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PubMed2003

The origins of RNA catalysis in ribozymes.

The discovery of RNA catalysis provided a paradigm shift in biology, insight into the evolution of life on the planet and a challenge to understand its mechanistic origins. RNA has limited catalytic resources that must be used to maximal effect. Consequently, RNA catalysis tends to be multifactorial, with several processes contributing to an overall significant enhancement of reaction rate. These include general acid-base catalysis, electrostatic effects, and substrate orientation and proximity. The main players are the RNA nucleobases and bound metal ions. Although most ribozymes carry out phosphoryl transfer, the same considerations appear to apply to peptidyl transfer in the ribosome.

Animals↗

PubMed2005

Conformational constraint as a means for understanding RNA-aminoglycoside specificity.

The lack of high RNA target selectivity displayed by aminoglycoside antibiotics results from both their electrostatically driven binding mode and their conformational adaptability. The inherent flexibility around their glycosidic bonds allows them to easily assume a variety of conformations, permitting them to structurally adapt to diverse RNA targets. This structural promiscuity results in the formation of aminoglycoside complexes with diverse RNA targets in which the antibiotics assume distinct conformations. Such differences suggest that covalently linking individual rings in an aminoglycoside could reduce its available conformations, thereby altering target selectivity. To explore this possibility, conformationally constrained neomycin and paromomycin analogues designed to mimic the A-site bound aminoglycoside structure have been synthesized and their affinities to the TAR and A-site, two therapeutically relevant RNA targets, have been evaluated. As per design, this constraint has minimal deleterious effect on binding to the A-site. Surprisingly, however, preorganizing these neomycin-class antibiotics into a TAR-disfavored structure has no deleterious effect on binding to this HIV-1 RNA sequence. We rationalize these observations by suggesting that the A-site and HIV TAR possess inherently different selectivities toward aminoglycosides. The inherent plasticity of the TAR RNA, coupled to the remaining flexibility within the conformationally constrained analogues, makes this RNA site an accommodating target for such polycationic ligands. In contrast, the deeply encapsulating A-site is a more discriminating RNA target. These observations suggest that future design of novel target selective RNA-based therapeutics will have to consider the inherent "structural" selectivity of the RNA target and not only the selectivity patterns displayed by the low molecular weight ligands.

Aminoglycosides↗

PubMed1997

Selectivity of F8-actinomycin D for RNA:DNA hybrids and its anti-leukemia activity.

