Analysis of sequence-specific binding of RNA to Hsp70 and its various homologs indicates the involvement of N- and C-terminal interactions.
Members of the 70-kDa family of molecular chaperones assist in a number of molecular interactions that are essential under both normal and stress conditions. These functions require ATP and co-chaperone molecules and are associated with a cyclic transition of intramolecular conformational changes. As a new putative function, we have previously shown that mammalian Hsp/Hsc70 as well as a distant relative, Hsp110, selectively bind certain RNA sequences via their N-terminal ATP-binding domain. To investigate this phenomenon in more detail, here we examined RNA-binding affinity and specificity of various deletion mutants of human Hsp70. We demonstrate, that, although the N-terminal ATPase domain alone is sufficient for RNA binding, its binding affinity is considerably reduced when compared to that of the full-length protein. Additionally, we provide evidence that binding of RNA to a membrane-immobilized protein partner results in complete loss of RNA sequence specificity. Using various Hsp70 homologs, we show distinct RNA-binding properties of these proteins judged by sequence specificity, ribopolymer sensitivity, and northwestern analysis. Finally, we present data disclosing that RNA binding by DnaK, the Escherichia coli homolog, is influenced by the activity of its co-chaperones, DnaJ and GrpE. We conclude that the RNA-binding capability of this class of molecular chaperones is a conserved feature and it is strongly influenced by the structural and conformational properties. Furthermore, the notion that RNA binding of some Hsp70 family members is influenced by co-chaperones suggests an RNA-binding cycle resembling the protein-binding property of the chaperones.
Zimmer
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C
Department of Molecular Biology, INTERCELL, Inc., Vienna, Austria.
von Gabain
A
A
Henics
T
T
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D017124:Conserved Sequence; D000091342:Endoplasmic Reticulum Chaperone BiP; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D018832:Molecular Chaperones / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; C494635:DnaJ protein, E coli; D000091342:Endoplasmic Reticulum Chaperone BiP; D029968:Escherichia coli Proteins; C068113:GrpE protein, Bacteria; C489400:GrpE protein, E coli; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D016601:RNA-Binding Proteins; D012313:RNA; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli