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) transcription. We demonstrate that SL1, the TBP-containing promoter-binding factor, is inactivated by cdc2/cyclin B-directed phosphorylation, and reactivated by dephosphorylation. Transcriptional inactivation in vitro is accompanied by phosphorylation of two subunits, e.g. TBP and hTAFI110. To distinguish whether transcriptional repression is due to phosphorylation of TBP, hTAFI110 or both, SL1 was purified from two HeLa cell lines that express either full-length or the core domain of TBP only. Both TBP-TAFI complexes exhibit similar activity and both are repressed at mitosis, indicating that the variable N-terminal domain which contains multiple target sites for cdc2/cyclin B phosphorylation is dispensable for mitotic repression. Protein-protein interaction studies reveal that mitotic phosphorylation impairs the interaction 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.
Heix
J
J
Division of Molecular Biology of the Cell II, German Cancer Research Center, D-69120 Heidelberg, Germany.
Vente
A
A
Voit
R
R
Budde
A
A
Michaelidis
T M
TM
Grummt
I
I
D000595:Amino Acid Sequence; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002474:Cell-Free System; D019926:Cyclin B / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010766:Phosphorylation; D035463:Pol1 Transcription Initiation Complex Proteins; D011485:Protein Binding; D012318:RNA Polymerase I / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000096:biosynthesis*; D035381:TATA-Binding Protein Associated Factors; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014158:Transcription, Genetic
D019926:Cyclin B; D004268:DNA-Binding Proteins; D035463:Pol1 Transcription Initiation Complex Proteins; D012335:RNA, Ribosomal; C486319:TAF1C protein, human; D035381:TATA-Binding Protein Associated Factors; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID; D014157:Transcription Factors; C063844:transcription factor UBF; C063660:transcription initiation factor TIF-IB; D016203:CDC2 Protein Kinase; D012318:RNA Polymerase I