Proteolytic turnover of the Gal4 transcription factor is not required for function in vivo.
Transactivator-promoter complexes are essential intermediates in the activation of eukaryotic gene expression. Recent studies of these complexes have shown that some are quite dynamic in living cells owing to rapid and reversible disruption of activator-promoter complexes by molecular chaperones, or a slower, ubiquitin-proteasome-pathway-mediated turnover of DNA-bound activator. These mechanisms may act to ensure continued responsiveness of activators to signalling cascades by limiting the lifetime of the active protein-DNA complex. Furthermore, the potency of some activators is compromised by proteasome inhibition, leading to the suggestion that periodic clearance of activators from a promoter is essential for high-level expression. Here we describe a variant of the chromatin immunoprecipitation assay that has allowed direct observation of the kinetic stability of native Gal4-promoter complexes in yeast. Under non-inducing conditions, the complex is dynamic, but on induction the Gal4-promoter complexes 'lock in' and exhibit long half-lives. Inhibition of proteasome-mediated proteolysis had little or no effect on Gal4-mediated gene expression. These studies, combined with earlier data, show that the lifetimes of different transactivator-promoter complexes in vivo can vary widely and that proteasome-mediated turnover is not a general requirement for transactivator function.
Nalley
Kip
K
Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, Texas 75390-9185, USA.
Johnston
Stephen Albert
SA
Kodadek
Thomas
T
D047369:Chromatin Immunoprecipitation; D004268:DNA-Binding Proteins; D015966:Gene Expression Regulation, Fungal; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D011499:Protein Processing, Post-Translational; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; D017405:Herpes Simplex Virus Protein Vmw65; D007976:Leupeptins; D061988:Proteasome Inhibitors; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde