The essential transcription factor Reb1p interacts with the CLB2 UAS outside of the G2/M control region

Regulation of CLB2 is important both for completion of the normal vegetative cell cycle in Saccharo myces cerevisiae and for departure from the vegetative cell cycle upon nitrogen deprivation. Cell cycle-regulated transcription of CLB2 in the G(2)/M phase is known to be brought about by a set of proteins including Mcm1p, Fkh2/1p and Ndd1p that associate with a 35 bp G(2)/M-specific sequence common to a set of co-regulated genes. CLB2 transcription is regulated by additional signals, including by nitrogen levels, by positive feedback from the Clb2-Cdc28 kinase, and by osmotic stress, but the corresponding regulatory sequences and proteins have not been identified. We have found that the essential Reb1 transcription factor binds with high affinity to a sequence upstream of CLB2, within a region implicated previously by others in regulated expression, but upstream of the known G(2)/M-specific site. CLB2 sequence from the region around the Reb1p site blocks activation by the Gal4 protein when positioned downstream of the Gal4-binding site. Since a mutation in the Reb1p site abrogates this effect, we suggest that Reb1p is likely to occupy this site in vivo.