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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 cells1 owing to rapid and reversible disruption of activator–promoter complexes by molecular chaperones2,3,4,5,6, or a slower, ubiquitin–proteasome-pathway-mediated turnover of DNA-bound activator7,8,9. 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 expression8,10,11,12. 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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Figure 1: The competition ChIP assay as applied to the yeast transcription factor Gal4.
Figure 2: Kinetic stability of Gal4–promoter complexes under inducing and non-inducing conditions.
Figure 3: Effect of MG132, an inhibitor of 26S proteasome-mediated proteolysis, on Gal4-mediated gene expression.


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This research was supported by the National Institutes of Health and the NHLBI Proteomics Initiative of the National Heart, Lung and Blood Institute, NIH. K.N. was supported by an NIH Cardiology Training Grant Fellowship. ER(LBD)-encoding plasmids were a gift from D. Picard.

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Correspondence to Thomas Kodadek.

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Nalley, K., Johnston, S. & Kodadek, T. Proteolytic turnover of the Gal4 transcription factor is not required for function in vivo. Nature 442, 1054–1057 (2006).

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