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RNA Complexes (rna + complex)
Selected AbstractsA mutagenic analysis of the RNase mechanism of the bacterial Kid toxin by mass spectrometryFEBS JOURNAL, Issue 17 2009Elizabeth Diago-Navarro Kid, the toxin of the parD (kis, kid) maintenance system of plasmid R1, is an endoribonuclease that preferentially cleaves RNA at the 5, of A in the core sequence 5,-UA(A/C)-3,. A model of the Kid toxin interacting with the uncleavable mimetic 5,-AdUACA-3, is available. To evaluate this model, a significant collection of mutants in some of the key residues proposed to be involved in RNA binding (T46, A55, T69 and R85) or RNA cleavage (R73, D75 and H17) were analysed by mass spectrometry in RNA binding and cleavage assays. A pair of substrates, 5,-AUACA-3,, and its uncleavable mimetic 5,-AdUACA-3,, used to establish the model and structure of the Kid,RNA complex, were used in both the RNA cleavage and binding assays. A second RNA substrate, 5,-UUACU-3, efficiently cleaved by Kid both in vivo and in vitro, was also used in the cleavage assays. Compared with the wild-type protein, mutations in the residues of the catalytic site abolished RNA cleavage without substantially altering RNA binding. Mutations in residues proposed to be involved in RNA binding show reduced binding efficiency and a corresponding decrease in RNA cleavage efficiency. The cleavage profiles of the different mutants were similar with the two substrates used, but RNA cleavage required much lower protein concentrations when the 5,-UUACU-3, substrate was used. Protein synthesis and growth assays are consistent with there being a correlation between the RNase activity of Kid and its inhibitory potential. These results give important support to the available models of Kid RNase and the Kid,RNA complex. [source] Crystallization of a ZRANB2,RNA complexACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2008Fionna E. Loughlin ZRANB2 is a zinc-finger protein that has been shown to influence alternative splice-site selection. The protein comprises a C-terminal arginine/serine-rich domain that interacts with spliceosomal proteins and two N-terminal RanBP2-type zinc fingers that have been implicated in RNA recognition. The second zinc finger bound to a six-nucleotide single-stranded RNA target sequence crystallized in the hexagonal space group P6522 or P6122, with unit-cell parameters a = 54.52, b = 54.52, c = 48.07,Å; the crystal contains one monomeric complex per asymmetric unit. This crystal form has a solvent content of 39% and diffracted to 1.4,Å resolution using synchrotron radiation. [source] Structure of the ribosomal protein L1,mRNA complex at 2.1,Å resolution: common features of crystal packing of L1,RNA complexesACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2006S. Tishchenko The crystal structure of a hybrid complex between the bacterial ribosomal protein L1 from Thermus thermophilus and a Methanococcus vannielii mRNA fragment containing an L1-binding site was determined at 2.1,Å resolution. It was found that all polar atoms involved in conserved protein,RNA hydrogen bonds have high values of density in the electron-density map and that their hydrogen-bonding capacity is fully realised through interactions with protein atoms, water molecules and K+ ions. Intermolecular contacts were thoroughly analyzed in the present crystals and in crystals of previously determined L1,RNA complexes. It was shown that extension of the RNA helices providing canonical helix stacking between open,open or open,closed ends of RNA fragments is a common feature of these and all known crystals of complexes between ribosomal proteins and RNAs. In addition, the overwhelming majority of complexes between ribosomal proteins and RNA molecules display crystal contacts formed by the central parts of the RNA fragments. These contacts are often very extensive and strong and it is proposed that they are formed in the saturated solution prior to crystal formation. [source] Do electrostatic interactions destabilize protein,nucleic acid binding?BIOPOLYMERS, Issue 2 2007Sanbo Qin Abstract The negatively charged phosphates of nucleic acids are often paired with positively charged residues upon binding proteins. It was thus counter-intuitive when previous Poisson,Boltzmann (PB) calculations gave positive energies from electrostatic interactions, meaning that they destabilize protein,nucleic acid binding. Our own PB calculations on protein,protein binding have shown that the sign and the magnitude of the electrostatic component are sensitive to the specification of the dielectric boundary in PB calculations. A popular choice for the boundary between the solute low dielectric and the solvent high dielectric is the molecular surface; an alternative is the van der Waals (vdW) surface. In line with results for protein,protein binding, in this article, we found that PB calculations with the molecular surface gave positive electrostatic interaction energies for two protein,RNA complexes, but the signs are reversed when the vdW surface was used. Therefore, whether destabilizing or stabilizing effects are predicted depends on the choice of the dielectric boundary. The two calculation protocols, however, yielded similar salt effects on the binding affinity. Effects of charge mutations differentiated the two calculation protocols; PB calculations with the vdW surface had smaller deviations overall from experimental data. © 2007 Wiley Periodicals, Inc. Biopolymers 86: 112,118, 2007. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Exploring Chemical Modifications for siRNA Therapeutics: A Structural and Functional OutlookCHEMMEDCHEM, Issue 3 2010Siddharth Shukla Abstract RNA interference (RNAi) is a post-transcriptional gene silencing mechanism induced by small interfering RNAs (siRNAs) and micro-RNAs (miRNAs), and has proved to be one of the most important scientific discoveries made in the last century. The robustness of RNAi has opened up new avenues in the development of siRNAs as therapeutic agents against various diseases including cancer and HIV. However, there had remained a lack of a clear mechanistic understanding of messenger RNA (mRNA) cleavage mediated by Argonaute2 of the RNA-induced silencing complex (RISC), due to inadequate structural data. The X-ray crystal structures of the Argonaute (Ago),DNA,RNA complexes reported recently have proven to be a breakthrough in this field, and the structural details can provide guidelines for the design of the next generation of siRNA therapeutics. To harness siRNAs as therapeutic agents, the prudent use of various chemical modifications is warranted to enhance nuclease resistance, prevent immune activation, decrease off-target effects, and to improve pharmacokinetic and pharmacodynamic properties. The focus of this review is to interpret the tolerance of various chemical modifications employed in siRNAs toward RNAi by taking into account the crystal structures and biochemical studies of Ago,RNA complexes. Moreover, the challenges and recent progress in imparting druglike properties to siRNAs along with their delivery strategies are discussed. [source] | ||||||||||||||||||||||