Assuntos
Acer , Ascomicetos/metabolismo , Doenças dos Cavalos/diagnóstico , Deficiência Múltipla de Acil Coenzima A Desidrogenase/veterinária , Acer/microbiologia , Ração Animal/análise , Animais , Ascomicetos/crescimento & desenvolvimento , Dieta/veterinária , Evolução Fatal , Doenças dos Cavalos/sangue , Doenças dos Cavalos/etiologia , Cavalos , Hipoglicinas/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Deficiência Múltipla de Acil Coenzima A Desidrogenase/sangue , Deficiência Múltipla de Acil Coenzima A Desidrogenase/diagnóstico , Deficiência Múltipla de Acil Coenzima A Desidrogenase/etiologia , Países Baixos , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Sementes/metabolismoRESUMO
The solution structure of a DNA three-way junction (3H) containing two unpaired thymidine bases at the branch site (3HT2), was determined by NMR. Arms A and B of the 3HT2 form a quasi-continuous stacked helix, which is underwound at the junction and has an increased helical rise. The unstacked arm C forms an acute angle of approximately 55 degrees with the unique arm A. The stacking of the unpaired thymidine bases on arm C resembles the folding of hairpin loops. From this data, combined with the reported stacking behavior of 23 other 3HS2 s, two rules are derived that together correctly reproduce their stacking preference. These rules predict, from the sequence of any 3HS2, its stacking preference. The structure also suggests a plausible mechanism for structure-specific recognition of branched nucleic acids by proteins.
Assuntos
Pareamento de Bases , DNA/química , DNA/genética , Timidina/metabolismo , Sequência de Bases , DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Prótons , RNA Catalítico/química , RNA Catalítico/genética , RNA Catalítico/metabolismo , Recombinação Genética/genética , Soluções , Especificidade por Substrato , Timidina/genéticaRESUMO
In budding yeast, MEC1 and RAD53 are essential for cell growth. Previously we reported that mec1 or rad53 lethality is suppressed by removal of Sml1, a protein that binds to the large subunit of ribonucleotide reductase (Rnr1) and inhibits RNR activity. To understand further the relationship between this suppression and the Sml1-Rnr1 interaction, we randomly mutagenized the SML1 open reading frame. Seven mutations were identified that did not affect protein expression levels but relieved mec1 and rad53 inviability. Interestingly, all seven mutations abolish the Sml1 interaction with Rnr1, suggesting that this interaction causes the lethality observed in mec1 and rad53 strains. The mutant residues all cluster within the 33 C-terminal amino acids of the 104-amino-acid-long Sml1 protein. Four of these residues reside within an alpha-helical structure that was revealed by nuclear magnetic resonance studies. Moreover, deletions encompassing the N-terminal half of Sml1 do not interfere with its RNR inhibitory activity. Finally, the seven sml1 mutations also disrupt the interaction with yeast Rnr3 and human R1, suggesting a conserved binding mechanism between Sml1 and the large subunit of RNR from different species.
Assuntos
Proteínas de Ciclo Celular , Inibidores Enzimáticos , Proteínas Fúngicas/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Ribonucleotídeo Redutases/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae , Supressão Genética , Quinase do Ponto de Checagem 2 , Cromossomos Fúngicos , Análise Mutacional de DNA , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Mutação de Sentido Incorreto , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Soluções , Especificidade da Espécie , Fator Trefoil-2 , Técnicas do Sistema de Duplo-HíbridoAssuntos
Proteínas de Homeodomínio/química , Espectroscopia de Ressonância Magnética/métodos , Proteínas do Tecido Nervoso , Animais , Sítios de Ligação , Isótopos de Carbono , Proteínas de Homeodomínio/metabolismo , Hidrogênio/química , Técnicas In Vitro , Proteínas com Homeodomínio LIM , Isótopos de Nitrogênio , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ratos , Fatores de TranscriçãoRESUMO
The circular DNA decamer 5'-d
Assuntos
DNA Circular/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação de Ácido Nucleico , PrótonsRESUMO
In recent years various examples of highly stable two-residue hairpin loops (miniloops) in DNA have been encountered. As the detailed structure and stability of miniloops appear to be determined not only by the nature and sequence of the two bases in the loop, but also by the closing base pair, it is desirable to carry out in-depth studies of especially designed small model DNA compounds. Therefore, a circular DNA dumbbell-like molecule is tailored to consist of a stem of three Watson-Crick base pairs, flanked on each side by a minihairpin loop. The resulting circular DNA decamer 5'-d
Assuntos
DNA Circular/química , Conformação de Ácido Nucleico , Sequência de Bases , Cinética , Dados de Sequência Molecular , TermodinâmicaRESUMO
The conformational behavior of DNA minihairpin loops is sensitive to the directionality of the base pair that closes the loop. Especially tailored circular dumbbells, consisting of a stem of three Watson-Crick base pairs capped on each side with a minihairpin loop, serve as excellent model compounds by means of which deeper insight is gained into the relative stability and melting properties of hairpin loops that differ only in directionality of the closing pair: C-G vs G-C. For this reason the thermodynamic properties of the circular DNA decamers 5'-d
Assuntos
DNA Circular/química , Conformação de Ácido Nucleico , Sequência de Bases , Fenômenos Químicos , Físico-Química , Dados de Sequência Molecular , TermodinâmicaRESUMO
Two heteronuclear proton-carbon NMR experiments are applied to the DNA-octamer d(TTGGCCAA)2 with carbon in natural abundance. They lead to a complete assignment of the carbon resonances of the sugars and bases. In addition, several heteronuclear coupling constants, proton-carbon as well as proton-phosphorous and phosphorous-carbon, were determined. The information can be obtained in a reasonable measuring time and offers valuable information for a detailed picture of DNA structure.
Assuntos
DNA/química , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Sequência de Bases , Espectroscopia de Ressonância Magnética/métodosRESUMO
The circular DNA decamer 5'-d [formula: see text] 3' is studied in solution by means of NMR spectroscopy. At low temperature the molecule adopts a dumbbell structure with three Watson-Crick C-G base pairs and two two-residue loops in opposite parts of the molecule. On raising the temperature another conformer appears, in which the closing C-G base pair in the 5'-GTTC-3' loop is disrupted, whereas the opposite 5'-CTTG-3' loop remains stable. The two conformers are in slow equilibrium over a limited temperature range.