Although many compounds have been found that bind to DNA in various ways and exhibit various biological activities, few compounds that specifically bind to RNA or RNA:DNA hybrids are known, even though such compounds are expected to have important biological properties. For example, one characteristic function of the retroviruses, which is generally not found in eukaryotic cells, is the production of an RNA:DNA hybrid in the viral replication phase. If an agent is designed to bind only to an RNA:DNA hybrid, and not to DNA or to RNA, such an agent might be able to inhibit specifically the RNase H activity of retroviral reverse transcriptase, and therefore suppress viral replication. Actinomycin D is known to bind to double-stranded DNA, but not to RNA, because steric hindrance between the 2-amino group of the phenoxazone ring and the 2'-hydroxyl group of RNA prevents intercalation of the compound. However, if the > C-H moiety at the 8-position of the phenoxazone ring is replaced by a > C-F, a possible hydrogen-bond acceptor, this analogue (8-fluoro-actinomycin D, F8AMD) might be able to bind intercalatively to an RNA:DNA hybrid by forming an additional hydrogen bond between F8 and the 2'-hydroxyl group of the guanosine ribose. To test this hypothesis, the crystal structure of d(GAAGCTTC)2-F8AMD has been determined at 3.0 A resolution. Based on this crystal structure, a model in which F8AMD binds into the hybrid r(GAAGCUUC):d(GAAGCTTC) has been built using molecular mechanics and dynamic methods. These structural studies indicate that F8AMD binds intercalatively to a B-form double-stranded DNA whereas the drug intercalates into an RNA:DNA hybrid taking an A-form conformation. In the RNA:DNA hybrid complex, the F8 atom is located so as to be able to interact to an O2' hydroxyl group with either an O-H...F hydrogen bond or H+...F- electrostatic interaction. This interaction might stabilize the F8AMD molecule in the RNA:DNA hybrid. A binding study indicates that both actinomycin D (AMD) and F8AMD bind intercalatively not only to double-stranded DNAs, but also to RNA:DNA hybrids. Although the overall binding capacity of F8AMD (k = 4.5 x 10(5) M-1) is reduced slightly in comparison with AMD itself (k = 1.8 x 10(6) M-1), F8AMD tends to bind relatively more favorably than AMD to the RNA:DNA hybrids. The drugs' effects on RNA synthesis in HeLa cells indicates that the binding capacities of AMD and F8AMD correlates strongly to their RNA synthesis inhibitory activities. F8AMD required a concentration of 78 nM to inhibit RNA polymerase activity in HeLa cells by 50%, whereas AMD reached the same inhibitory level at 30 nM. Surprisingly, F8AMD exhibits unique selectivity against leukemia cells as does another C8-derivatized AMD analogue, N8AMD. F8AMD inhibits 50% of leukemia cell growth at less than 1.0 nM whereas 10- to 130-fold-higher drug concentrations are required to inhibit the growth of other tumor cell lines by 50%. The GI50 value of F8AMD for leukemia cells is the lowest among the GI50 values for all other AMD derivatives tested. By contrast, AMD is quite potent and kills most cells at less than 50 nM concentration, but it does not show any selectivity for certain cell lines. This indicates that AMD should have very limited use as an antitumor agent. It is difficult to rationalize why F8AMD and N8AMD show such strong selectivity against leukemia cells. However, this study and our previous study (J. Am. Chem. Soc. 1994, 116, 7971) indicated that F8AMD and N8AMD tended to bind more favorably to RNA:DNA hybrids. Thus, the unique antileukemia selectivity shown by F8AMD and N8AMD might be used by the agents binding to RNA:DNA hybrids rather than to double-stranded DNA.

Antineoplastic Agents↗

PubMed1996

A docking and modelling strategy for peptide-RNA complexes: applications to BIV Tat-TAR and HIV Rev-RBE.

BACKGROUND: In spite of the great interest in the interaction between RNAs and proteins, no general protocol for modelling these complexes is presently available. This methodological vacuum is particularly acute because the structure of few such complexes is known. RESULTS: A general strategy for docking and modelling RNA-protein complexes has been developed. The docking procedure involves minimizing electrostatic and van der Waals' interaction energies of conformationally rigid structures during docking. After docking, libraries of amino acid sidechain conformations are searched to obtain the best interactions between the peptide and the RNA. Using this method, we have reproduced the structure of a bovine immunodeficiency virus (BIV) Tat peptide bound to BIV TAR RNA and have developed a model for the structure of the arginine-rich HIV-1 Rev peptide (Rev34-50) interacting with the Rev-binding element (RBE). CONCLUSIONS: The resulting model of the Rev34-50-RBE complex predicts that although no single arginine sidechain is responsible for complex formation, residues Arg2, Arg5 and Arg11 are more important for binding than the other arginine residues in the peptide. One model is supported by binding measurements performed on wild-type and mutant RBE molecules with the peptide.

Animals↗

PubMedDate not supplied

The energetics of small internal loops in RNA.

The energetics of small internal loops are important for prediction of RNA secondary and tertiary structure, selection of drug target sites, and understanding RNA structure-function relationships. Hydrogen bonding, base stacking, electrostatic interactions, backbone distortion, and base-pair size compatibility all contribute to the energetics of small internal loops. Thus, the sequence dependence of these energetics are idiosyncratic. Current approximations for predicting the free energies of internal loops consider size, asymmetry, closing base pairs, and the potential to form GA, GG, or UU pairs. The database of known three-dimensional structures allows for comparison with the models used for predicting stability from sequence.

Base Pairing↗

PubMed2000

Antitermination in bacteriophage lambda. The structure of the N36 peptide-boxB RNA complex.

