The Experts below are selected from a list of 318 Experts worldwide ranked by ideXlab platform
David E. Draper - One of the best experts on this subject based on the ideXlab platform.
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effects of osmolytes on rna secondary and Tertiary Structure stabilities and rna mg2 interactions
Journal of Molecular Biology, 2007Co-Authors: Dominic Lambert, David E. DraperAbstract:Osmolytes are small organic molecules accumulated by cells in response to osmotic stress. Although their effects on protein stability have been studied, there has been no systematic documentation of their influence on RNA. Here, the effects of nine osmolytes on the secondary and Tertiary Structure stabilities of six RNA Structures of differing complexity and stability have been surveyed. Using thermal melting analysis, m-values (change in ΔG° of RNA folding per molal concentration of osmolyte) have been measured. All the osmolytes destabilize RNA secondary Structure, although to different extents, probably because they favor solubilization of base surfaces. Osmolyte effects on Tertiary Structure, however, can be either stabilizing or destabilizing. We hypothesize that the stabilizing osmolytes have unfavorable interactions with the RNA backbone, which becomes less accessible to solvent in most Tertiary Structures. Finally, it was found that as a larger fraction of the negative charge of an RNA Tertiary Structure is neutralized by hydrated Mg2+, the RNA becomes less responsive to stabilizing osmolytes and may even be destabilized. The natural selection of osmolytes as protective agents must have been influenced by their effects on the stabilities of functional RNA Structures, though in general, the effects of osmolytes on RNA and protein stabilities do not parallel each other. Our results also suggest that some osmolytes can be useful tools for studying intrinsically unstable RNA folds and assessing the mechanisms of Mg2+-induced RNA stabilization.
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Stabilization of RNA Tertiary Structure by monovalent cations.
Journal of Molecular Biology, 2000Co-Authors: R Shiman, David E. DraperAbstract:Abstract The effects of monovalent cations (Li+, Na+, K+, Rb+, Cs+, and NH4+) on the thermal stability of RNA Tertiary Structure were investigated by UV melting. We show that with the RNA used here (nucleotides 1051-1108 of Escherichia coli 23 S rRNA with four base substitutions), monovalent cations and Mg2+ compete in stabilizing the RNA Tertiary Structure, and that the competition takes place between two boundaries: one where Mg2+ concentration is zero and the other where it is maximally stabilizing (“saturating”). The pattern of competition is the same for all monovalent cations and depends on the cation’s ability to displace Mg2+ from the RNA, its ability to stabilize Tertiary Structure in the absence of Mg2+, and its ability to stabilize Tertiary Structure at saturating Mg2+ concentrations. The stabilizing ability of a monovalent cation depends on its unhydrated ionic radius, and at a low monovalent cation concentration and saturating Mg2+, there is a (calculated) net release of a single monovalent cation/RNA molecule when Tertiary Structure is denatured. The implications are that under these conditions there is at least one binding site for monovalent cations on the RNA, the site is specifically associated with formation of stable Tertiary Structure, K+ is the most effective of the tested cations, and Mg2+ appears ineffective at this site. At high ionic strength, and in the absence of Mg2+, stabilization of Tertiary Structure is still monovalent-cation specific and ionic-radius dependent, but a larger number of cations (∼eight) are released upon RNA Tertiary Structure denaturation, and NH4+ appears to be the most effective cation in stabilizing Tertiary Structure under these conditions. In the majority of the experiments, methanol was added as a cosolvent to the buffer. Its use allowed the examination of the behavior of monovalent ions under conditions where their effects would otherwise have been too weak to be observed. Methanol stabilizes Tertiary but not secondary Structure of the RNA. There was no evidence that it either causes qualitative changes in cation-binding properties of the RNA or a change in the pattern of monovalent cation/Mg2+ competition.
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Affinities and selectivities of divalent cation binding sites within an RNA Tertiary Structure.
Journal of Molecular Biology, 1997Co-Authors: Yury V. Bukhman, David E. DraperAbstract:Abstract A 58 nucleotide fragment of Escherichia coli large subunit ribosomal RNA, nucleotides 1051 to 1108, adopts a specific Tertiary Structure normally requiring both monovalent (NH 4 + or K + ) and divalent (Mg 2+ ) ions to fold; this ion-dependent Structure is a prerequisite for recognition by ribosomal protein L11. Melting experiments have been used to show that a sequence variant of this fragment, GACG RNA, is able to adopt a stable Tertiary Structure in the presence of 1.6 M NH 4 Cl and absence of divalent ions. The similarity of this high-salt Structure to the Tertiary Structure formed under more typical salt conditions (0.1 M NH 4 Cl and several mM MgCl 2 ) was shown by its following properties: (i) an unusual ratio of hyperchromicity at 260 nm and 280 nm upon unfolding, (ii) selectivity for NH 4 + over K + or Na + , (iii) stabilization by L11 protein, and (iv) further stabilization by added Mg 2+ . Delocalized electrostatic interactions of divalent ions with nucleic acids should be very weak in the presence of >1 M monovalent salt; thus stabilization of the Tertiary Structure by low ( 2+ concentrations in these high-salt conditions suggests that Mg 2+ binds at specific site(s). GACG RNA Tertiary Structure unfolding in 1.6 M NH 4 Cl ( T m ≈ 39°C) is distinct from melting of the secondary Structure (centered at ∼72°C), and it has been possible to calculate the free energy of Tertiary Structure stabilization upon addition of various divalent cations. From these binding free energies, ion-RNA binding isotherms for Mn 2+ , Mg 2+ , Ca 2+ , Sr 2+ and Ba 2+ have been obtained. All of these ions bind at two sites: one site favors Mg 2+ and Ba 2+ and discriminates against Ca 2+ , while the other site favors binding of smaller ions over larger ones (Mg 2+ > Ca 2+ > Sr 2+ > Ba 2+ ). Weak cooperative or anticooperative interactions between the sites, also dependent on ion radius, may also be taking place.
