A major determinant of hnRNP C protein binding to RNA is a novel bZIP-like RNA binding domain.
The C protein tetramer of hnRNP complexes binds approximately 150-230 nt of RNA with high cooperativity (McAfee J et al., 1996, Biochemistry 35:1212-1222). Three contiguously bound tetramers fold 700-nt lengths of RNA into a 19S triangular intermediate that nucleates 40S hnRNP assembly in vitro (Huang M et al., 1994, Mol Cell Biol 14:518-533). Although it has been assumed that the consensus RNA recognition motif (RRM) of C protein (residues 8-87) is the primary determinant of RNA binding, we report here that a recombinant construct containing residues 1-115 has very low affinity for RNA at physiological ionic strength (100 mM NaCl). Moreover, we demonstrate that an N-terminal deletion construct lacking the consensus RRM but containing residues 140-290 binds RNA with an affinity sufficient to account for the total free energy change observed for the binding of intact protein. Like native C protein, the 140-290 construct is a tetramer in solution and binds RNA stoichiometrically in a salt-resistant manner in 100-300 mM NaCl. Residues 140-179 of the N-terminal truncated variant contain 11 basic and 2 acidic residues, whereas residues 180-207 specify a leucine zipper motif that directs dimer assembly. Elements within the 50-residue carboxy terminus of C protein are required for tetramer assembly. A basic region followed by a leucine zipper is identical to the domain organization of the basic-leucine zipper (bZIP) class of DNA binding proteins. Sequence homologies with other proteins containing RRMs and the bZIP motif suggest that residues 140-207 represent a conserved bZIP-like RNA binding motif (designated bZLM). The steric orientation of four high-affinity RNA binding sites about rigid leucine zipper domains may explain in part C protein's asymmetry, its large occluded site size, and its RNA folding activity.
McAfee
J G
JG
Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA.
Shahied-Milam
L
L
Soltaninassab
S R
SR
LeStourgeon
W M
WM
D000595:Amino Acid Sequence; D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors; D001665:Binding Sites / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051790:G-Box Binding Factors; D006367:HeLa Cells; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D016350:Leucine Zippers / Q000235:genetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors
D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D051790:G-Box Binding Factors; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D014157:Transcription Factors; D012313:RNA