The solution structure of a 15-mer nutRboxB RNA hairpin complexed with the 36-mer N-terminal peptide of the N protein (N36) from bacteriophage lambda was determined by 2D and 3D homonuclear and heteronuclear magnetic resonance spectroscopy. These 36 amino acids include the arginine-rich motif of the N protein involved in transcriptional antitermination of phage lambda. Upon complex formation with boxB RNA, the synthetic N36 peptide binds tightly to the major groove of the boxB hairpin through hydrophobic and electrostatic interactions forming a bent alpha helix. Four nucleotides of the GAAAA pentaloop of the boxB RNA adopt a GNRA-like tetraloop fold in the complex. The formation of a GAAA tetraloop involves a loop-closing sheared base pair (G6-A10), base stacking of three adenines (A7, A8, and A10), and extrusion of one nucleotide (A9) from the loop, as observed previously for the complex of N(1-22) peptide and the nutLboxB RNA [Legault, P., Li, J., Mogridge, J., Kay, L.E. & Greenblatt, J. (1998) Cell 93, 289-299]. Stacking of the bases is extended by the indole-ring of Trp18 which also forms hydrophobic contacts to the side-chains of Leu24, Leu25, and Val26. Based on the structure of the complex, three mutant peptides were synthesized and investigated by CD and NMR spectroscopy in order to determine the role of particular residues for complex formation. These studies revealed very distinct amino-acid requirements at positions 3, 4, and 8, while replacement of Trp18 with tyrosine did not result in any gross structural changes.

Amino Acid Sequence↗

PubMed2006

Role of metal ions in the tetraloop-receptor complex as analyzed by NMR.

Metal ions are critical for the proper folding of RNA, and the GAAA tetraloop-receptor is necessary for the optimal folding and function of many RNAs. We have used NMR to investigate the role of metal ions in the structure of the tetraloop-receptor in solution. The NMR data indicate native tertiary structure is formed under a wide range of ionic conditions. The lack of conformational adaptation in response to very different ionic conditions argues against a structural role for divalent ions. Nuclear Overhauser effects to cobalt hexammine and paramagnetic relaxation enhancement induced by manganese ions were used to determine the NMR structures of the tetraloop receptor in association with metal ions, providing the first atomic-level view of these interactions in the solution state. Five manganese and two cobalt hexammine ions could be localized to the RNA surface. The locations of the associated metal ions are similar, but not identical to, those of previously determined crystal structures. The sites of association are in general agreement with nonlinear Poisson-Boltzmann calculations of the electrostatic surface, emphasizing the general importance of diffusely associated ions in RNA tertiary structure.

Base Sequence↗

PubMed2004

The hairpin ribozyme.

The hairpin ribozyme is a naturally occurring RNA that catalyzes sequence-specific cleavage and ligation of RNA. It has been the subject of extensive biochemical and structural studies, perhaps the most detailed for any catalytic RNA to date. Comparison of the structures of its constituent domains free and fully assembled demonstrates that the RNA undergoes extensive structural rearrangement. This rearrangement results in a distortion of the substrate RNA that primes it for cleavage. This ribozyme is known to achieve catalysis employing exclusively RNA functional groups. Metal ions or other catalytic cofactors are not used. Current experimental evidence points to a combination of at least four mechanistic strategies by this RNA: (1) precise substrate orientation, (2) preferential transition state binding, (3) electrostatic catalysis, and (4) general acid base catalysis.

Binding Sites↗

PubMed2004

Electrostatic contribution of serine phosphorylation to the Drosophila SLBP--histone mRNA complex.

Unlike all other metazoan mRNAs, mRNAs encoding the replication-dependent histones are not polyadenylated but end in a unique 26 nucleotide stem-loop structure. The protein that binds the 3' end of histone mRNA, the stem-loop binding protein (SLBP), is essential for histone pre-mRNA processing, mRNA translation, and mRNA degradation. Using biochemical, biophysical, and nuclear magnetic resonance (NMR) experiments, we report the first structural insight into the mechanism of SLBP-RNA recognition. In the absence of RNA, phosphorylated and unphosphorylated forms of the RNA binding and processing domain (RPD) of Drosophila SLBP (dSLBP) possess helical secondary structure but no well-defined tertiary fold. Drosophila SLBP is phosphorylated at four out of five potential serine or threonine sites in the sequence DTAKDSNSDSDSD at the extreme C-terminus, and phosphorylation at these sites is necessary for histone pre-mRNA processing. Here, we provide NMR evidence for serine phosphorylation of the C-terminus using (31)P direct-detect experiments and show that both serine phosphorylation and RNA binding are necessary for proper folding of the RPD. The electrostatic effect of protein phosphorylation can be partially mimicked by a mutant form of SLBP wherein four C-terminal serines are replaced with glutamic acids. Hence, both RNA binding and protein phosphorylation are necessary for stabilization of the SLBP RPD.

