Contributions of the RNA-binding and linker domains and RNA structure to the specificity and affinity of the nucleolin RBD12/NRE interaction.
Nucleolin is a multidomain phosphoprotein involved in ribosome biogenesis. In vitro selection and binding studies with pre-rRNA fragments have shown that the first two RNA-binding domains (RBDs) in nucleolin (RBD12) recognize the consensus sequence (U/G)CCCG(A/G) in the context of a stem-loop structure (nucleolin-recognition element = NRE). Structural studies of nucleolin RBD12 in complex with an in vitro selected NRE (sNRE) and a natural pre-rRNA NRE (b2NRE) have revealed that sequence-specific binding of the consensus NRE is achieved in a similar manner in both complexes using residues in both RBDs as well as the linker connecting them. Using fluorescence anisotropy (FA) and nuclear magnetic resonance (NMR), we demonstrate the importance of the linker for NRE affinity by showing that only the individual RBDs with the linker attached retain the ability to specifically bind, albeit weakly, to sNRE and b2NRE. Binding of RBD1 and RBD2 to the NREs in trans is not detected even when one of the RBDs has the linker attached, which suggests that the linker also contributes to the affinity by tethering the two RBDs. To determine if binding of nucleolin RBD12 to natural NREs is dependent on a specific RNA stem-loop structure, as was the case for the sNRE, we conducted FA and NMR binding assays with nucleolin RBD12 and a single-stranded NRE. The results show that nucleolin RBD12 sequence-specifically binds a single-stranded NRE with an affinity similar to that for b2NRE, indicating that a stem-loop structure is not required for the nucleolin RBD12/pre-rRNA NRE interaction.
Finger
L David
LD
Department of Chemistry and Biochemistry, and Molecular Biology Institute, University of California, Los Angeles, California 90095-1569, USA.
Johansson
Carina
C
Rinaldi
Bruno
B
Bouvet
Philippe
P
Feigon
Juli
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D006224:Cricetinae; D005454:Fluorescence Polarization; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012322:RNA Precursors / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000097796:Nucleolin
D010750:Phosphoproteins; D012313:RNA; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D000097796:Nucleolin