Recruitment of the transcriptional machinery through GAL11P: structure and interactions of the GAL4 dimerization domain

Genes Dev. 2001 Apr 15;15(8):1007-20. doi: 10.1101/gad.873901.

Abstract

The GAL4 dimerization domain (GAL4-dd) is a powerful transcriptional activator when tethered to DNA in a cell bearing a mutant of the GAL11 protein, named GAL11P. GAL11P (like GAL11) is a component of the RNA-polymerase II holoenzyme. Nuclear magnetic resonance (NMR) studies of GAL4-dd revealed an elongated dimer structure with C(2) symmetry containing three helices that mediate dimerization via coiled-coil contacts. The two loops between the three coiled coils form mobile bulges causing a variation of twist angles between the helix pairs. Chemical shift perturbation analysis mapped the GAL11P-binding site to the C-terminal helix alpha3 and the loop between alpha1 and alpha2. One GAL11P monomer binds to one GAL4-dd dimer rendering the dimer asymmetric and implying an extreme negative cooperativity mechanism. Alanine-scanning mutagenesis of GAL4-dd showed that the NMR-derived GAL11P-binding face is crucial for the novel transcriptional activating function of the GAL4-dd on GAL11P interaction. The binding of GAL4 to GAL11P, although an artificial interaction, represents a unique structural motif for an activating region capable of binding to a single target to effect gene expression.

Publication types

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

MeSH terms

  • Alanine / metabolism
  • Amino Acid Sequence
  • Binding Sites
  • DNA / metabolism
  • DNA-Binding Proteins
  • Dimerization
  • Fungal Proteins / chemistry*
  • Fungal Proteins / metabolism
  • Magnetic Resonance Spectroscopy
  • Mediator Complex
  • Models, Genetic
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Protein Folding
  • Protein Structure, Tertiary
  • RNA Polymerase II / metabolism
  • Saccharomyces cerevisiae / chemistry
  • Saccharomyces cerevisiae Proteins*
  • Sequence Homology, Amino Acid
  • Temperature
  • Trans-Activators / chemistry*
  • Trans-Activators / metabolism
  • Transcription Factors / chemistry*
  • Transcription Factors / metabolism
  • Transcription, Genetic

Substances

  • DNA-Binding Proteins
  • Fungal Proteins
  • GAL11 protein, S cerevisiae
  • GAL4 protein, S cerevisiae
  • Mediator Complex
  • Saccharomyces cerevisiae Proteins
  • Trans-Activators
  • Transcription Factors
  • DNA
  • RNA Polymerase II
  • Alanine