Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Ácidos Nucleicos/química , Sequência de Bases , Sítios de Ligação , Cátions Monovalentes , DNA/química , Concentração de Íons de Hidrogênio , Modelos Moleculares , Estrutura Molecular , Conformação de Ácido Nucleico , Compostos de Amônio Quaternário , RNA/química , RNA Catalítico/química , Soluções , Temperatura , Tálio , TermodinâmicaRESUMO
Nucleolin is an abundant nucleolar protein which is essential for ribosome biogenesis. The first two of its four tandem RNA-binding domains (RBD12) specifically recognize a stem-loop structure containing a conserved UCCCGA sequence in the loop called the nucleolin-recognition element (NRE). We have determined the structure of the consensus SELEX NRE (sNRE) by NMR spectroscopy. In both the free and bound RNA the top part of the stem forms a loop E (or S-turn) motif. In the absence of protein, the structure of the hairpin loop is not well defined due to conformational heterogeneity, and appears to be in equilibrium between two families of conformations. Titrations of RBD1, RBD2, and RBD12 with the sNRE show that specific binding requires RBD12. In complex with RBD12, the hairpin loop interacts specifically with the protein and adopts a well-defined structure which shares some of the features of the free form. The loop E motif also has specific interactions with the protein. Implications of these findings for the mechanism of recognition of RNA structures by modular proteins are discussed.
Assuntos
Conformação de Ácido Nucleico , Fosfoproteínas/química , Fosfoproteínas/metabolismo , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Animais , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Sequência Consenso/genética , Humanos , Camundongos , Modelos Moleculares , Mutação/genética , Ressonância Magnética Nuclear Biomolecular , Nucleotídeos/química , Nucleotídeos/genética , Nucleotídeos/metabolismo , Maleabilidade , Ligação Proteica , Estrutura Terciária de Proteína , Precursores de RNA/química , Precursores de RNA/genética , Precursores de RNA/metabolismo , Estabilidade de RNA , RNA Ribossômico/genética , Sequências Reguladoras de Ácido Nucleico/genética , Especificidade por Substrato , Termodinâmica , Titulometria , NucleolinaRESUMO
Many bacteria, including several pathogens of plants and humans, use a pheromone called an autoinducer to regulate gene expression in a cell density-dependent manner. Agrobacterium autoinducer [AAI, N-(3-oxo-octanoyl)-L-homoserine lactone] of A. tumefaciens is synthesized by the Tral protein, which is encoded by the tumor-inducing plasmid. Purified hexahistidinyl-Tral (H6-Tral) used S-adenosylmethionine to make the homoserine lactone moiety of AAI, but did not use related compounds. H6-Tral used 3-oxo-octanoyl-acyl carrier protein to make the 3-oxo-octanoyl moiety of AAI, but did not use 3-oxo-octanoyl-coenzyme A. These results demonstrate the enzymatic synthesis of an autoinducer through the use of purified substrates.
