Domain Organization and Quaternary Structure of the Saccharomyces cerevisiae Silent Information Regulator 3 Protein, Sir3p

Abstract

The silent information regulator protein 3 (Sir3p) functions in the initiation, propagation, and maintenance of transcriptionally silenced chromatin in Saccharomyces cerevisiae. To better understand the physicochemical basis for its effects on chromatin architecture, recombinant full-length S. cerevisiae Sir3p has been purified to near homogeneity on the large-scale and characterized by circular dichroism, limited protease digestion, and analytical ultracentrifugation. Results indicate that the Sir3p monomer has a unique tripartite domain organization, including a nearly 300-amino-acid residue stretch of intrinsically disordered residues that lies internal to its structured N- and C-terminal regions. Sir3p self-associates extensively in moderate salt and at micromolar protein concentrations producing a broad range of oligomers that sediment from 8 to in excess of 85 S. These results provide new insight into Sir3p domain organization and quaternary structure and support a nucleosome bridging model for Sir3p-dependent regulation of chromatin architecture.

The Silent Information Regulator 3 Protein, SIR3p, Binds to Chromatin Fibers and Assembles a Hypercondensed Chromatin Architecture in the Presence of Salt

“…To properly interpret the resulting biochemical and EM data, one must take into account the complex quaternary structure of SIR3p when free in solution. The 113-kDa full-length SIR3p monomer sediments at 6S (39). Under physiological salt conditions and at submicromolar protein concentrations, the 6S monomer forms a stable 8S dimer (39), most likely arranged in an antiparallel orientation (33).…”

“…The 113-kDa full-length SIR3p monomer sediments at 6S (39). Under physiological salt conditions and at submicromolar protein concentrations, the 6S monomer forms a stable 8S dimer (39), most likely arranged in an antiparallel orientation (33). When the SIR3p concentration is raised into the micromolar range, the dimers reversibly and specifically self-associate into 13S tetramers and a series of higher-order oligomers that sediment from 16 to Ͼ80S.…”

“…Sf9 insect cells were infected with a recombinant baculovirus expressing full-length rSIR3p fused to six tandem C-terminal histidines. The protein was purified by conventional ion-exchange chromatography as previously described (39). The resulting protein was Ͼ95% pure, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie staining.…”

“…Self-association of SIR3p in solution has been widely reported (16,36,42). The self-association behavior of the fulllength protein has recently been characterized in detail and shown to be quite complicated (39). The most stable form of SIR3p is an 8S dimer.…”

“…The most stable form of SIR3p is an 8S dimer. However, in low-micromolar-concentration solutions and under physiological salt conditions, the 8S SIR3p dimers are able to self-associate indefinitely in a reversible, concentration-dependent manner to produce unusually large oligomers that sediment in excess of 70 to 80S (39). While such oligomerization properties could help explain SIR3p-dependent assembly and the spreading of silent chromatin architecture, the biochemical properties and morphology of SIR3p-bound chromatin fibers have yet to be investigated in detail.…”