We are interested in studying position-dependent but gene independent transcriptional silencing in the yeast, S. cerevisiae. This method of silencing is achieved through the action of a group of proteins known as Silent Information Regulators. One particular protein, Silent Information Regulator 3, or SIR3, is an essential component of the S. cerevisiae silencing complex that functions at telomeres and the silent mating type loci, HMR and HML. In order for SIR3 to function, the presence of its N-terminus is crucial, as a protein missing the first 252 amino acids of the N-terminus does not complement a SIR3 deletion. The requirement for the presence of SIR3N is clearly delineated when transcomplementation occurs following the coexpression of an N-terminal deletion of SIR3 with the N-terminus of SIR3. Transcomplementation also suggests that SIR3N can function as its own domain, and thus is a critical unit in this particular method of silencing in yeast. To elucidate the function of the N-terminus of SIR3 and to better understand its role in silencing, we wanted to determine if the N-terminal domain of SIR3 interacted with any other yeast proteins. By utilizing the two-hybrid system, we tested whether or not SIR3N interacted with any of the proteins expressed from a GAD yeast genomic library.
The two-hybrid system allows for the identification of interacting proteins. It utilizes two plasmid-borne gene fusions that are co-transformed into a host yeast strain containing inducible reporter genes. Activation of the reporter genes occurs only if the two fusion proteins interact. To test whether SIR3 interacts with any other proteins expressed from the GAD yeast genomic library, we fused the DNA sequence coding for SIR3N, our protein of interest, or "bait," to Lex A, a DNA binding protein, and coexpressed the fusion complex with a GAD yeast genomic library in L40 (a strain used routinely for two-hybrid studies.) L40 contains two reporter genes for the two-hybrid system, HIS3 and lacZ, both under the control of a series of Lex A binding sites. An interaction between SIR3N and a GAD fusion is assessed by selection on -HIS media, and by an assay for beta-galactosidase activity, the product of the lacZ gene. Out of 100,000 colonies plated, 9 were HIS +. Of these nine, one was also positive for beta-galactosidase, implying the activity of an interaction with SIR3N and this yet unknown protein. Currently, we are trying to isolate the two-hybrid candidate, i.e. the GAD fusion plasmidp. Following isolation, we will test the candidate for specificity. If the candidate interacts with SIR3N-Lex A only, then the plasmid will be sequenced and the identity of the fusion protein be determined. Sequencing will thus enable us to identify the first protein documented to interact with the N-terminus of SIR3.