RESUMEN
Non-coding RNA polymerase II transcripts are processed by the poly(A)-independent termination pathway that requires the Nrd1 complex. The Nrd1 complex includes two RNA-binding proteins, the nuclear polyadenylated RNA-binding (Nab) 3 and the nuclear pre-mRNA down-regulation (Nrd) 1 that bind their specific termination elements. Here we report the solution structure of the RNA-recognition motif (RRM) of Nab3 in complex with a UCUU oligonucleotide, representing the Nab3 termination element. The structure shows that the first three nucleotides of UCUU are accommodated on the ß-sheet surface of Nab3 RRM, but reveals a sequence-specific recognition only for the central cytidine and uridine. The specific contacts we identified are important for binding affinity in vitro as well as for yeast viability. Furthermore, we show that both RNA-binding motifs of Nab3 and Nrd1 alone bind their termination elements with a weak affinity. Interestingly, when Nab3 and Nrd1 form a heterodimer, the affinity to RNA is significantly increased due to the cooperative binding. These findings are in accordance with the model of their function in the poly(A) independent termination, in which binding to the combined and/or repetitive termination elements elicits efficient termination.
Asunto(s)
Proteínas Nucleares/química , Oligonucleótidos/química , Proteínas de Unión al ARN/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Transcripción Genética , Secuencia de Bases , Sitios de Unión , Espectroscopía de Resonancia Magnética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oligonucleótidos/metabolismo , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , SolucionesRESUMEN
1-alkynyl-2-deoxy-D-riboses 7 and 8 were independently synthesized and subsequently used to generate several novel C-nucleosides.
RESUMEN
Nuclear polyadenylated RNA-binding (Nab)3 protein is an RNA-binding protein that is involved in the poly(A) independent termination pathway. Here, we report the NMR spectral assignments of RNA-recognition motif (RRM) of Nab3. The assignment will allow performing NMR structural and RNA-binding studies of Nab3 with the aim to investigate its role in the poly(A) independent termination pathway.
Asunto(s)
Proteínas Nucleares/química , Proteínas de Unión al ARN/química , Proteínas de Saccharomyces cerevisiae/química , Secuencias de Aminoácidos , Isótopos de Carbono/química , Hidrógeno/química , Isótopos de Nitrógeno/química , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Saccharomyces cerevisiaeRESUMEN
A new tripodal receptor for the recognition of monosaccharides is described. The prototypical host 1 features a 1,3,5-substituted 2,4,6-triethylbenzene scaffold bearing three convergent H-bonding units. The binding ability of the t-octyl derivative 1a toward a set of octylglycosides of biologically relevant monosaccharides, including Glc, Gal, Man, and GlcNAc, was investigated by 1H NMR in CDCl3. A protocol for the correct evaluation of binding affinities was established, which can be generally applied for the recognition of monosaccharides by 1H NMR spectroscopy. A three-constant equilibrium model, including 1:1 and 2:1 host-guest association and dimerization of the receptor, was ascertained for the interaction of 1a with all the investigated glycosides. An affinity index, which we defined median binding concentration BC50 in analogy to the IC50 parameter, intended to address the general issue of comparing dimensionally heterogeneous binding data, and a limiting BC0(50)quantity describing intrinsic binding affinities were developed for evaluating the results. BC0(50) values for 1a range from 1 to 6 mM, indicating an intrinsic binding affinity in the millimolar range and a selectivity factor of 5 toward the investigated glycosides. The treatment has been extended to include any generic host-guest system involved in single or multiple binding equilibria.