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...

We purified xUBF on the basis of its ability to specifically bind the enhancer elements of the Xenopus laevis rRNA genes. xUBF also binds to both upstream and downstream regions of the X. laevis ribosomal gene promoter and is essential for polymerase I transcription. Unexpectedly, xUBF binds to both upstream and downstream regions of the human ribosomal gene promoter, producing footprints that are indistinguishable from the footprints produced by hUBF, a previously described polymerase I transcription factor isolated from human cells. Despite extensive sequence divergence of vertebrate polymerase I promoters, these data suggest an evolutionary conservation of the primary DNA-protein interaction.

No abstract

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.

Little is known about what enzyme complexes or mechanisms control global lysine acetylation in the amino-terminal tails of the histones. Here, we show that glucose induces overall acetylation of H3 K9, 18, 27 and H4 K5, 8, 12 in quiescent yeast cells mainly by stimulating two KATs, Gcn5 and Esa1. Genetic and pharmacological perturbation of carbon metabolism, combined with 1H-NMR metabolic profiling, revealed that glucose induction of KAT activity directly depends on increased glucose catabolism. Glucose-inducible Esa1 and Gcn5 activities predominantly reside in the picNuA4 and SAGA complexes, respectively, and act on chromatin by an untargeted mechanism. We conclude that direct metabolic regulation of globally acting KATs can be a potent driving force for reconfiguration of overall histone acetylation in response to a physiological cue.

The hydrophilic amino-terminal sequences of histones H3 and H4 extend from the highly structured nucleosome core. Here we examine the importance of the amino termini and their position in the nucleosome with regard to both nucleosome assembly and gene regulation. Despite previous conclusions based on nonphysiological nucleosome reconstitution experiments, we find that the histone amino termini are important for nucleosome assembly in vivo and in vitro. Deletion of both tails, a lethal event, alters micrococcal nuclease-generated nucleosomal ladders, plasmid superhelicity in whole cells, and nucleosome assembly in cell extracts. The H3 and H4 amino-terminal tails have redundant functions in this regard because the presence of either tail allows assembly and cellular viability. Moreover, the tails need not be attached to their native carboxy-terminal core. Their exchange re-establishes both cellular viability and nucleosome assembly. In contrast, the regulation of GALl and the silent mating loci by the H3 and H4 tails is highly disrupted by exchange of the histone amino termini.

Histone H4 can be acetylated at N-terminal lysines K5, K8, K12, and K16, but newly synthesized H4 is diacetylated at K5͞K12 in diverse organisms. This pattern is widely thought to be important for histone deposition onto replicating DNA. To investigate the importance of K5͞K12 we have mutagenized these lysines in yeast and assayed for nucleosome assembly. Assaying was done in the absence of the histone H3 N terminus, which has functions redundant with those of H4 in histone deposition. Nucleosome assembly was assayed by three methods. Because nucleosome depletion may be lethal, we examined cell viability. We also analyzed nucleosome assembly in vivo and in vitro by examining plasmid superhelicity density in whole cells and supercoiling in yeast cell extracts. All three approaches demonstrate that mutagenizing K5 and K12 together does not prevent cell growth and histone deposition in vivo or in vitro. Therefore, K5͞K12 cannot be required for nucleosome assembly in yeast. It is only when the first three sites of acetylation-K5, K8, and K12-are mutagenized simultaneously that lethality occurs and assembly is most strongly decreased both in vivo and in vitro. These data argue for the redundancy of sites K5, K8, and K12 in the deposition of yeast histone H4.Nucleosome assembly involves the deposition of a tetramer of histones H3 and H4 onto DNA, followed by the association of two histone H2A͞H2B dimers. In this process, acetylation of histone H4 is likely to play a key role. Newly synthesized histone H4 was shown by pulse-labeling to be diacetylated at lysine residues 5 and 12 (K5͞K12), a conserved feature in Tetrahymena, flies, and humans (1-3). This distinct nonrandom pattern of acetylation among the four acetylatable lysines (K5, K8, K12, and K16) has led to the suggestion that K5͞K12 diacetylation serves a unique role in targeting newly synthesized histone H4 for assembly (4). Another argument for the importance of H4 acetylation in nucleosome assembly derives from the study of the human multiprotein complex (CAF-1, for chromatin assembly factor) that enables H3 and H4 assembly onto replicating DNA in a simian virus 40-based cell-free system (5-7). CAF-1 deposits newly synthesized histones but not those extracted from bulk chromatin onto DNA (7), a result which is consistent with the finding that CAF-1 associates preferentially with histone H4 acetylated in a specific manner (8). However, H4 associated with CAF-1 is not uniquely diacetylated at K5 and K12 but is heterogeneously acetylated at K5, K8, and K12. Moreover, some 33% of H4 in the complex is not acetylated. In addition, much of the H3 in the complex is monoacetylated, whereas some 60% of H3 is unacetylated (9).The yeast (Saccharomyces cerevisiae) histone H4 N-terminal sequence and the location of its acetylated lysines are extremely conserved in evolution. While it is not known whether newly synthesized yeast H4 is diacetylated or whether acetylated H4 is associated with a yeast chromatin assembly factor, we set out to ask whether K5͞K12 is requi...

Previous work has shown that rRNA synthesis is strongly inhibited in yeast topl-top2 double mutants. Here, we show that inactivation of yeast topoisomerases can have paradoxical effects on transcription by RNA polymerase I. For example, transcription of ribosomal minigenes on extrachromosomal plasmids is greatly stimulated in topl-top2 cells while accumulation of full-length endogenous rRNA is strongly inhibited. We present evidence for a mechanism that can partly account for these opposing effects on transcription. On the one hand, transcription initiation can be stimulated owing to an accumulation of negative superhelicity because polymerase I prefers to initiate on negatively supercoiled templates. Conversely, synthesis of full-length rRNA is inhibited owing to the fact that chain elongation requires a DNA relaxing activity.