Accessible chromatin is important for RNA polymerase II recruitment and transcription initiation at eukaryotic promoters. We investigated the mechanistic links between promoter DNA sequence, nucleosome positioning, and transcription. Our results indicate that positioning of the transcription start site-associated +1 nucleosome in yeast is critical for efficient TBP binding and is driven by two key factors, the essential chromatin remodeler RSC and a small set of ubiquitous general regulatory factors (GRFs). Our findings indicate that the strength and directionality of RSC action on promoter nucleosomes depends on the arrangement and proximity of two specific DNA motifs. This, together with the effect on nucleosome position observed in double depletion experiments, suggests that, despite their widespread co-localization, RSC and GRFs predominantly act through independent signals to generate accessible chromatin. Our results provide mechanistic insight into how the promoter DNA sequence instructs trans-acting factors to control nucleosome architecture and stimulate transcription initiation.

Chromosome dynamics are recognized to be intimately linked to genomic transactions, yet the physical principles governing spatial fluctuations of chromatin are still a matter of debate. Using high-throughput single-particle tracking, we recorded the movements of nine fluorescently labeled chromosome loci located on chromosomes III, IV, XII, and XIV of Saccharomyces cerevisiae over an extended temporal range spanning more than four orders of magnitude (10 -2 -10 3 sec). Spatial fluctuations appear to be characterized by an anomalous diffusive behavior, which is homogeneous in the time domain, for all sites analyzed. We show that this response is consistent with the Rouse polymer model, and we confirm the relevance of the model with Brownian dynamics simulations and the analysis of the statistical properties of the trajectories. Moreover, the analysis of the amplitude of fluctuations by the Rouse model shows that yeast chromatin is highly flexible, its persistence length being qualitatively estimated to <30 nm. Finally, we show that the Rouse model is also relevant to analyze chromosome motion in mutant cells depleted of proteins that bind to or assemble chromatin, and suggest that it provides a consistent framework to study chromatin dynamics. We discuss the implications of our findings for yeast genome architecture and for target search mechanisms in the nucleus.

Ribosome biogenesis is a complex and energy-demanding process requiring tight coordination of ribosomal RNA (rRNA) and ribosomal protein (RP) production. Given the extremely high level of RP synthesis in rapidly growing cells, alteration of any step in the ribosome assembly process may impact growth by leading to proteotoxic stress. Although the transcription factor Hsf1 has emerged as a central regulator of proteostasis, how its activity is coordinated with ribosome biogenesis is unknown. Here, we show that arrest of ribosome biogenesis in the budding yeast Saccharomyces cerevisiae triggers rapid activation of a highly specific stress pathway that coordinately upregulates Hsf1 target genes and downregulates RP genes. Activation of Hsf1 target genes requires neo-synthesis of RPs, which accumulate in an insoluble fraction and presumably titrate a negative regulator of Hsf1, the Hsp70 chaperone. RP aggregation is also coincident with that of the RP gene activator Ifh1, a transcription factor that is rapidly released from RP gene promoters. Our data support a model in which the levels of newly synthetized RPs, imported into the nucleus but not yet assembled into ribosomes, work to continuously balance Hsf1 and Ifh1 activity, thus guarding against proteotoxic stress during ribosome assembly.

Rpa34 and Rpa49 are nonessential subunits of RNA polymerase I, conserved in species from Saccharomyces cerevisiae and Schizosaccharomyces pombe to humans. Rpa34 bound an N-terminal region of Rpa49 in a two-hybrid assay and was lost from RNA polymerase in an rpa49 mutant lacking this Rpa34-binding domain, whereas rpa34⌬ weakened the binding of Rpa49 to RNA polymerase. rpa34⌬ mutants were caffeine sensitive, and the rpa34⌬ mutation was lethal in a top1⌬ mutant and in rpa14⌬, rpa135(L656P), and rpa135(D395N) RNA polymerase mutants. These defects were shared by rpa49⌬ mutants, were suppressed by the overexpression of Rpa49, and thus, were presumably mediated by Rpa49 itself. rpa49 mutants lacking the Rpa34-binding domain behaved essentially like rpa34⌬ mutants, but strains carrying rpa49⌬ and rpa49-338::HIS3 (encoding a form of Rpa49 lacking the conserved C terminus) had reduced polymerase occupancy at 30°C, failed to grow at 25°C, and were sensitive to 6-azauracil and mycophenolate. Mycophenolate almost fully dissociated the mutant polymerase from its ribosomal DNA (rDNA) template. The rpa49⌬ and rpa49-338::HIS3 mutations had a dual effect on the transcription initiation factor Rrn3 (TIF-IA). They partially impaired its recruitment to the rDNA promoter, an effect that was bypassed by an N-terminal deletion of the Rpa43 subunit encoded by rpa43-35,326, and they strongly reduced the release of the Rrn3 initiation factor during elongation. These data suggest a dual role of the Rpa49-Rpa34 dimer during the recruitment of Rrn3 and its subsequent dissociation from the elongating polymerase.Fast-growing Saccharomyces cerevisiae and Schizosaccharomyces pombe cells mobilize some 70% of their transcriptional capacity to produce the 6.8-kb precursor of the 18S, 5.8S, and 25S rRNAs by transcribing the highly repeated ribosomal DNA (rDNA) locus (44, 50). Genetic studies (43) have established that this is the only role or, at least, the only essential role of yeast RNA polymerase I (Pol I). rDNA transcription starts with the binding of Pol I to its specificity factor Rrn3 (7, 39-41, 48, 57). The Rrn3-Pol I dimer is then directed to a preinitiation complex residing at the rDNA promoter region. This complex combines the TATA box-binding protein (also operating in the Pol II and Pol III systems) with the core and upstream activation factors that are specific to the Pol I system (31, 32). In mammals, Pol I first binds transcription initiation factor IA, akin to Rrn3 and functionally interchangeable with that protein in vivo (7,40,41). The Pol I-transcription initiation factor IA dimer is then targeted to the rDNA promoter by interacting with SL1, a factor made of the TATA box-binding protein associated with four protein subunits, TAF I 95, TAF I 68, TAF I 48, and TAF I 41 (11,22,23,40,59); TAF I 68 is distantly related to the yeast core factor at the level of the core factor's Rrn7/TAF I 68 subunit (9). Pol I is also associated with the upstream binding factor (UBF; mammals) and Hmo1 (yeast) HMG box proteins, which stimulate rDNA t...