RNA polymerase II senses obstruction in the DNA minor groove via a conserved sensor motif

RNA polymerase II (pol II) encounters numerous barriers during transcription elongation, including DNA strand breaks, DNA lesions, and nucleosomes. Pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA with programmable sequence specificity and high affinity. Previous studies suggest that Py-Im polyamides can prevent transcription factor binding, as well as interfere with pol II transcription elongation. However, the mechanism of pol II inhibition by Py-Im polyamides is unclear. Here we investigate the mechanism of how these minor-groove binders affect pol II transcription elongation. In the presence of site-specifically bound Py-Im polyamides, we find that the pol II elongation complex becomes arrested immediately upstream of the targeted DNA sequence, and is not rescued by transcription factor IIS, which is in contrast to pol II blockage by a nucleosome barrier. Further analysis reveals that two conserved pol II residues in the Switch 1 region contribute to pol II stalling. Our study suggests this motif in pol II can sense the structural changes of the DNA minor groove and can be considered a “minor groove sensor.” Prolonged interference of transcription elongation by sequence-specific minor groove binders may present opportunities to target transcription addiction for cancer therapy

RNA polymerase II senses obstruction in the DNA minor groove via a conserved sensor motif

RNA polymerase II (pol II) encounters numerous barriers during transcription elongation, including DNA strand breaks, DNA lesions, and nucleosomes. Pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA with programmable sequence specificity and high affinity. Previous studies suggest that Py-Im polyamides can prevent transcription factor binding, as well as interfere with pol II transcription elongation. However, the mechanism of pol II inhibition by Py-Im polyamides is unclear. Here we investigate the mechanism of how these minor-groove binders affect pol II transcription elongation. In the presence of site-specifically bound Py-Im polyamides, we find that the pol II elongation complex becomes arrested immediately upstream of the targeted DNA sequence, and is not rescued by transcription factor IIS, which is in contrast to pol II blockage by a nucleosome barrier. Further analysis reveals that two conserved pol II residues in the Switch 1 region contribute to pol II stalling. Our study suggests this motif in pol II can sense the structural changes of the DNA minor groove and can be considered a “minor groove sensor.” Prolonged interference of transcription elongation by sequence-specific minor groove binders may present opportunities to target transcription addiction for cancer therapy

Transcription errors induce proteotoxic stress and shorten cellular lifespan

Transcription errors occur in all living cells; however, it is unknown how these errors affect cellular health. To answer this question, we monitored yeast cells that were genetically engineered to display error-prone transcription. We discovered that these cells suffer from a profound loss in proteostasis, which sensitizes them to the expression of genes that are associated with protein-folding diseases in humans; thus, transcription errors represent a new molecular mechanism by which cells can acquire disease. We further found that the error rate of transcription increases as cells age, suggesting that transcription errors affect proteostasis particularly in aging cells. Accordingly, transcription errors accelerate the aggregation of a peptide that is implicated in Alzheimer’s disease, and shorten the lifespan of cells. These experiments reveal a novel, basic biological process that directly affects cellular health and aging

Interconversion of Yeast Mating Types III. Action of the Homothallism (HO) Gene in Cells Homozygous for the Mating Type Locus

Mating type interconversion in homothallic Saccharomyces cerevisiae has been studied in diploids homozygous for the mating type locus produced by sporulation of a/a/a/α and a/a/α/α tetraploid strains. Mating type switches have been analyzed by techniques including direct observation of cells for changes in α-factor sensitivity. Another method of following mating type switching exploits the observation that a/α cells exhibit polar budding and a/a and α/α cells exhibit medial budding.—These studies indicate the following: (1) The allele conferring the homothallic life cycle (HO) is dominant to the allele conferring the heterothallic life cycle (ho). (2) The action of the HO gene is controlled by the mating type locus—active in a/a and α/α cells but not in a/α cells. (3) The HO (or HO-controlled) gene product can act independently on two mating type alleles located on separate chromosomes in the same nucleus. (4) A switch in mating type is observed in pairs of cells, each of which has the same change

Healing of mat mutations and control of mating type interconversion by the mating type locus in Saccharomyces cerevisiae

Homothallic yeasts switch cell types (mating types a and α) at high frequency by changing the alleles of the mating type locus, MATa and MATα. We have proposed in the cassette model that yeast cells contain silent MATa and MATα blocs (“cassettes”), copies of which can be substituted at the mating type locus for the resident information. The existence of silent cassettes was originally proposed to explain efficient switching of a defective MATα locus (matα) to a functional MATα locus. We report here that this “healing” of mat mutations is a general property of the mating type interconversion system and is not specific to the class of matα mutations studied earlier: a defective MATa (mata1) switches readily to MATa and various matα loci switch readily to MATα. These observations satisfy the prediction of the cassette model that all mutations within MATa and MATα be healed. These studies also identify MAT functions that control the switching process: the same functions known to promote sporulation and prevent mating in a/α cells also inhibit the switching system in a/α cells. Finally, we present additional characterization of a natural variant of MATα, MATα-inc [Takano, I., Kusumi, T. & Oshima, Y. (1973) Mol. Gen. Genet. 126, 19-28] that is insensitive to switching. Our observation that MATα-inc acts in cis suggests that it may be altered in a site concerned with excision of MATα-inc or its replacement by another cassette