The macrolide antibiotic rapamycin inhibits cellular proliferation by interfering with the highly conserved TOR (for target of rapamycin) signaling pathway. Growth arrest of budding yeast cells treated with rapamycin is followed by the program of molecular events that characterizes entry into G 0 (stationary phase), including the induction of polymerase (Pol) II genes typically expressed only in G 0 . Normally, progression into G 0 is characterized by transcriptional repression of the Pol I and III genes. Here, we show that rapamycin treatment also causes the transcriptional repression of Pol I and III genes. The down-regulation of Pol III transcription is TOR dependent. While it coincides with translational repression by rapamycin, transcriptional repression is due in part to a translation-independent effect that is evident in extracts from a conditional tor2 mutant. Biochemical experiments reveal that RNA Pol III and probably transcription initiation factor TFIIIB are targets of repression by rapamycin. In view of previous evidence that TFIIIB and Pol III are inhibited when protein phosphatase 2A (PP2A) function is impaired, and that PP2A is a component of the TOR pathway, our results suggest that TOR signaling regulates Pol I and Pol III transcription in response to nutrient growth signals.Gradual nutrient depletion in Saccharomyces cerevisiae provokes a broad spectrum of morphological and biochemical changes that result in a terminal cell cycle arrest phenotype called G 0 or stationary phase (reviewed in references 44 and 45). Stationary-phase cells have a 1n DNA content, are uniformly large and unbudded, and display a prominent vacuole. The G 0 state is further characterized by reduced protein synthesis, and the pattern of RNA polymerase (Pol) II transcripts is distinct in cycling and G 0 cells. Thus, more than 95% of Pol II genes are repressed in G 0 , and a subset of Pol II genes whose products promote survival under conditions of nutrient limitation are massively induced at the transcriptional level (8). Induction of the G 0 pattern of Pol II transcription in yeast accompanies the repression of transcription of the large rRNA genes by Pol I and the tRNA and 5S rRNA genes by Pol III (9,26,33,36). Since tRNA and rRNA synthesis accounts for about 70% of nuclear transcription, this regulatory mechanism may enhance survival in G 0 by limiting the energetically costly production of relatively stable RNA products not immediately required for viability.While there is striking repression of translation in G 0 , some critical aspects of the stationary-phase response are not simply downstream consequences of a decreased rate of protein synthesis. For example, treatment of cultures with cycloheximide does not cause the accumulation of large unbudded cells or cells with a 1n DNA content (6), and in some strains there is no inhibition of Pol I or Pol III transcription in extracts from cells treated with cycloheximide (9). Key physiological steps in the differentiation of a stationary-phase cell are therefore likely to...

Here we report that RNA polymerase (pol) III transcription is repressed in response to DNA damage by downregulation of TFIIIB, the core component of the pol III transcriptional machinery. Protein kinase CK2 transduces this stress signal to TFIIIB. CK2 associates with and normally activates the TATA binding protein (TBP) subunit of TFIIIB. The beta regulatory subunit of CK2 binds to TBP and is required for high TBP-associated CK2 activity and pol III transcription in unstressed cells. Transcriptional repression induced by DNA damage requires CK2 and coincides with downregulation of TBP-associated CK2 and dissociation of catalytic subunits from TBP-CK2 complexes. Therefore, CK2 is the terminal effector in a signaling pathway that represses pol III transcription when genome integrity is compromised.

The highly conserved protein kinase casein kinase II (CKII) is required for efficient Pol III transcription of the tRNA and 5S rRNA genes in Saccharomyces cerevisiae. Using purified factors from wild-type cells to complement transcription extracts from a conditional lethal mutant of CKII we show that TFIIIB is the CKII-responsive component of the Pol III transcription machinery. Dephosphorylation of TFIIIB eliminated its ability to complement CKII-depleted extract, and a single TFIIIB subunit, the TATA-binding protein (TBP), is a preferred substrate of CKII in vitro. Recombinant TBP purified fromEscherichia coli is phosphorylated efficiently by CKII and, in the presence of a limiting amount of CKII, is able to substantially rescue transcription in CKII-deficient extract. Our results establish that TBP is a key component of the pathway linking CKII activity and Pol III transcription and suggest that TBP is the target of a CKII-mediated regulatory mechanism that can modulate Pol III transcription.

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We have investigated the molecular basis of the requirement for protein kinase CK2 in nuclear transcription in Saccharomyces cerevisiae. In vivo and in vitro analysis has demonstrated that CK2 is required for efficient transcription of the tRNA and 55 rRNA genes by RNA polymerase III. This suggests that a component of the pol III transcription machinery is regulated by CK2. We tested this possibility by a biochemical complementation approach in which components of the pol III transcription machinery from wild type cells were tested for their ability to rescue transcription in extract from a conditionally CK2-deficient mutant. We found that pol III transcription initiation factor IIIB (TFIIIB) fully restores transcription in CK2-deficient extract. Further in vitro studies revealed that TFIIIB must be phosphorylated to be active, that a single subunit of wild type TFIIIB, the TATA binding protein (TBP), is efficiently phosphorylated by CK2, and that recombinant TBP and a limiting amount of CK2 rescues transcription in CK2-deficient extract. We conclude that TBP is the physiological target of CK2 among the components of the pol III transcription machinery. The implications of this result are discussed in the context of previous data concerning the regulation of TFIIIB.