An antiparallel four-helix bundle orients the high-affinity RNA binding sites in hnRNP C: A mechanism for RNA chaperonin activity

Lillian Shahied, Emory H. Braswell, Wallace M. LeStourgeon, Andrzej M. Krezel

Research output: Contribution to journalArticle

14 Scopus citations

Abstract

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 Å 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.

Original languageEnglish
Pages (from-to)817-828
Number of pages12
JournalJournal of Molecular Biology
Volume305
Issue number4
DOIs
StatePublished - Jan 26 2001
Externally publishedYes

Keywords

  • Leucine zipper
  • NMR
  • RNA binding
  • Ribonucleoprotein
  • hnRNP

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