Importance of the Sir3 N terminus and its acetylation for yeast transcriptional silencing

Abstract

The N-terminal alanine residues of the silencing protein Sir3 and of Orc1 are acetylated by the NatA Nalpha-acetyltransferase. Mutations demonstrate that the N terminus of Sir3 is important for its function. Sir3 and, perhaps, also Orc1 are the NatA substrates whose lack of acetylation in ard1 and nat1 mutants explains the silencing defect of those mutants.

Figure 1.—

Mutations that alter the Ala2 residue of SIR3 affect silencing. Plasmids encoding wild-type Sir3 or the various sir3 mutations were introduced into the indicated strains. Details about the plasmids and how they were made are available upon request. (A) Silencing at HML as measured by mating of an MATa sir3Δ strain (JCY3) and an MATa sir3Δ sir1Δ strain (JCY8). Both patch mating and quantitative mating results are shown. (B) Silencing at HMR as measured by mating of an MATα sir3Δ strain (JCY4) and an MATα sir3Δ sir1Δ strain (JCY9). (C) Silencing at telomeres assessed in a sir3Δ strain (XRY16) with a URA3 reporter gene near a telomere. Tenfold serial dilutions of the cells were spotted on −trp medium to show the total number of cells plated and on 5-FOA medium to show the fraction of cells in which the URA3 reporter gene is silenced. Good growth on the 5-FOA plates indicates good telomeric silencing.

Figure 1.—

Mutations that alter the Ala2 residue of SIR3 affect silencing. Plasmids encoding wild-type Sir3 or the various sir3 mutations were introduced into the indicated strains. Details about the plasmids and how they were made are available upon request. (A) Silencing at HML as measured by mating of an MATa sir3Δ strain (JCY3) and an MATa sir3Δ sir1Δ strain (JCY8). Both patch mating and quantitative mating results are shown. (B) Silencing at HMR as measured by mating of an MATα sir3Δ strain (JCY4) and an MATα sir3Δ sir1Δ strain (JCY9). (C) Silencing at telomeres assessed in a sir3Δ strain (XRY16) with a URA3 reporter gene near a telomere. Tenfold serial dilutions of the cells were spotted on −trp medium to show the total number of cells plated and on 5-FOA medium to show the fraction of cells in which the URA3 reporter gene is silenced. Good growth on the 5-FOA plates indicates good telomeric silencing.

Figure 1.—

Mutations that alter the Ala2 residue of SIR3 affect silencing. Plasmids encoding wild-type Sir3 or the various sir3 mutations were introduced into the indicated strains. Details about the plasmids and how they were made are available upon request. (A) Silencing at HML as measured by mating of an MATa sir3Δ strain (JCY3) and an MATa sir3Δ sir1Δ strain (JCY8). Both patch mating and quantitative mating results are shown. (B) Silencing at HMR as measured by mating of an MATα sir3Δ strain (JCY4) and an MATα sir3Δ sir1Δ strain (JCY9). (C) Silencing at telomeres assessed in a sir3Δ strain (XRY16) with a URA3 reporter gene near a telomere. Tenfold serial dilutions of the cells were spotted on −trp medium to show the total number of cells plated and on 5-FOA medium to show the fraction of cells in which the URA3 reporter gene is silenced. Good growth on the 5-FOA plates indicates good telomeric silencing.