RESUMEN
The conformation of a hammerhead ribozyme domain, formed between a 35-mer ribozyme and its 14-mer substrate, was studied under cleavage conditions with non-cleavable substrate analogues. Each analogue substrate contained a single 2'-deoxy-4-thiouridine that formed specific intermolecular crosslinks within the ribozyme-substrate complex upon irradiation with 365 nm light. The residues at positions 7 and 8 to 9 (the cleavage site) were found in contact with several bases of the ribozyme 5' conserved region regardless of whether the substrate was all-RNA, with a single deoxynucleotide at the cleavage site, or all-DNA. These contacts were observed in the presence of either 20 mM Mg2+ or 200 mM Na+. The multiple crosslinks generated between the ribozyme central core and each of the three substrates suggest the existence of several folded conformers of the ribozyme. A ribozyme mutation (A28U), which abolishes the catalytic activity, was shown to strongly affect the pattern of crosslinks; this argues for the presence of multiple folded conformers of the ribozyme some of which may be catalytically inactive.
Asunto(s)
Conformación de Ácido Nucleico , ARN Catalítico/química , ARN Catalítico/metabolismo , ARN Viral/metabolismo , Secuencia de Bases , Reactivos de Enlaces Cruzados , ADN/síntesis química , ADN/química , ADN/metabolismo , Desoxiuridina/análogos & derivados , VIH/genética , Datos de Secuencia Molecular , Ácidos Nucleicos Heterodúplex/análisis , ARN Catalítico/síntesis química , ARN Catalítico/genética , ARN Viral/química , ARN Viral/genética , Tiouridina/análogos & derivados , Rayos UltravioletaRESUMEN
The conformation in solution of a small bipartite I-III hammerhead ribozyme has been deduced from the photo-crosslinks formed between cleavable ribo-deoxysubstrates appropriately substituted with the probe deoxy-4-thiouridine and ribozyme residues. The ribozyme-substrate complex is able to adopt a Y-like structure with stems I and II in close proximity in the presence of 400 mM Na+ only. Indeed, a cross-link joining stem I (1.6) to loop II (AL2.4) forms in significant amount under these conditions. This cross-linked complex furthermore elicits, upon Mg2+ addition, a catalytic activity similar to that exhibited by the complexes cross-linked at the distal ends of either stem I or stem III or of the non-substituted bipartite complex. This shows that the reaction mechanism is fully compatible with a strong structural constraint between stems I and II and that sodium ions at high concentration (400 mM) are able to promote a proper folding of hammerhead ribozymes. None of the multiple cross-links formed within the ribozyme core (probe in position 16.1 or 1.1) was found catalytically active. The cross-link patterns nevertheless indicate a higher flexibility of the core in Na+ than in Mg2+. While most of the cross-links can be accommodated by the Y solution structure, some of them (16.1 to U4 and 2.1) definitely can not, suggesting that additional alternative inactive conformations exist in solution.
Asunto(s)
ARN Catalítico/metabolismo , Tampones (Química) , Hidrólisis , Cinética , Modelos Moleculares , Conformación de Ácido Nucleico , Concentración Osmolar , Fotoquímica , Conformación Proteica , Sondas ARN , ARN Catalítico/químicaRESUMEN
Antisense oligodeoxynucleotides directed against a 24-mer RNA derived from the long terminal repeat (LTR) region of HIV were linked to proto- and methylpyrroporphyrin and their zinc derivatives. The oligonucleotide-porphyrin conjugates were tested for their ability to induce photodamage on the target RNA. Upon hybridization followed by irradiation at 405 nm, the photochemical reaction led to photocross-linking of the antisense derivative to the RNA substrate. The protoporphyrin exhibited a much higher cross-linking yield than the methylpyrroporphyrin while the Zn-porphyrin derivatives were found to be less efficient than their corresponding nonmetallated congeners. The specificity of the photocross-linking reaction between the porphyrin-oligomer and its target RNA was demonstrated by the following evidence: (1) hybrid formation was required for photocross-linking to occur, (2) the sites of cross-linking on the target RNA were identified at G residues located in close proximity to the porphyrin photoactive center in the hybrid and (3) addition of bulk calf liver RNA did not affect the photocross-linking efficiency.
Asunto(s)
Duplicado del Terminal Largo de VIH , VIH/genética , Oligodesoxirribonucleótidos , Oligonucleótidos Antisentido , Oligorribonucleótidos/química , Porfirinas , Secuencia de Bases , Cartilla de ADN , Datos de Secuencia Molecular , Oligorribonucleótidos/efectos de la radiación , Fotoquímica , ARN Viral/genéticaRESUMEN
We report studies on the interaction of some zinc(II) and copper(II) complexes of amines and amino acids with poly(dC-dG) and poly(dm5C-dG). Of the zinc complexes the species zinc-tris(2-aminoethyl) amine is found to be the most efficient for inducing Z-DNA giving a mid point at low ionic strength of 1.4 microM (poly(dC-dG] and 44nM (poly(dm5C-dG). While an antagonistic effect on raising the ionic strength is observed, the transition occurs at only 2 microM for poly(dm5C-dG) at 150mM NaCl. The most efficient copper(II) complex is that of diethylene triamine, though copper(II) complexes are generally less efficient than zinc(II) complexes. We also report kinetic and thermodynamic studies upon the B-Z transition induced by these complexes. A model is proposed for the interaction of one of the zinc complexes which involves not only direct zinc-DNA binding but also the formation of hydrogen bonds between the metal bond amine groups and the residues adjacent to the coordination site.
