Substrate dependence of conformational changes in the RNA-binding domain of tristetraprolin assessed by fluorescence spectroscopy of tryptophan mutants.

ABSTRACT: Association of tristetraprolin (TTP) with mRNAs containing selected AU-rich mRNA-destabilizing elements (AREs) initiates rapid cytoplasmic degradation of these transcripts. The RNA-binding activity of TTP is mediated by an internal tandem zinc finger domain that preferentially recognizes U-rich RNA ligands containing adjacent UUAU half-sites and is accompanied by conformational changes within the peptide. Here, we have used analogues of the TTP RNA-binding domain containing specific tryptophan substitutions to probe the Zn2+ and RNA substrate dependence of conformational events within individual zinc fingers. Fluorescence methods demonstrate that the N-terminal, but not C-terminal, zinc finger domain adopts a stably folded conformation in the presence of Zn2+. Denaturant titrations suggest that both the N- and C-terminal zinc fingers exhibit limited structural heterogeneity in the absence of RNA substrates, although this is more pronounced for the C-terminal finger. Binding to a cognate ARE substrate induced significant conformational changes within each zinc finger, which also included increased resistance to chemical denaturation. Studies with mutant ARE ligands revealed that a single UUAU half-site was sufficient to induce structural modulation of the N-terminal finger. However, RNA-dependent folding of the C-terminal zinc finger was only observed in the presence of tandem UUAU half-sites, suggesting that the conformation of this domain is linked not only to RNA substrate recognition but also to the ligand occupancy and/or conformational status of the N-terminal finger. Coupled with previous structural and thermodynamic analyses, these data provide a mechanistic framework for discrimination of RNA substrates involving ligand-dependent conformational adaptation of both zinc fingers within the TTP RNA-binding domain.

SUBMITTER: Brewer BY 

PROVIDER: S-EPMC1640280 | biostudies-literature | 2006 Nov

REPOSITORIES: biostudies-literature