Zinc is required for assembly and function of the anti-trp RNA-binding attenuation protein, AT

J Biol Chem. 2002 Dec 13;277(50):48574-8. doi: 10.1074/jbc.M208980200. Epub 2002 Oct 16.

Abstract

The anti-TRAP protein (AT) of Bacillus subtilis regulates expression of the trp operon and other genes concerned with tryptophan metabolism. AT acts by inhibiting the tryptophan-activated trp RNA-binding attenuation protein (TRAP). AT is an oligomer of identical 53-residue polypeptides; it is produced in response to the accumulation of uncharged tRNA(Trp). Each AT polypeptide has two cysteine-rich clusters that correspond to the signature motif of the cysteine-rich zinc-binding domain of the chaperone protein DnaJ. Here we characterize the putative zinc-binding domain of AT and establish the importance of zinc for AT assembly and activity. AT is shown to contain Zn(II) at a ratio of one ion per monomer. Bound zinc is necessary for maintenance of the quaternary structure of AT; the removal of zinc converts the AT complex into inactive monomers. All four cysteine residues in the AT polypeptide are involved in Zn(II) coordination. Chemical cross-linking analyses indicate that the AT functional oligomer is a hexamer composed of two trimers. Substituting alanine for any cysteine residue of AT results in rapid degradation of the mutant protein in vivo. We propose a model for the AT trimer in which three AT chains are held together by three zinc atoms, each coordinated by the N-terminal segment and the C-terminal segment of separate AT polypeptides.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Electrophoresis, Polyacrylamide Gel
  • Mutagenesis
  • Protein Conformation
  • RNA-Binding Proteins / chemistry
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*
  • RNA-Binding Proteins / physiology
  • Zinc / physiology*

Substances

  • RNA-Binding Proteins
  • Zinc