Recognition of pre-formed and flexible elements of an RNA stem-loop by nucleolin.
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.
Bouvet
P
P
Laboratoire de Pharmacologie et de Biologie Structurale, 205 route de Narbonne, Toulouse Cedex, 31077, France.
Allain
F H
FH
Finger
L D
LD
Dieckmann
T
T
Feigon
J
J
D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence / Q000235:genetics; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D018583:Pliability; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020871:RNA Stability; D012335:RNA, Ribosomal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D013379:Substrate Specificity; D013816:Thermodynamics; D017186:Titrimetry; D000097796:Nucleolin
D009711:Nucleotides; D010750:Phosphoproteins; D012322:RNA Precursors; D012335:RNA, Ribosomal; D016601:RNA-Binding Proteins; D000097796:Nucleolin