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On the role of rRNA Tertiary Structure in recognition of ribosomal protein L11 and thiostrepton
Nucleic Acids Research, 1995Co-Authors: Ming Lu, David E. DraperAbstract:Ribosomal protein L11 and an antibiotic, thiostrepton, bind to the same highly conserved region of large subunit ribosomal RNA and stabilize a set of NH4(+)-dependent Tertiary interactions within the domain. In vitro selection from partially randomized pools of RNA sequences has been used to ask what aspects of RNA Structure are recognized by the ligands. L11-selected RNAs showed little sequence variation over the entire 70 nucleotide randomized region, while thiostrepton required a slightly smaller 58 nucleotide domain. All the selected mutations preserved or stabilized the known secondary and Tertiary Structure of the RNA. L11-selected RNAs from a pool mutagenized only around a junction Structure yielded a very different consensus sequence, in which the RNA Tertiary Structure was substantially destabilized and L11 binding was no longer dependent on NH4+. We propose that L11 can bind the RNA in two different 'modes', depending on the presence or absence of the NH4(+)-dependent Tertiary Structure, while thiostrepton can only recognize the RNA Tertiary Structure. The different RNA recognition mechanisms for the two ligands may be relevant to their different effects on protein synthesis.
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A conformational switch in a regulated mRNA involves Tertiary Structure.
Nucleic Acids Symposium Series, 1995Co-Authors: Thomas C. Gluick, Resi B. Gerstner, David E. DraperAbstract:: The E. coli alpha operon mRNA is autogenously regulated by binding the repressor ribosomal protein S4. Repression occurs via a novel mechanism in which S4 traps the mRNA in a conformation that prevents formation of the complete initiation complex. The conformations have similar stabilities but separated by a high activation energy which is a criterion for a conformational switch. The conformation switch is likely to involve alteration in Tertiary Structure as indicated by gel electrophoresis and thermal denaturation experiments. It was found that Mg2+ stabilizes the repressed form and Tertiary Structure, and H+ stabilizes the translated form and have complicated effects on Tertiary Structure.
Martin Weigt - One of the best experts on this subject based on the ideXlab platform.
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direct coupling analysis of nucleotide coevolution facilitates rna secondary and Tertiary Structure prediction
Nucleic Acids Research, 2015Co-Authors: Eleonora De Leonardis, Benjamin Lutz, Sebastian Ratz, Simona Cocco, Remi Monasson, Alexander Schug, Martin WeigtAbstract:Despite the biological importance of non-coding RNA, their structural characterization remains challenging. Making use of the rapidly growing sequence databases, we analyze nucleotide coevolution across homologous sequences via Direct-Coupling Analysis to detect nucleotide-nucleotide contacts. For a representative set of riboswitches, we show that the results of Direct-Coupling Analysis in combination with a generalized Nussinov algorithm systematically improve the results of RNA secondary Structure prediction beyond traditional covariance approaches based on mutual information. Even more importantly, we show that the results of Direct-Coupling Analysis are enriched in Tertiary Structure contacts. By integrating these predictions into molecular modeling tools, systematically improved Tertiary Structure predictions can be obtained, as compared to using secondary Structure information alone.
R Welty - One of the best experts on this subject based on the ideXlab platform.
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ribosomal protein l11 selectively stabilizes a Tertiary Structure of the gtpase center rrna domain
Journal of Molecular Biology, 2020Co-Authors: R Welty, Suzette A Pabit, Graeme L Conn, Lois Pollack, Kathleen B HallAbstract:Abstract The GTPase Center (GAC) RNA domain in bacterial 23S rRNA is directly bound by ribosomal protein L11 and this complex is essential to ribosome function. Previous co-crystal Structures of the 58-nucleotide GAC RNA bound to L11 revealed the intricate Tertiary fold of the RNA domain, with one monovalent and several divalent ions located in specific sites within the Structure. Here, we report a new crystal Structure of the free GAC that is essentially identical to the L11-bound Structure, and which retains many common sites of divalent ion occupation. This new Structure demonstrates that RNA alone folds into its Tertiary Structure with bound divalent ions. In solution, we find that this Tertiary Structure is not static, but rather is best described as an ensemble of states. While L11 protein cannot bind to the GAC until the RNA has adopted its Tertiary Structure, new experimental data show that L11 binds to Mg2+-dependent folded states which we suggest lie along the folding pathway of the RNA. We propose that L11 stabilizes a specific GAC RNA Tertiary state, corresponding to the crystal Structure, and that this Structure reflects the functionally critical conformation of the rRNA domain in the fully assembled ribosome.
