Summary Mating type switching in yeast occurs through gene conversion between the MAT locus and one of two silent loci (HML or HMR) on opposite ends of the chromosome. MATa cells choose HML as template, while MATα cells use HMR. The Recombination Enhancer (RE), located on the left arm regulates this process. One long-standing hypothesis is that switching is guided by mating type-specific, and possibly RE-dependent chromosome folding. Here we use Hi-C, 5C, and live cell imaging to characterize the conformation of chromosome III in both mating types. We discovered a mating type-specific conformational difference in the left arm. Deletion of a 1 kb subregion within the RE, which is not necessary during switching, abolished mating type-dependent chromosome folding. The RE is therefore a composite element with one subregion essential for donor selection during switching, and a separate region involved in modulating chromosome conformation.

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Most transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I. Previous studies provided insight in the involvement of Rpa49 in initiation, elongation, docking and releasing of Rrn3, an essential Pol I transcription factor. Here, we took advantage of the spontaneous occurrence of extragenic suppressors of the growth defect of the rpa49 null mutant to better understand the activity of Pol I. Combining genetic approaches, biochemical analysis of rRNA synthesis and investigation of the transcription rate at the individual gene scale, we characterized mutated residues of the Pol I as novel extragenic suppressors of the growth defect caused by the absence of Rpa49. When mapped on the Pol I structure, most of these mutations cluster within the jaw-lobe module, at an interface formed by the lobe in Rpa135 and the jaw made up of regions of Rpa190 and Rpa12. In vivo , the suppressor allele RPA135-F301S restores normal rRNA synthesis and increases Pol I density on rDNA genes when Rpa49 is absent. Growth of the Rpa135-F301S mutant is impaired when combined with exosome mutation rrp6Δ and it massively accumulates pre-rRNA. Moreover, Pol I bearing Rpa135-F301S is a hyper-active RNA polymerase in an in vitro tailed-template assay. We conclude that RNA polymerase I can be engineered to produce more rRNA in vivo and in vitro . We propose that the mutated area undergoes a conformational change that supports the DNA insertion into the cleft of the enzyme resulting in a super-active form of Pol I.

Most transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I. Previous studies provided insight in the involvement of Rpa49 in initiation, elongation, docking and releasing of Rrn3, an essential Pol I transcription factor. Here, we took advantage of the spontaneous occurrence of extragenic suppressors of the growth defect of the rpa49 null mutant to better understand the activity of Pol I. Combining genetic approaches, biochemical analysis of rRNA synthesis and investigation of the transcription rate at the individual gene scale, we characterized mutated residues of the Pol I as novel extragenic suppressors of the growth defect caused by the absence of Rpa49. When mapped on the Pol I structure, most of these mutations cluster within the jaw-lobe module, at an interface formed by the lobe in Rpa135 and the jaw made up of regions of Rpa190 and Rpa12. In vivo, the suppressor allele RPA135-F301S restores normal rRNA synthesis and increases Pol I density on rDNA genes when Rpa49 is absent. Growth of the Rpa135-F301S mutant is impaired when combined with exosome mutation rrp6Δ and it massively accumulates pre-rRNA. Moreover, Pol I bearing Rpa135-F301S is a hyper-active RNA polymerase in an in vitro tailed-template assay. We conclude that wild-type RNA polymerase I can be engineered to produce more rRNA in vivo and in vitro. We propose that the mutated area undergoes a conformational change that supports the DNA insertion into the cleft of the enzyme resulting in a super-active form of Pol I.Author summaryThe nuclear genome of eukaryotic cells is transcribed by three RNA polymerases. RNA polymerase I (Pol I) is a multimeric enzyme specialized in the synthesis of ribosomal RNA. Deregulation of the Pol I function is linked to the etiology of a broad range of human diseases. Understanding the Pol I activity and regulation represents therefore a major challenge. We chose the budding yeast Saccharomyces cerevisiae as a model, because Pol I transcription apparatus is genetically amenable in this organism. Analyses of phenotypic consequences of deletion/truncation of Pol I subunits-coding genes in yeast indeed provided insights into the activity and regulation of the enzyme. Here, we characterized mutations in Pol I that can alleviate the growth defect caused by the absence of Rpa49, one of the subunits composing this multi-protein enzyme. We mapped these mutations on the Pol I structure and found that they all cluster in a well-described structural element, the jaw-lobe module. Combining genetic and biochemical approaches, we showed that Pol I bearing one of these mutations in the Rpa135 subunit is able to produce more ribosomal RNA in vivo and in vitro. We propose that this super-activity is explained by structural rearrangement of the Pol I jaw/lobe interface.

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The ParB-parS partition complexes that bacterial replicons use to ensure their faithful inheritance also find employment in visualization of DNA loci, as less intrusive alternatives to fluorescent repressor-operator systems. The ability of ParB molecules to interact via their N-terminal domains and to bind to non-specific DNA enables expansion of the initial complex to a size both functional in partition and, via fusion to fluorescent peptides, visible by light microscopy. We have investigated whether it is possible to dispense with the need to insert parS in the genomic locus of interest, by determining whether ParB fused to proteins that bind specifically to natural DNA sequences can still assemble visible complexes. In yeast cells, coproduction of fusions of ParB to a fluorescent peptide and to a TALE protein targeting an endogenous sequence did not yield visible foci; nor did any of several variants of these components. In E.coli, coproduction of fusions of SopB (F plasmid ParB) to fluorescent peptide, and to dCas9 together with specific guide RNAs, likewise yielded no foci. The result of coproducing analogous fusions of SopB proteins with distinct binding specificities was also negative. We conclude that in order to assemble higher order partition complexes, ParB proteins need specific activation through binding to their cognate parS sites.

No abstract

The ParB-parS partition complexes that bacterial replicons use to ensure their faithful inheritance also find employment in visualization of DNA loci, as less intrusive alternatives to fluorescent repressor-operator systems. The ability of ParB molecules to interact via their Nterminal domains and to bind to non-specific DNA enables expansion of the initial complex to a size both functional in partition and, via fusion to fluorescent peptides, visible by light microscopy. We have investigated whether it is possible to dispense with the need to insert parS in the genomic locus of interest, by determining whether ParB fused to proteins that bind specifically to natural DNA sequences can still assemble visible complexes. In yeast cells, coproduction of fusions of ParB to a fluorescent peptide and to a TALE protein targeting an endogenous sequence did not yield visible foci; nor did any of several variants of these components. In E.coli, coproduction of fusions of SopB (F plasmid ParB) to fluorescent peptide, and to dCas9 together with specific guide RNAs, likewise yielded no foci. The result of coproducing analogous fusions of SopB proteins with distinct binding specificities was also negative. Our observations imply that in order to assemble higher order partition complexes, ParB proteins need specific activation through binding to their cognate parS sites.