A Reb1p-CLB2 UAS complex is modulated by an activity associated with glutaminyl tRNA synthetase that is likely a protease.
Lakmal Kotelawala, Jodi Reynolds, Eric Phizicky, Elizabeth J. Grayhack
Biochemistry & Biophysics, University of Rochester, 601 Elmwood Avenue, Rochester, NY, 14642, USA
Regulation of the CLB2 gene is important for passage through the cell cycle, and in the transition from vegetative growth to pseudohyphal development that is triggered by low nitrogen. In examining regulation of CLB2 transcription, we have found that the essential Reb1p forms a high affinity complex with the CLB2 UAS both in vitro and in vivo. We have also sought yeast proteins that modulate the Reb1p-CLB2 UAS complex by assaying a genomic collection of purified GST-ORF fusion proteins for changes in the complex on EMSA. Surprisingly, we have found that an activity associated with glutaminyl tRNA synthetase (Gln4p) alters, but does not eliminate, this complex. Three observations underscore our interest in this activity: the activity co-purifies with Gln4p through two affinity purification steps from chromosomally TAP-tagged Gln4p; the concentration of Gln4p required to alter the Reb1p-CLB2 complex (0.5 nM) is well below its cellular concentration; and the activity is specific for the Reb1p-DNA complex compared to a control complex such as the Gal4DBDp-GAL UAS complex. We have recently found two lines of evidence that the activity that acts on the Reb1p-CLB2 UAS complex is due to a protease: Gln4p co-purifies with a latent protease activity that can be activated by SDS; and the activity that affects the Reb1p-CLB2 complex is inhibited by GST-Pbi2p, which is known to associate with, and inhibit, protease Prb1p.
Return to YGM 2004 Home at SGD