RESUMEN
The TPsiC stem and loop (TSL) of tRNA contains highly conserved nucleoside modifications, m(5)C(49), T(54), Psi(55)and m(1)A(58). U(54)is methylated to m(5)U (T) by m(5)U(54)methyltransferase (RUMT); A(58)is methylated to m(1)A by m(1)A(58)tRNA methyltransferase (RAMT). RUMT recognizes and methylates a minimal TSL heptadecamer and RAMT has previously been reported to recognize and methylate the 3'-half of the tRNA molecule. We report that RAMT can recognize and methylate a TSL heptadecamer. To better understand the sensitivity of RAMT and RUMT to TSL conformation, we have designed and synthesized variously modified TSL constructs with altered local conformations and stabilities. TSLs were synthesized with natural modifications (T(54)and Psi(55)), naturally occurring modifications at unnatural positions (m(5)C(60)), altered sugar puckers (dU(54)and/or dU(55)) or with disrupted U-turn interactions (m(1)Psi(55)or m(1)m(3)Psi(55)). The unmodified heptadecamer TSL was a substrate of both RAMT and RUMT. The presence of T(54)increased thermal stability of the TSL and dramatically reduced RAMT activity toward the substrate. Local conformation around U(54)was found to be an important determinant for the activities of both RAMT and RUMT.
Asunto(s)
Escherichia coli/enzimología , Conformación de Ácido Nucleico , ARN de Transferencia de Fenilalanina/metabolismo , Tetrahymena pyriformis/enzimología , ARNt Metiltransferasas/metabolismo , Animales , Cinética , Espectroscopía de Resonancia Magnética , Metilación , Nucleósidos/química , Nucleósidos/genética , Nucleósidos/metabolismo , Estabilidad del ARN , ARN de Transferencia de Fenilalanina/síntesis química , ARN de Transferencia de Fenilalanina/química , ARN de Transferencia de Fenilalanina/genética , Especificidad por Sustrato , Temperatura , Termodinámica , Levaduras/genéticaRESUMEN
The structure of an analogue of the yeast tRNAPhe T Psi C stem-loop has been determined by NMR spectroscopy and restrained molecular dynamics. The molecule contained the highly conserved modification ribothymidine at its naturally occurring position. The ribothymidine-modified T Psi C stem-loop is the product of the m5U54-tRNA methyltransferase, but is not a substrate for the m1A58-tRNA methyltransferase. Site-specific substitutions and 15N labels were used to confirm the assignment of NOESY cross-peaks critical in defining the global fold of the molecule. The structure is unusual in that the loop folds far over into the major groove of the curved stem. This conformation is stabilized by both stacking interactions and hydrogen bond formation. Furthermore, this conformation appears to be unique among RNA hairpins of similar size. There is, however, a considerable resemblance to the analogous domain in the crystal structure of the full-length yeast tRNAPhe. We believe, therefore, that the structure we have determined may represent an intermediate in the folding pathway during the maturation of tRNA.
Asunto(s)
Conformación de Ácido Nucleico , Seudouridina/química , ARN de Hongos/química , ARN de Transferencia de Fenilalanina/química , Saccharomyces cerevisiae/química , Uridina/análogos & derivados , Anticodón/química , Secuencia de Bases , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular/métodos , Soluciones , Uridina/químicaRESUMEN
The three-dimensional structure of a pyrimidine-purine-pyrimidine DNA triplex containing an N7-glycosylated guanine (7G) in the third strand has been determined by NMR spectroscopy, restrained molecular dynamics calculations, and complete relaxation matrix refinement. Glycosylation of the guanine at the N7 position permits it to adopt a conformation such that the Hoogsteen face of the base mimics the arrangement of hydrogen bond donors seen in protonated cytosine. The NMR data confirm the previously proposed hydrogen bonding scheme of the 7G x G x C triplet. The three-dimensional structure of the triplex accommodates the 7G with less distortion of the phosphodiester backbone than would be required for an N9-glycosylated guanine in the same sequence position, but some changes in the positions of the phosphodiester backbone are present compared to a C+ x G x C triplet. The structure provides a rationale for the observations that 7G binds to Watson-Crick G x C base pairs with higher specificity and affinity than guanine, but with a lower stability at pH 5.2 than would be provided by a canonical C+ x G x C triplet.
Asunto(s)
Citosina/química , ADN/química , Guanina/química , Ácidos Nucleicos Heterodúplex/química , Composición de Base , ADN/genética , Glicosilación , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/genética , Protones , Soluciones , Espectrofotometría UltravioletaRESUMEN
We have used NMR spectroscopy to study a pyrimidine-purine-pyrimidine DNA triplex containing a non-natural base, 1-(2-deoxy-beta-D-ribofuranosyl)-4-(3-benzamido)phenylimidazole (D3), in the third strand. The D3 base has been previously shown to specifically recognize T-A and C-G base-pairs via intercalation on the 3' side (with respect to the purine strand) of the target base pair, instead of forming sequence-specific hydrogen bonds. 1H resonance assignments have been made for the D3 base and most of the non-loop portion of the triplex. The solution structure of the triplex was calculated using restrained molecular dynamics and complete relaxation matrix refinement. The duplex portion of the triplex has an over-all helical structure that is more similar to B-DNA than to A-DNA. The three aromatic rings of the D3 base stack on the bases of all three strands and mimic a triplet. The conformation of the D3 base and its sequence specificity are discussed.
