STRUCTURAL BASIS FOR RECOGNITION OF THE INTRON BRANCH SITE RNA BY SPLICING FACTOR 1
Yeast and mammalian consensus BPS sequence
GAMATRVSDKVMIPQDEYPEINFVGLLIGPRGNTLKNIEKECNAKIMIRGKGSVKEGKVGRKDGQMLPGEDEPLHALVTA NTMENVKKAVEQIRNILKQGIETPEDQNDLRKMQLRELARLNGTLREDDNR
Backbone and side chain 1H, 15N and 13C resonances were assigned using standard triple resonance experiments. Distance restraints were derived from 13C- and 15N-edited 3D NOESY experiments. Assignments for the RNA were obtained from 2D isotope-filtered experiments. Intermolecular distance restraints were measured in 3D 13C-edited/filtered experiments. Dihedral angle restraints for the backbone angle phi were derived from 3J(HN,Ha) coupling constants measured in an HNHA-J experiment, additional phi/psi restraints were derived from TALOS. Hydrogen bond restraints for secondary structure elements in the protein were defined from slowly exchanging amide protons, identified after exchange of the H2O buffer to D2O.
10 lowest energy structures consistent with experimental distance and dihedral angle restraints
STRUCTURES WERE CALCULATED WITH A MIXED TORSION AND CARTESIAN ANGLE DYNAMICS PROTOCOL USING ARIA/CNS. A REDUCED RELAXATION MATRIX APPROACH WAS USED FOR THE NOE CALIBRATION. THE STRUCTURES ARE CURRENTLY BEING REFINED USING RESIDUAL DIPOLAR COUPLINGS AND WILL BE UPDATED IN THE FUTURE.
restrained molecular dynamics, simulated annealing
1 mM U-15N,13C SF1 KH-QUA2/unlabeled BPS in 20 mM phosphate buffer NA pH 6.5, 50 mM NaCl, 2 mM DTT
1 mM U-15N SF1 KH-QUA2/unlabeled BPS in 20 mM phosphate buffer NA pH 6.5, 50 mM NaCl, 2 mM DTT
F. Delaglio, S. Grzesiek, G. Vuister, G. Zhu, J. Pfeifer, & A. Bax
Ch. Bartels, T.-H. Xia, M. Billeter, P. Gntert and K. Wthrich
Splicing, branch point sequence, protein/RNA recognition, complex E, KH domain, QUA2 homology, STAR proteins, GENE REGULATION-RNA COMPLEX
KPPATRVSDKVMIPQDEYPEINFVGLLIGPRGNTLKNIEKECNAKIMIRGKGSVKEGKVGRKDGQMLPGEDEPLHALVTA NTMENVKKAVEQIRNILKQGIETPEDQNDLRKMQLRELARLNGTLREDDNRILRPWQSSETRSITNTTVCTKCGGAGHIA SDCKFQRPGDPQSAQDKARMDKEYLSLMAELGEAPVPASVGSTSGPATTPLASAPRPAAPANNPPPPSLMSTTQSRPPWM NSGPSESRPYHGMHGGGPGGPGGGPHSFPHPLPSLTGGHGGHPMQHNPNGPPPPWMQPPPPPMNQGPHPPGHHGPPPMDQ YLGSTPVGSGVYRLHQGKGMMPPPPMGMMPPPPPPPSGQPPPPPSGPLPPWQQQQQQPPPPPPPSSSMASSTPLPWQQNT TTTTTSAGTGSIPPWQQQQAAAAASPGAPQMQGNPTMVPLPPGVQPPLPPGAPPPPPPPPPGSAGMMIPPRGGDGPSHES EDFPRPLVTLPGRQPQQRPWWTGWFGKAA