Amino Acid Sequence↗

PubMed1984

RNA-protein interactions and secondary structures of cowpea chlorotic mottle virus for in vitro assembly.

Laser Raman spectroscopy of the cowpea chlorotic mottle virus (CCMV) in native (pH 5.0) and partially swollen (pH 7.5) states reveals the presence of small percentages of protonated adenine (less than 15%) and cytosine (less than 7%) bases in the encapsidated RNA molecule of the native virion. The protonated bases are titrated with pH-induced swelling of the virus. Titration of putative COOH groups of aspartic and glutamic side chains of the virion subunit cannot be detected over the same pH range, which suggests that carboxyl anions (CO-2) and protonated bases are both available at pH 5 to stabilize the ribonucleoprotein particles by electrostatic interactions. The highly (95%) ordered secondary structure of encapsidated RNA may undergo a small additional increase (less than 3%) in ordered structure with release from the virion, suggesting at most a marginal structure-distorting influence from protein contacts in the native particle. The Raman spectra of the virion are also compared by difference spectroscopy with spectra of capsids (empty shells devoid of RNA), subunit dimers, and protein-free RNA. The results indicate that the subunit structure is altered by the release of RNA from the virion, as well as by the swelling of the virion. Amino acid residues and protein secondary structures that are affected in these in vitro assembly and disassembly processes are identified from their characteristic Raman lines. Two classes of cysteinyl SH groups, solvent exposed and solvent protected, are revealed for the capsid and virion subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed2002

Transition state stabilization by a catalytic RNA.

The hairpin ribozyme catalyzes sequence-specific cleavage of RNA through transesterification of the scissile phosphate. Vanadate has previously been used as a transition state mimic of protein enzymes that catalyze the same reaction. Comparison of the 2.2 angstrom resolution structure of a vanadate-hairpin ribozyme complex with structures of precursor and product complexes reveals a rigid active site that makes more hydrogen bonds to the transition state than to the precursor or product. Because of the paucity of RNA functional groups capable of general acid-base or electrostatic catalysis, transition state stabilization is likely to be an important catalytic strategy for ribozymes.

Binding Sites↗

PubMed2005

Quantitation of HIV-1 RNA in breast milk by real time PCR.

HIV-1 RNA in breast milk is a strong predictor of HIV-1 transmission through breastfeeding. In the present report, breast milk samples from HIV-1 uninfected donors were spiked with dilution of quantified culture supernatant from HIV-1(NDK) infected PBMC. Two RNA extraction techniques based on silica extraction, Nuclisens (BioMerieux) and Triazol (Qiagen), two techniques based on guanidine thiocynanate/chloroforme extraction, TRIzol (Life Technologie) and Amplicor HIV-1 Monitor (Roche Diagnostic Systems), and one technique based on electrostatic adsorption on iron oxide micro beads (Promega) were compared. HIV-1 RNA was quantitated by real time PCR (LTR gene) and Amplicor HIV-1 Monitor. Combining magnetic micro beads extraction and real time PCR quantitation allowed to correctly quantify breast milk HIV-1 RNA, with a difference between the expected and measured HIV-1 RNA levels always lower than 0.3 log copies/ml. The same combination was confirmed on 25 breast milk samples from HIV-1 infected women collected in Kwazulu-Natal, South Africa, by comparing measurements with those obtained by the Amplicor HIV-1 Monitor (r(2)=0.88). Nucleic acid extraction by magnetic micro beads followed by real time PCR is a reliable, sensitive, rapid and simple procedure to quantify HIV-1 RNA in breast milk and allows for PCR inhibitors found frequently in these samples.

