Concerted activities of the RNA recognition and the glycine-rich C-terminal domains of nucleolin are required for efficient complex formation with pre-ribosomal RNA.
Nucleolin is an abundant nucleolar protein which is involved in the early stages of ribosome assembly. The central 40-kDa domain of nucleolin comprises four RNA recognition motifs (RRM) which are presumed to be involved in specific interactions with pre-rRNA. In order to examine in detail the role of this central domain and the contribution of the N-terminal and C-terminal domains of nucleolin to RNA binding, we have used an Escherichia coli expression system to synthezise polypeptides corresponding to various combinations of the three domains and their subdomains. By means of an in-vitro binding assay and a synthetic RNA corresponding to a specific recognition site in pre-rRNA we have been able to demonstrate conclusively that the central 40-kDa domain is indeed responsible for the specificity of RNA recognition and that the N-terminal domain can be removed without affecting RNA binding. Most interestingly, it appears that the C-terminal 10-kDa domain, which is rich in glycine and arginine residues, is essential for efficient binding of nucleolin to RNA, but does not itself contribute to the specificity of the interaction. Circular dichroic spectroscopic probing of the RNA component shows that the C-terminal domain significantly modifies the RNA-binding properties of the central RRM core. Finally, infrared spectroscopic studies reveal that the central 40-kDa domain is structured in alpha helices and beta sheets and that the interaction with the specific pre-rRNA site induces subtle changes in the beta sheet conformation.
Ghisolfi
L
L
Laboratoire de Biologie Moléculaire Eucaryote, Institut de Biologie Cellulaire et de Génétique du CNRS, Toulouse, France.
Kharrat
A
A
Joseph
G
G
Amalric
F
F
Erard
M
M
D001665:Binding Sites; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005998:Glycine; D007700:Kinetics; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011487:Protein Conformation; D012322:RNA Precursors / Q000378:metabolism*; D016601:RNA-Binding Proteins; D015183:Restriction Mapping; D013053:Spectrophotometry; D014158:Transcription, Genetic; D000097796:Nucleolin
D005998:Glycine; D009687:Nuclear Proteins; D010750:Phosphoproteins; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D000097796:Nucleolin