Asunto(s)
Cobre/metabolismo , ADN , Conformación de Ácido Nucleico , Polidesoxirribonucleótidos/metabolismo , Zinc/metabolismo , Dicroismo Circular , Glicol de Etileno , Glicoles de Etileno/farmacología , Cinética , Concentración Osmolar , TermodinámicaRESUMEN
The synthetic polynucleotide poly(dA-dC).poly(dG-dT) at low ionic strength is shown to undergo conformational changes in the presence of [tris(2-aminoethyl)amine]zinc(II) chloride (ZnN4). At 100 microM ZnN4, circular dichroism and 31P NMR spectra show the formation of Z DNA. With an increase of the concentration up to 600 microM, an A-like form is obtained, and at still higher concentration, the polynucleotide reverts to the original B form. Experiments on polynucleotide samples in which some sequence errors were observed showed that spermine was necessary as well as ZnN4 to induce the Z form. At higher concentrations of spermine and ZnN4, a second Z form (Z*) is observed. Raising the ionic strength inhibits the formation of the Z form, whereas the presence of ethylene glycol favors it.
Asunto(s)
ADN , Conformación de Ácido Nucleico , Polidesoxirribonucleótidos , Dicroismo Circular , Espectroscopía de Resonancia MagnéticaRESUMEN
The hammerhead ribozyme is a small catalytic RNA motif made up of 3 base-paired stems connected by conserved sequences which are essential for catalysis. We have modelled its 3 dimensional structure, taking advantage of proximity data between several substrate and ribozyme residues determined by photo-crosslinking experiments. It is characterized by an Y shape of the 3 stems stabilized in the central core by a network of hydrogen bonds involving in particular 2 non Watson-Crick G:A base-pairs. The 5' conserved sequence CUGA makes a sharp turn, the G residue exchanging hydrogen bonds with a conserved base-pair of stem III. The substrate is stretched at the cleavage site. Overall this structure is consistent with that deduced from X-ray crystallography but differences are observed at the level of the CUGA turn.
Asunto(s)
Reactivos de Enlaces Cruzados/química , Modelos Moleculares , Conformación de Ácido Nucleico , ARN Catalítico/química , Fotoquímica , Programas InformáticosRESUMEN
The low-salt Z form of poly(dm5C-dG) reported in the literature was shown to be due to minute amounts of bivalent cations. If the polymer was purified by dialysis against 1 mM EDTA, poly(dm5C-dG) showed the normal B form. No change in this B form was observed up to 0.8 M NaCl. Very small amounts of Mg2+ or Ca2+ (10(-6) to 10(-5) M) induced the characteristic Z form CD spectrum, which could easily and rapidly be reverted to B-DNA by addition of EDTA.
Asunto(s)
ADN , Conformación de Ácido Nucleico , Dicroismo Circular , Cinética , Concentración Osmolar , Polidesoxirribonucleótidos , Sodio , Espectrofotometría UltravioletaRESUMEN
We have previously observed that double-stranded DNA fragments containing a tract of the tandemly repeated sequence poly(CA). poly(TG) can associate in vitro to form stable complexes of low electrophoretic mobility, which are recognized with high specificity by proteins HMG1 and HMG2. The formation of such complexes has since been observed to depend on interactions of DNA with polypropylene surfaces, with the suggestion that the formation of low mobility complexes might be the result of strand dissociation followed by misaligned reassociation of the repetitive sequences. The data presented here show that at high ionic strength the interactions of DNA with polypropylene are sufficiently strong for DNA to remain bound to the polypropylene surface, which suggests that DNA might also be involved in interactions with hydrophobic molecules in vivo. Under such conditions, low-mobility complexes are found only in the material adsorbed to the polypropylene surface, and all DNA fragments are able to form low-mobility structures, whether or not they contain repetitive sequences. Preventing the separation of strands by ligating hairpin loop oligonucleotides at both ends of the fragments does not prevent the formation of low-mobility complexes. Our results suggest two different pathways for the formation of complexes. In the first, dissociation is followed by misaligned reassociation of repetitive sequences, yielding duplexes with single-stranded end regions that associate to form multimeric complexes. In the second, repetitive as well as nonrepetitive DNA molecules bound to polypropylene adopt a conformation with locally unwound regions, which allows interactions between neighboring duplexes adsorbed on the surface, resulting in the formation of low-mobility complexes.
Asunto(s)
ADN/química , Polipropilenos/química , ADN/ultraestructura , Escherichia coli/química , Conformación de Ácido Nucleico , Plásmidos , Poli A/química , Poli C/química , Poli G/química , Poli T/químicaRESUMEN
The Escherichia coli mismatch repair system does not recognize and/or repair all mismatched base pairs with equal efficiency: whereas transition mismatches (G X T and A X C) are well repaired, the repair of some transversion mismatches (e.g. A X G or C X T) appears to depend on their position in heteroduplex DNA of phage lambda. Undecamers were synthesized and annealed to form heteroduplexes with a single base-pair mismatch in the centre and with the five base pairs flanking each side corresponding to either repaired or unrepaired heteroduplexes of lambda DNA. Nuclear magnetic resonance (n.m.r.) studies show that a G X A mismatch gives rise to an equilibrium between fully helical and a looped-out structure. In the unrepaired G X A mismatch duplex the latter predominates, while the helical structure is predominant in the case of repaired G X A and G X T mismatches. It appears that the E. coli mismatch repair enzymes recognize and repair intrahelical mismatched bases, but not the extrahelical bases in the looped-out structures.