Female↗

PubMed2006

RNA binding small molecules: studies on t-RNA binding by cytotoxic plant alkaloids berberine, palmatine and the comparison to ethidium.

The interaction of two natural protoberberine plant alkaloids berberine and palmatine with t-RNA(phe) was studied using various biophysical techniques and the data was compared with the binding of the classical DNA intercalator, ethidium. The results of optical thermal melting, differential scanning calorimetry and circular dichroism characterized the native cloverleaf structure of t-RNA under the conditions of the study. The strong binding of the alkaloids and ethidium to t-RNA was revealed from the absorption and fluorescence studies. The salt dependence of the binding constants enabled the dissection of the binding free energy to electrostatic and non-electrostatic contributions. This analysis revealed a surprisingly large favourable component of the non-electrostatic contribution to the binding of these charged alkaloids and ethidium to t-RNA. Isothermal titration calorimetric studies revealed that the binding of both the alkaloids is driven by a moderately favourable enthalpy decrease and a moderately favourable entropy increase while that of ethidium is driven by a large favourable enthalpy decrease. Taken together, the results suggest that the binding of these alkaloid molecules on the t-RNA structure appears to be mostly by partial intercalation while ethidium intercalates to the t-RNA. These results reveal the molecular aspects on the interaction of these alkaloids to t-RNA.

Alkaloids↗

PubMed1989

Ribosomal protein L7/L12 has a helix-turn-helix motif similar to that found in DNA-binding regulatory proteins.

Inspection of the structure of the C-terminal domain of ribosomal protein L7/L12 (1) reveals a helix-turn-helix motif similar to the one found in many DNA-binding regulatory proteins (2-5). The 19 alpha-carbon atoms of the L7/L12 alpha-helices superimpose on the DNA binding helices of CAP and cro with root-mean-square distances between corresponding alpha carbons of 1.45 and 1.55 A, respectively. These helices in L7/L12 are within a patch of highly conserved residues on the surface of L7/L12 whose role is as yet uncertain. We raise the possibility that they may constitute a binding site for nucleic acids, most probably RNA. Consistent with this hypothesis are calculations of the electrostatic charge potential surrounding the protein, which show a region of positive potential centered on the first of these helices.

Bacterial Proteins↗

PubMed1998

Equilibrium and kinetic parameters of the sequence-specific interaction of Escherichia coli RNA polymerase with nontemplate strand oligodeoxyribonucleotides.

The specific recognition by Escherichia coli RNA polymerase of single-stranded oligodeoxyribonucleotides (oligos) with the sequence of the -10 promoter region on the nontemplate strand has been studied. Binding was monitored by observing the increase in fluorescence of 2-aminopurine residues incorporated in the oligos. The effects of salt on the rates of formation and dissociation of RNA polymerase.oligo complexes are relatively small, from which we conclude that electrostatic interactions contribute minimally to the favorable binding free energy. From the convex temperature dependence of ln Ka (Ka is the equilibrium association constant), we infer that a large apparent negative heat capacity, of 1-2 kcal M-1 K-1, accompanies complex formation, which is interpreted as due to a conformational change in RNA polymerase. Contrary to expectation, the forward rate constant for binding of oligos is more than 10-fold smaller than that for open complex formation at strong promoters. This suggests that in comparison to an oligo, promoter DNA may be better able to accelerate this required conformational change in the RNA polymerase. Oligo binding was shown to compete with the interaction between RNA polymerase and promoters, indicating that the two bind to overlapping sites on the RNA polymerase

Base Sequence↗

PubMed1990

Switching in the self-assembly of tobacco mosaic virus.

