Fareed Aboul-ela

Base-base mismatches. Thermodynamics of double helix formation for dCA3XA3G + dCT3YT3G (X, Y = A,C,G,D

Thermodynamic parameters for double strand formation have been measured for the sixteen double helices of the sequence dCA3XA3G.dCT3YT3G, with each of the bases A, C, G and T at the positions labelled X and Y. The results are analyzed in terms of nearest-neighbors and are compared with thermodynamic parameters for RNA secondary structure. At room temperature the sequence (Formula: see text) is more stable than (Formula: see text) and is similar in stability to (Formula: see text) and (Formula: see text) are least stable. At higher temperatures the sequences containing a G.C base pair become more stable than those containing only A.T. All molecules containing mismatches are destabilized with respect to those with only Watson-Crick pairing, but there is a wide range of destabilization. At room temperature the most stable mismatches are those containing guanine (G.T, G.G, G.A); the least stable contain cytosine (C.A, C.C). At higher temperatures pyrimidine-pyrimidine mismatches become the least stable.

Crystal structures of complexes between aminoglycosides and decoding A site oligonucleotides: role of the number of rings and positive charges in the specific binding leading to miscoding

The crystal structures of six complexes between aminoglycoside antibiotics (neamine, gentamicin C1A, kanamycin A, ribostamycin, lividomycin A and neomycin B) and oligonucleotides containing the decoding A site of bacterial ribosomes are reported at resolutions between 2.2 and 3.0 Å. Although the number of contacts between the RNA and the aminoglycosides varies between 20 and 31, up to eight direct hydrogen bonds between rings I and II of the neamine moiety are conserved in the observed complexes. The puckered sugar ring I is inserted into the A site helix by stacking against G1491 and forms a pseudo base pair with two H-bonds to the Watson–Crick sites of the universally conserved A1408. This central interaction helps to maintain A1492 and A1493 in a bulged-out conformation. All these structures of the minimal A site RNA complexed to various aminoglycosides display crystal packings with intermolecular contacts between the bulging A1492 and A1493 and the shallow/minor groove of Watson–Crick pairs in a neighbouring helix. In one crystal, one empty A site is observed. In two crystals, two aminoglycosides are bound to the same A site with one bound specifically and the other bound in various ways in the deep/major groove at the edge of the A sites.

A conserved RNA structure within the HCV IRES eIF3-binding site

The hepatitis C virus (HCV) internal ribosome entry site (IRES) is recognized specifically by the small ribosomal subunit and eukaryotic initiation factor 3 (eIF3) before viral translation initiation. Using extensive mutagenesis and structure probing analysis, we show that the eIF3-binding domain of the HCV IRES contains an internal loop structure (loop IIIb) and an adjacent mismatched helix that are important for IRES-dependent initiation of translation. NMR studies reveal a unique three-dimensional structure for this internal loop that is conserved between viral isolates of varying primary sequence in this region. These data indicate that internal loop IIIb may be an attractive target for structure-based design of new antiviral agents.

Structural Basis for Contrasting Activities of Ribosome Binding Thiazole Antibiotics

Thiostrepton and micrococcin inhibit protein synthesis by binding to the L11 binding domain (L11BD) of 23S ribosomal RNA. The two compounds are structurally related, yet they produce different effects on ribosomal RNA in footprinting experiments and on elongation factor-G (EF-G)-dependent GTP hydrolysis. Using NMR and an assay based on A1067 methylation by thiostrepton-resistance methyltransferase, we show that the related thiazoles, nosiheptide and siomycin, also bind to this region. The effect of all four antibiotics on EF-G-dependent GTP hydrolysis and EF-G-GDP-ribosome complex formation was studied. Our NMR and biochemical data demonstrate that thiostrepton, nosiheptide, and siomycin share a common profile, which differs from that of micrococcin. We have generated a three-dimensional (3D) model for the interaction of thiostrepton with L11BD RNA. The model rationalizes the differences between micrococcin and the thiostrepton-like antibiotics interacting with L11BD.

[5] Two-dimensional gel electrophoresis of circular DNA topoisomers

Publisher Summary This chapter discusses the two-dimensional gel electrophoresis of circular DNA topoisomers. The major problem with one-dimensional gel electrophoresis is the relatively limited range over which migration is a function of topology. Two-dimensional gel electrophoresis increases this range by a considerable degree, and it provides a simple way to reveal topology-dependent structural transitions and measure some parameters for the process. The geometric distortion of a supercoiled molecule means that the average shape of the molecule is changed as a result of its topology and the result is altered frictional properties. This can be done by agarose gel electrophoresis. This shows the result of electrophoresing a series of topoisomers of the 3658-bp plasmid pAT153 in 1% agarose. A ladder of species is observed, each member of which is a single topoisomer differing from its neighbors by a linking difference of ±. The differences in shape among the different topoisomers results in a progressive change in electrophoretic mobility—that is, the more supercoiled the species, the faster the migration. The resolving power of agarose gels for topoisomers is impressive, but the range is limited so that the more highly supercoiled species comigrate as a broad band at the lower end of the gel. The resolution can be improved for smaller DNA molecules by the use of composite polyacrylamide–agarose gels.

