Mitotic silencing of human rRNA synthesis: inactivation of the promoter selectivity factor SL1 by cdc2 cyclin B-mediated phosphorylation

We have used a reconstituted cell-free transcription system to investigate the molecular basis of mitotic repression of RNA polymerase I (pol I) trans cription. We demonstrate that SL1, the TBP-containing promoter-binding fact or, is inactivated by cdc2/cyclin B-directed phosphorylation, and reactivat ed by dephosphorylation. Transcriptional inactivation in vitro is accompani ed by phosphorylation of two subunits, e.g. TBP and hTAF(I)110. To distingu ish whether transcriptional repression is due to phosphorylation of TBP, hT AFI110 or both, SL1 was purified from two HeLa cell lines that express eith er full-length or the core domain of TBP only. Both TBP-TAF(I) complexes ex hibit similar activity and both are repressed at mitosis, indicating that t he variable N-terminal domain which contains multiple target sites for cdc2 /cyclin B phosphorylation is dispensable for mitotic repression. Protein-pr otein interaction studies reveal that mitotic phosphorylation impairs the i nteraction of SL1 with UBF, The results suggest that phosphorylation of SL1 is used as a molecular switch to prevent pre-initiation complex formation and to shut down rDNA transcription at mitosis.