Regulation of rRNA Synthesis by TATA-Binding Protein-Associated Factor Mot1

Osheim

et al. 2013

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Ribosomal DNA (rDNA) genes in eukaryotes are organized into multicopy tandem arrays and transcribed by RNA polymerase I. During cell proliferation, ϳ50% of these genes are active and have a relatively open chromatin structure characterized by elevated accessibility to psoralen cross-linking. In Saccharomyces cerevisiae, transcription of rDNA genes becomes repressed and chromatin structure closes when cells enter the diauxic shift and growth dramatically slows. In this study, we found that nucleosomes are massively depleted from the active rDNA genes during log phase and reassembled during the diauxic shift, largely accounting for the differences in psoralen accessibility between active and inactive genes. The Rpd3L histone deacetylase complex was required for diauxic shift-induced H4 and H2B deposition onto rDNA genes, suggesting involvement in assembly or stabilization of the entire nucleosome. The Spt16 subunit of FACT, however, was specifically required for H2B deposition, suggesting specificity for the H2A/H2B dimer. Miller chromatin spreads were used for electron microscopic visualization of rDNA genes in an spt16 mutant, which was found to be deficient in the assembly of normal nucleosomes on inactive genes and the disruption of nucleosomes on active genes, consistent with an inability to fully reactivate polymerase I (Pol I) transcription when cells exit stationary phase.

Section: Methodsmentioning

confidence: 99%

Rpd3- and Spt16-Mediated Nucleosome Assembly and Transcriptional Regulation on Yeast Ribosomal DNA Genes

Johnson

Osheim

et al. 2013

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“…Chromatin immunoprecipitation (ChIPs) were performed as previously described (19). Exponentially growing cells (optical density at 600 nm [OD 600 ] of ϳ1.0) in 100 ml of YPD medium were cross-linked with 1% formaldehyde for 20 min and then washed twice with 10 ml of cold TBS (300 mM NaCl, 40 mM Tris-HCl [pH 7.5]).…”

Section: Methodsmentioning

confidence: 99%

Distinguishing the Roles of Topoisomerases I and II in Relief of Transcription-Induced Torsional Stress in Yeast rRNA Genes

Sikes

Hontz

et al. 2011

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To better understand the role of topoisomerase activity in relieving transcription-induced supercoiling, yeast genes encoding rRNA were visualized in cells deficient for either or both of the two major topoisomerases. In the absence of both topoisomerase I (Top1) and topoisomerase II (Top2) activity, processivity was severely impaired and polymerases were unable to transcribe through the 6.7-kb gene. Loss of Top1 resulted in increased negative superhelical density (two to six times the normal value) in a significant subset of rRNA genes, as manifested by regions of DNA template melting. The observed DNA bubbles were not R-loops and did not block polymerase movement, since genes with DNA template melting showed no evidence of slowed elongation. Inactivation of Top2, however, resulted in characteristic signs of slowed elongation in rRNA genes, suggesting that Top2 alleviates transcription-induced positive supercoiling. Together, the data indicate that torsion in front of and behind transcribing polymerase I has different consequences and different resolution. Positive torsion in front of the polymerase induces supercoiling (writhe) and is largely resolved by Top2. Negative torsion behind the polymerase induces DNA strand separation and is largely resolved by Top1.Eukaryotic cells have two major topoisomerases that are capable of efficiently relaxing torsionally stressed DNA: topoisomerase I (Top1) and topoisomerase II (Top2) (75). They are both abundant nuclear proteins with roles in many DNA activities, and since they both can relax positive and negative torsion, they can substitute for each other in most situations (11,28,29,35,62). In spite of this partial functional redundancy, they control DNA topology by very different mechanisms (65). Top1 (a type IB topoisomerase) makes transient single-strand breaks in torsionally stressed DNA (recognizing the torque in such DNA), followed by controlled rotation of the nicked strand and resealing of the DNA in a more relaxed state (38). Top2 (a type IIA topoisomerase) recognizes juxtaposed DNA helices (as in supercoiled DNA) and passes one DNA helix through the other by making a transient doublestrand break in one of the helices (61, 65

“…ChIPs were performed as previously described (11), with some modifications. Yeast cells were grown in 10 ml YPD overnight and then reinoculated into 100 ml fresh YPD at an OD 600 of 0.2 and shaken for 4 h at 30°C to an OD 600 of ϳ1.…”

Section: Methodsmentioning

confidence: 99%

Transcription of Multiple Yeast Ribosomal DNA Genes Requires Targeting of UAF to the Promoter by Uaf30

Hontz¹,

Oakes³

et al. 2008

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Upstream activating factor (UAF) is a multisubunit complex that functions in the activation of ribosomal DNA (rDNA) transcription by RNA polymerase I (Pol I). Cells lacking the Uaf30 subunit of UAF reduce the rRNA synthesis rate by ϳ70% compared to wild-type cells and produce rRNA using both Pol I and Pol II. Miller chromatin spreads demonstrated that even though there is an overall reduction in rRNA synthesis in uaf30 mutants, the active rDNA genes in such strains are overloaded with polymerases. This phenotype was specific to defects in Uaf30, as mutations in other UAF subunits resulted in a complete absence of rDNA genes with high or even modest Pol densities. The lack of Uaf30 prevented UAF from efficiently binding to the rDNA promoter in vivo, leading to an inability to activate a large number of rDNA genes. The relatively few genes that did become activated were highly transcribed, apparently to compensate for the reduced rRNA synthesis capacity. The results show that Uaf30p is a key targeting factor for the UAF complex that facilitates activation of a large proportion of rDNA genes in the tandem array.