Novel three-dimensional 1H−13C−31P triple resonance experiments for sequential backbone correlations in nucleic acids

SummaryBackbone-driven assignment methods that utilize covalent connectivities have greatly facilitated spectral assignments of proteins. In nucleic acids, 1H−13C−31P correlations could play a similar role, and several related experiments (HCP) have recently been presented for backbone-driven sequential assignments in RNA. The three-dimensional extension of 1H−31P Het-Cor (P,H-COSY-H,C-HMQC) and Het-TOCSY (P,H-TOCSY-H,C-HMQC) experiments presented here complements HCP experiments as tools for spectral assignments and extraction of dihydral angle constraints. By relying on 1H−31P rather than 13C−31P couplings to generate cross peaks, the strongest connectivities are observed in different spectral regions, increasing the likelihood of resolving spectral overlap. In addition, semiquantitative estimates of 1H−31P and 13C−31P couplings provide dihedral angle constraints for RNA structure determination.

RNA as a drug target

In the antiviral and antibacterial area, increasing drug resistance means that there is an ever growing need for novel approaches towards structures and mechanisms which avoid the current problems. The huge increase in high resolution structural data is set to make a dramatic impact on targeting RNA as a drug target. The examples of the RNA binding antibiotics, particularly, the totally synthetic oxazolidinones, should help persuade the skceptics that clinically useful, selective drugs can be obtained from targeting RNA directly.

The Impact of a Ligand Binding on Strand Migration in the SAM-I Riboswitch

Riboswitches sense cellular concentrations of small molecules and use this information to adjust synthesis rates of related metabolites. Riboswitches include an aptamer domain to detect the ligand and an expression platform to control gene expression. Previous structural studies of riboswitches largely focused on aptamers, truncating the expression domain to suppress conformational switching. To link ligand/aptamer binding to conformational switching, we constructed models of an S-adenosyl methionine (SAM)-I riboswitch RNA segment incorporating elements of the expression platform, allowing formation of an antiterminator (AT) helix. Using Anton, a computer specially developed for long timescale Molecular Dynamics (MD), we simulated an extended (three microseconds) MD trajectory with SAM bound to a modeled riboswitch RNA segment. Remarkably, we observed a strand migration, converting three base pairs from an antiterminator (AT) helix, characteristic of the transcription ON state, to a P1 helix, characteristic of the OFF state. This conformational switching towards the OFF state is observed only in the presence of SAM. Among seven extended trajectories with three starting structures, the presence of SAM enhances the trend towards the OFF state for two out of three starting structures tested. Our simulation provides a visual demonstration of how a small molecule (<500 MW) binding to a limited surface can trigger a large scale conformational rearrangement in a 40 kDa RNA by perturbing the Free Energy Landscape. Such a mechanism can explain minimal requirements for SAM binding and transcription termination for SAM-I riboswitches previously reported experimentally.

Novel techniques in nuclear magnetic resonance for nucleic acids

Recent techniques for the efficient preparation of isotopically labelled RNA of desired sequence represent a dramatic step forward for NMR of nucleic acids. Three- and four-dimensional NMR experiments greatly facilitate spectral analysis and quantification of structural constraints. Backbone-driven assignment procedures have been introduced to parallel the powerful assignment methods introduced for work with proteins. Additional structural information to complement interproton distances, namely scalar coupling constants defining the backbone conformation, can be obtained using isotopically labelled oligonucleotides. The additional interproton distance and dihedral angle constraints resolved in higher-dimensional spectra will enable the determination of larger DNA and RNA structures and also increase accuracy and precision.

Structure of Parallel-Stranded Guanine Tetraplexes

Oligonucleotides rich in guanine possess a distinctive ability to self-associate, even in the absence of any sequence complementarity of the Watson-Crick kind (see review by Sundquist 1991 in Volume 5 of this Series). At the macroscopic level, this property is manifested in a propensity to form semicrystalline gels, which are even formed by the free guanine mononucleoside. It is now appreciated that this macroscopic property originates from the ability of guanine bases to form base-base interactions through multidentate hydrogen bonding interactions. More than 30 years ago (Geliert et al. 1962), it was proposed that this ability may permit four guanine bases to selfassociate by forming cyclic tetrads (sometimes termed G-quartets). The tetrad arrangement brings four strands together into a quadruple helix, and such structures have been characterised by fibre diffraction studies (Tougard et al. 1973; Arnott et al. 1974; Zimmerman et al. 1975). The tetrad model also explained the observation that proton exchange rates are very slow in guanine gels (Pinnavaia et al. 1975).