An antiparallel four-helix bundle orients the high-affinity RNA binding sites in hnRNP C: a mechanism for RNA chaperonin activity.
Previous studies have shown that the C protein of 40 S hnRNP complexes contains a leucine-zipper domain, residues 180-207, and that a 40 residue highly basic domain, immediately preceding the zipper, is responsible for almost all of the free energy of RNA binding to C protein. Because this domain arrangement is like that seen in the bZIP transcription factors it has been termed the bZIP-like-motif or bZLM. We report here that the zipper domain drives C protein oligomerization through its spontaneous assembly into an anti-parallel four-helix bundle approximately 50 A in length. The anti-parallel nature of the four-helix bundle positions the tetramer's four high-affinity RNA binding domains at opposing ends of a rigid core formed by the helix bundle. This domain topology is ideally suited to accommodate and direct a double wrapping of RNA around the tetramer and is fully consistent with C protein's ability to bind and order 230 nt lengths of pre-mRNA through a highly cooperative RNA binding mode. We have used a novel sequence-specific 13C/15N labeling strategy and multidimensional NMR spectroscopy to define the anti-parallel orientation of the four-helix bundle and its molecular dimensions. In vitro reconstitution and hydrodynamic studies on native C protein, on several C protein fragments, and on various synthetic peptides, are consistent with the proposed model and indicate that C protein's canonical RNA recognition motifs probably function in tetramer-tetramer interactions during 40 S hnRNP assembly.
Shahied
L
L
Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235-1634, USA.
Braswell
E H
EH
LeStourgeon
W M
WM
Krezel
A M
AM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D016350:Leucine Zippers; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000378:metabolism*; D016415:Sequence Alignment; D013816:Thermodynamics
D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D018832:Molecular Chaperones; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D012313:RNA