1. Departments of Internal Medicine and Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, Texas 75390-9185, USA
2. †Present address: Center for Innovations in Medicine, Biodesign Institute, Arizona State University, 1001 S. McAllister Ave, Tempe, Arizona 85287-5001, USA

Correspondence to: Thomas Kodadek¹ Correspondence and requests for materials should be addressed to T.K. (Email: Thomas.Kodadek@utsouthwestern.edu).

Abstract

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 cells¹ owing to rapid and reversible disruption of activator–promoter complexes by molecular chaperones^{2, 3, 4, 5, 6}, or a slower, ubiquitin–proteasome-pathway-mediated turnover of DNA-bound activator^{7, 8, 9}. 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^{8, 10, 11, 12}. 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.

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