
Large ribozymes fold on a 'glacial' timescale compared to the folding of their protein counterparts. The sluggish folding exhibited by large RNAs results from the formation of kinetically trapped, misfolded intermediates, which are nonessential features of the folding mechanism. Newly developed mutant ribozymes that avoid kinetic traps should facilitate the study of the RNA folding problem.
Animals↗
A novel loop-loop recognition motif in the yeast ribosomal protein L30 autoregulatory RNA complex.
The yeast Saccharomyces cerevisiae ribosomal protein L30 negatively autoregulates its production by binding to a helix-loop-helix structure formed in its pre-mRNA and its mRNA. A three-dimensional solution structure of the L30 protein in complex with its regulatory RNA has been solved using NMR spectroscopy. In the complex, the helix-loop-helix RNA adopts a sharply bent conformation at the internal loop region. Unusual RNA features include a purine stack, a reverse Hoogsteen base pair (G11anti-G56syn) and highly distorted backbones. The L30 protein is folded in a three-layer alpha/beta/alpha sandwich topology, and three loops at one end of the sandwich make base-specific contacts with the RNA internal loop. The protein-RNA binding interface is divided into two clusters, including hydrophobic and aromatic stacking interactions centering around G56, and base-specific hydrogen-bonding contacts to A57, G58 and G10-U60 wobble base pair. Both the protein and the RNA exhibit a partially induced fit for binding, where loops in the protein and the internal loop in the RNA become more ordered upon complex formation. The specific interactions formed between loops on L30 and the internal loop on the mRNA constitute a novel loop-loop recognition motif where an intimate RNA-protein interface is formed between regions on both molecules that lack regular secondary structure.
Allosteric Regulation↗
Activation of ERK by Ca2+ store depletion in rat liver epithelial cells.
In rat liver epithelial (WB) cells, Ca2+ pool depletion induced by two independent methods resulted in activation of extracellular signal-regulated protein kinase (ERK). In the first method, Ca2+ pool depletion by thapsigargin increased the activity of ERK, even when rise in cytosolic Ca2+ was blocked with the Ca2+ chelator BAPTA-AM. For the second method, addition of extracellular EGTA at a concentration shown to deplete intracellular Ca2+ pools also increased ERK activity. In each instance, ERK activation, as measured by an immunocomplex kinase assay, was greatly reduced by the tyrosine kinase inhibitor genistein, suggesting that Ca2+ store depletion increased ERK activity through a tyrosine kinase pathway. The intracellular Ca2+-releasing agent thapsigargin increased Fyn activity, which was unaffected by BAPTA-AM pretreatment, suggesting that Fyn activity was unaffected by increased cytosolic free Ca2+. Furthermore, depletion of intracellular Ca2+ with EGTA caused inactivation of protein phosphatase 2A and protein tyrosine phosphatases. ANG II-induced activations of Fyn, Raf-1, and ERK were augmented in cells pretreated with BAPTA-AM, but ANG II-induced expression of the dual-specificity phosphatase mitogen-activated protein kinase phosphatase-1 was blocked by BAPTA-AM pretreatment. Together these results indicate that ERK activity is regulated by the balance of phosphorylation vs. dephosphorylation reactions in intact cells and that the amount of Ca2+ stored in intracellular pools plays an important role in this regulation.
Angiotensin II↗
Resistance to TNF-alpha cytotoxicity can be achieved through different signaling pathways in rat mesangial cells.
We reported previously that Ro-318220 blocked expression of mitogen-activated protein kinase phosphatase-1 (MKP-1) induced by tumor necrosis factor-alpha (TNF-alpha) and subsequently caused apopotosis in mesangial cells (Y.-L. Guo, B. Kang, and J. R. Williamson. J. Biol. Chem. 273: 10362-10366, 1998). These data support our hypothesis that a TNF-alpha-inducible phosphatase may be responsible for preventing sustained activation of c-Jun NH2-terminal protein kinase (JNK) and consequent cell death in these cells (Y.-L. Guo, K. Baysal, B. Kang, L.-J. Yang, and J. R. Williamson. J. Biol. Chem. 273: 4027-4034, 1998). In this study, we investigated the involvement of protein kinase C (PKC) in regulation of MKP-1 expression in mesangial cells together with effects on viability. Although originally characterized as a PKC inhibitor, Ro-318220 inhibited TNF-alpha-induced MKP-1 expression through a mechanism other than blocking the PKC pathway. Furthermore, inhibition of the PKC pathway neither significantly affected TNF-alpha-induced MKP-1 expression nor made cells susceptible to toxic effect of TNF-alpha. Thus PKC activation is not essential for cells to achieve the resistance to TNF-alpha cytotoxicity displayed by normal mesangial cells. However, activation of PKC by phorbol 12-myristate 13-acetate (PMA) dramatically increased cellular resistance to the apoptotic effect of TNF-alpha. Coincidentally, PMA stimulated MKP-1 expression and suppressed JNK activation. Therefore, PMA-induced MKP-1 expression may contribute to the protective effect of PMA. These results provide a mechanistic explanation for previous documentation that PKC activation can rescue some cells from apopotosis.
Animals↗
Tumor necrosis factor-alpha and ceramide induce cell death through different mechanisms in rat mesangial cells.
It has been proposed that ceramide acts as a cellular messenger to mediate tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis. Based on this hypothesis, it was postulated that resistance of some cells to TNF-alpha cytotoxicity was due to an insufficient production of ceramide on stimulation by TNF-alpha. The present study was initiated to investigate whether this was the case in mesangial cells, which normally are insensitive to TNF-alpha-induced apoptosis. Our results indicate that although C2 ceramide was toxic to mesangial cells, the cell death it induced differed both morphologically and biochemically from that induced by TNF-alpha in the presence of cycloheximide (CHX). The most apparent effect of C2 ceramide was to cause cells to swell, followed by disruption of the cell membrane. It is evident that C2 ceramide caused cell death by necrosis, whereas TNF-alpha in the presence of CHX killed the cells by apoptosis. C2 ceramide did not mimic the effects of TNF-alpha on the activation of c-Jun NH2-terminal protein kinase and nuclear factor-kappaB transcription factor. Although mitogen-activated protein kinase [extracellular signal-related kinase (ERK)] was activated by both C2 ceramide and TNF-alpha, such activation appeared to be mediated by different mechanisms as judged from the kinetics of ERK activation. Furthermore, the cleavage of cytosolic phospholipase A2 during cell death induced by C2 ceramide and by TNF-alpha in the presence of CHX showed distinctive patterns. The present study provides evidence that apoptosis and necrosis use distinctive signaling machinery to cause cell death.
Animals↗
PACE analysis of RNA-peptide interactions.
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Autoradiography↗
Role for nitric oxide in the hyperpermeability and hemodynamic changes induced by intravenous VEGF.
PURPOSE: To explore the effects of brief intravenous (IV) infusion of vascular endothelial growth factor (VEGF) on vascular albumin permeability, blood flow, and vascular conductance (blood flow normalized to arterial blood pressure) in ocular tissues and brain and to assess the role of nitric oxide in mediating these changes. METHODS: A quantitative, double-tracer, radiolabeled albumin permeation method was combined with radiolabeled microspheres for assessment of changes in vascular permeability and blood flow, respectively, induced in ocular tissues by IV infusion of recombinant human VEGF165 for 20 minutes (80-450 picomoles/kg body weight). An inhibitor of nitric oxide synthase (NOS), NG-monomethyl-L-arginine (L-NMMA; 50 micromoles/kg body weight infused simultaneously with VEGF), was used to explore the role of nitric oxide in mediating the vascular changes induced by VEGF. RESULTS: Infusion of VEGF165 in thiopental-anesthetized rats dose-dependently increased 125I albumin permeation in the retina, anterior uvea, and choroid/sclera and in brain, aorta, lung, kidney, small intestine, and peripheral nerve. Mean arterial blood pressure, cardiac output, and stroke volume were decreased only at the highest dose of VEGF, whereas heart rate remained unchanged. Blood flow was increased in the anterior uvea, and vascular conductance was increased in retina, anterior uvea, choroid/sclera, and brain at the highest dose of VEGF. The NOS inhibitor, L-NMMA, blocked VEGF-induced vascular hyperpermeability in all ocular and nonocular tissues, prevented the increase in vascular conductance in all ocular tissues, and blocked the decrease in mean arterial blood pressure, cardiac output, and stroke volume. Infusion of L-NMMA alone decreased vascular conductance in choroid/sclera and kidney, slightly increased mean arterial blood pressure, and in general, did not affect 125I-albumin permeation. (L-NMMA slightly decreased albumin permeation in the retina and increased it in the brain.) CONCLUSIONS: Intravenous infusion of VEGF can acutely impair endothelial cell barrier functional integrity and relax resistance arterioles in ocular tissues and brain through a mechanism involving activation of NOS.
Animals↗