The Ty5 TD is located at the C terminus of integrase. (A) Ty5 is 5,375 bp in length. It expresses a full-length protein of 182 kDa, which is processed by protease (PR) into Gag, IN, and RT (22). The cleavage sites, based on the mobilities of mature proteins by SDS-PAGE, are shown by dashed lines. The black bar marks the position of TD. The BspEI and PflMI sites define the region of IN used in the mutagenesis experiment. pIP19, pWW32, and pWW59 carry Ty5 elements that were modified by an RGS-H₆ tag. The tag replaced TD (pWW59) or was inserted either into the middle of IN (pWW32) or at the end of RT (pIP19) (22). (B) The modified Ty5 elements were expressed in yeast, and an anti-RGS-H₆ antibody was used to identify IN or RT on immunoblots. The partially processed and mature protein species are indicated. (C) The Ty5 IN fragments used throughout this paper are shown. INC and inc are 258 aa long (the small letters indicate the version with the S1094L mutation). TD and td represent the wild-type and mutant TD plus 3 flanking aa from Ty5. (D) Western blots demonstrating that the wild-type and mutant GBD fusion proteins are expressed at comparable levels in the test strains (YSB2 and UCC3505). GBD-INC and GBD-TD have molecular masses of approximately 47 and 19 kDa, respectively.

The Ty5 TD nucleates silent chromatin. (A) A cartoon depicting the tethered silencing assay. Yeast strains were used with deletions in two of the protein binding sites in HMR-E (A, E, and B) and three copies of UAS_G, binding sites for Gal4p (10). In the example depicted, the E and B binding sites are deleted (e, b), resulting in derepression of transcription at HMR. Expression of a fusion protein between GBD and the Ty5 TD (GBD-TD) was tested for its ability to recruit components of silent chromatin and restore silencing. Silencing is measured by expression of the adjacent TRP1 marker gene. (B) Silencing was established when GBD-INC and GBD-TD were tethered to the weakened HMR locus by the triple UAS_G. Serial, 10-fold dilutions of cells were plated onto control (SC-Ura) or test (SC-Ura-Trp) medium to measure silencing of the TRP1 reporter gene at HMR. (C) A point mutation that abolishes targeting fails to restore silencing for both fusion proteins (GBD-inc and GBD-td) in both test strains.

Silencing conferred by the TD is Sir dependent. The assay system is described in the legend to Fig. 2. In strains with deletions of SIR2, SIR3, or SIR4, GBD-TD fails to establish silencing at a weakened HMR locus. Silencing, however, does not require the SIR1 gene. The Ade⁻ phenotype of the sir2Δ, sir3Δ, and sir4Δ strains confers their dark color; the sir1Δ stain is Ade⁺.

Overexpression of the Ty5 TD disrupts telomeric silencing and loss of silencing is complemented by overexpression of Sir3p. Two reporter genes, URA3 and ADE2, are located at telomeres VIIL and VR, respectively (16). URA3 expression was measured by growth of the yeast cells on selective media. ADE2 expression is indicated by colony color; when ADE2 is repressed, the colonies are red or pink. (A) Overexpression of GBD-TD disrupts telomeric silencing (i.e., causes URA3 expression), in contrast to what occurs in its mutant form (GBD-td) and with GBD alone. (B) Overexpression of Sir2p and Sir3p strengthen silencing, and Sir4p breaks silencing, as previously reported (23, 39). This is demonstrated in the GBD control strains and GBD-td strains. When the TD and SIR genes are overexpressed in the same strains, Sir3p, but not Sir2p or Sir4p, restores silencing.

The Ty5 INC interacts with the C terminus of Sir4p. (A) Yeast two-hybrid assays reveal an interaction between GAD-INC and LexA-SIR4C. Liquid cultures expressing the various LexA and GAD proteins were serially diluted 10-fold and spotted onto plates. A positive two-hybrid interaction was measured by transcriptional activation of the HIS3 reporter, which allowed for growth on selective media (SC-Trp-Leu-His with 5 mM 3-amino-1,2,4-triazole [3AT]). Activation of the HIS3 marker requires both SIR4C and the wild-type TD. (B) SIR4C interacts with GBD-TD in vitro. SIR4C was expressed and labeled with [³⁵S]methionine by coupled transcription and translation. GBD-TD and GBD-td were expressed in yeast and immunoaffinity purified with anti-GBD agarose beads. The top lanes indicate the amount of labeled SIR4C bound by the various GBD proteins. The bottom lanes are from an immunoblot performed with anti-GBD antibodies, and they indicate the levels of GBD proteins in the extract used for immunoaffinity purification.