Kevin M. Weeks - One of the best experts on this subject based on the ideXlab platform.
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RNA Tertiary Structure analysis by 2'-hydroxyl molecular interference.
Biochemistry, 2014Co-Authors: P. Homan, Arpit Tandon, Greggory M. Rice, Feng Ding, Nikolay V. Dokholyan, Kevin M. WeeksAbstract:We introduce a melded chemical and computational approach for probing and modeling higher-order intramolecular Tertiary interactions in RNA. 2'-Hydroxyl molecular interference (HMX) identifies nucleotides in highly packed regions of an RNA by exploiting the ability of bulky adducts at the 2'-hydroxyl position to disrupt overall RNA Structure. HMX was found to be exceptionally selective for quantitative detection of higher-order and Tertiary interactions. When incorporated as experimental constraints in discrete molecular dynamics (DMD) simulations, HMX information yielded accurate three-dimensional models, emphasizing the power of molecular interference to guide RNA Tertiary Structure analysis and fold refinement. In the case of a large, multi-domain RNA, the Tetrahymena group I intron, HMX identified multiple distinct sets of Tertiary Structure interaction groups in a single, concise experiment.
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on the significance of an rna Tertiary Structure prediction
RNA, 2010Co-Authors: Christine E Hajdin, Feng Ding, Nikolay V. Dokholyan, Kevin M. WeeksAbstract:Tertiary Structure prediction is important for understanding Structure–function relationships for RNAs whose Structures are unknown and for characterizing RNA states recalcitrant to direct analysis. However, it is unknown what root-mean-square deviation (RMSD) corresponds to a statistically significant RNA Tertiary Structure prediction. We use discrete molecular dynamics to generate RNA-like folds for Structures up to 161 nucleotides (nt) that have complex Tertiary interactions and then determine the RMSD distribution between these decoys. These distributions are Gaussian-like. The mean RMSD increases with RNA length and is smaller if secondary Structure constraints are imposed while generating decoys. The compactness of RNA molecules with true Tertiary folds is intermediate between closely packed spheres and a freely jointed chain. We use this scaling relationship to define an expression relating RMSD with the confidence that a Structure prediction is better than that expected by chance. This is the prediction significance, and corresponds to a P-value. For a 100-nt RNA, the RMSD of predicted Structures should be within 25 A u of the accepted Structure to reach the P £ 0.01 level if the secondary Structure is predicted de novo and within 14 A u if secondary Structure information is used as a constraint. This significance approach should be useful for evaluating
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Assembly of a Ribonucleoprotein Catalyst by Tertiary Structure Capture
Science, 1996Co-Authors: Kevin M. Weeks, Thomas R CechAbstract:CBP2 is an RNA Tertiary Structure binding protein required for efficient splicing of a yeast mitochondrial group I intron. CBP2 must wait for folding of the two RNA domains that make up the catalytic core before it can bind. In a subsequent step, association of the 5′ domain of the RNA is stabilized by additional interactions with the protein. Thus, CBP2 functions primarily to capture otherwise transient RNA Tertiary Structures. This simple one-RNA, one-protein system has revealed how the kinetic pathway of RNA folding can direct the assembly of a specific ribonucleoprotein complex. There are parallels to steps in the formation of a much more complex ribonucleoprotein, the 30S ribosomal subunit.
David Baker - One of the best experts on this subject based on the ideXlab platform.
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coupled prediction of protein secondary and Tertiary Structure
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Jens Meiler, David BakerAbstract:The strong coupling between secondary and Tertiary Structure formation in protein folding is neglected in most Structure prediction methods. In this work we investigate the extent to which nonlocal interactions in predicted Tertiary Structures can be used to improve secondary Structure prediction. The architecture of a neural network for secondary Structure prediction that utilizes multiple sequence alignments was extended to accept low-resolution nonlocal Tertiary Structure information as an additional input. By using this modified network, together with Tertiary Structure information from native Structures, the Q3-prediction accuracy is increased by 7–10% on average and by up to 35% in individual cases for independent test data. By using Tertiary Structure information from models generated with the rosetta de novo Tertiary Structure prediction method, the Q3-prediction accuracy is improved by 4–5% on average for small and medium-sized single-domain proteins. Analysis of proteins with particularly large improvements in secondary Structure prediction using Tertiary Structure information provides insight into the feedback from Tertiary to secondary Structure.