Experimental observations on the structure and physicochemical properties of TMV protein assemblies have led to a fundamental switch in the model of the self-assembly process: rather than being nucleated by the hypothetical two-layer disk, virus assembly appears to be initiated by interaction of the specific RNA sequence with a short helical aggregate of the coat protein arranged as in the virus. Formation of the 20s nucleating aggregate involves the binding of an average of half a proton per protein subunit. This proton-binding site can be identified with the carboxyl-carboxylate pair that is formed between top and bottom protein surfaces at a radius of 58 A in the virus helix. Because the 20s aggregate consists of about two helical turns, only one carboxyl-carboxylate pair will be formed between each top-bottom pair of protein subunits. Limitation of the length of the 20s helical aggregate at neutral pH can be accounted for by disorder of the inner loop of the protein chain, due to electrostatic repulsion among the carboxyl groups that form the anomalous proton-binding site at 25 A radius in the ordered virus structure. To grow beyond two to three turns, inner loops of the protein at the interior of the helix must be ordered in the close-packed arrangement. The electrostatic repulsion opposing this ordering can be overcome by binding of the viral RNA at neutral pH, by calcium binding, or by proton binding in slightly acid solution. Virus disassembly upon infection appears to result from the low intracellular calcium and proton concentration compared to the extracellular environment, which increases the electrostatic repulsion among the negatively charged groups involved in calcium and proton binding, thereby allowing cellular ribosomes to competitively bind the viral RNA. Disk aggregates of TMV protein, which form at high ionic strength in alkaline solution, do not appear to be involved in virus assembly. The stacked-disc aggregate, which was previously presumed to be built of a polar stack of the hypothetical polar two-layer aggregate, is, in fact, a bipolar structure. Because the bonding between turns of the disc structures is different from that of the virus helix, direct switching between these structures by the postulated dislocation does not occur. TMV assembly does appear to involve conservation of bonding specificity, as initially presumed, but only in helical packing arrangements of the protein subunits. Switching from disordered to ordered conformations of the protein, dependent on changes in the electrostatic interactions among the protein subunits, appears to be critical in controlling the assembly process.

Microscopy, Electron↗

PubMed2007

Ribonucleocapsid formation of severe acute respiratory syndrome coronavirus through molecular action of the N-terminal domain of N protein.

Conserved among all coronaviruses are four structural proteins: the matrix (M), small envelope (E), and spike (S) proteins that are embedded in the viral membrane and the nucleocapsid phosphoprotein (N), which exists in a ribonucleoprotein complex in the lumen. The N-terminal domain of coronaviral N proteins (N-NTD) provides a scaffold for RNA binding, while the C-terminal domain (N-CTD) mainly acts as oligomerization modules during assembly. The C terminus of the N protein anchors it to the viral membrane by associating with M protein. We characterized the structures of N-NTD from severe acute respiratory syndrome coronavirus (SARS-CoV) in two crystal forms, at 1.17 A (monoclinic) and at 1.85 A (cubic), respectively, resolved by molecular replacement using the homologous avian infectious bronchitis virus (IBV) structure. Flexible loops in the solution structure of SARS-CoV N-NTD are now shown to be well ordered around the beta-sheet core. The functionally important positively charged beta-hairpin protrudes out of the core, is oriented similarly to that in the IBV N-NTD, and is involved in crystal packing in the monoclinic form. In the cubic form, the monomers form trimeric units that stack in a helical array. Comparison of crystal packing of SARS-CoV and IBV N-NTDs suggests a common mode of RNA recognition, but they probably associate differently in vivo during the formation of the ribonucleoprotein complex. Electrostatic potential distribution on the surface of homology models of related coronaviral N-NTDs suggests that they use different modes of both RNA recognition and oligomeric assembly, perhaps explaining why their nucleocapsids have different morphologies.

Amino Acid Sequence↗

PubMed2004

Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells.

We have used cationic oligopeptide polyarginine-12mer (POA) to deliver double-stranded RNA (dsRNA), prepared in vitro, to tobacco (Nicotiana tabacum) suspension cells. POA interacts electrostatically with dsRNA to form a complex. When dsRNA for the GUS or NPTII gene was delivered into cells carrying the same genes, the corresponding mRNA was degraded. Using RNase protection assay we were able to detect 21-bp small interfering RNA in dsRNA/POA-treated cells. These results demonstrate that POA can be used to deliver dsRNA to induce post-transcriptional gene silencing in plant cells.

Blotting, Northern↗