Multiple RRMs contribute to RNA binding specificity and affinity for polypyrimidine tract binding protein.
Polypyrimidine tract binding protein (PTB) is a 57 kD hnRNP protein (hnRNP I) that binds to the pyrimidine tract typically found near the 3' end of introns. Primary sequence analysis suggests that PTB contains four RNA recognition motifs (RRMs). Data from comparative structural and deletional analysis of PTB are consistent with the presence of a four reiterated domain structure. Since PTB exists in solution as a homodimer, it contains an oligomeric array of eight RRMs. Though the function of RRMs in a monomeric context has been addressed, the significance of their presence in an oligomeric context has not been investigated. To correlate structural motifs with function, we have analyzed the RNA binding properties of wild-type and deletion constructs of PTB that contain RRMs in both an oligomeric and monomeric context. These studies indicate that there is not a strong correlation between the RNA binding affinity and specificity upon oligomerization. However, the mode of RNA interaction and dimerization is linked. We have also found that the primary contributor to the free energy of PTB binding and the primary determinant for RNA binding specificity resides in RRM 3, while the primary contributor to dimer stabilization coincides with residues in RRM 2.
Pérez
I
I
Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA.
McAfee
J G
JG
Patton
J G
JG
D000818:Animals; D001665:Binding Sites; D002522:Chlorocebus aethiops; D019281:Dimerization; D038941:Polypyrimidine Tract-Binding Protein; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism*; D017423:Sequence Analysis, RNA; D014709:Vero Cells
D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D038941:Polypyrimidine Tract-Binding Protein; D012313:RNA