Of Protein-Protein and DNA-Protein Interactions (URA3/Negative Selection/5-Fluoroorotic Acid) MARC VIDAL*T, RAINER K
Total Page:16
File Type:pdf, Size:1020Kb
Proc. Natl. Acad. Sci. USA Vol. 93, pp. 10315-10320, September 1996 Genetics Reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions (URA3/negative selection/5-fluoroorotic acid) MARC VIDAL*t, RAINER K. BRACHMANNt§, ALI FATrAEY1, ED HARLOW*, AND JEF D. BOEKEt *Massachusetts General Hospital Cancer Center, Building 149, 13th Street, Charlestown, MA 02129; tDepartment of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, §Johns Hopkins Oncology Center, Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, MD 21287; and 1Onyx Pharmaceuticals, 3031 Research Drive, Richmond, CA 94806 Contributed by Ed Harlow, May 14, 1996 ABSTRACT Macromolecular interactions define many function relationships of an interaction requires the identifi- biological phenomena. Although genetic methods are avail- cation of dissociating mutations in the two partners. (ii) able to identify novel protein-protein and DNA-protein in- Important regulatory mechanisms can be unraveled by the teractions, no genetic system has thus far been described to identification of molecules that mediate their function by identify molecules or mutations that dissociate known inter- dissociation of particular protein-protein interactions (see actions. Herein, we describe genetic systems that detect such below). Neither the two-hybrid system nor the one-hybrid events in the yeast Saccharomyces cerevisiae. We have engi- system allows detection of such dissociation events; instead neered yeast strains in which the interaction of two proteins they are limited to positive selection for protein-protein expressed in the context of the two-hybrid system or the association events. interaction between a DNA-binding protein and its binding In this report, we describe "reverse" two- and one-hybrid site in the context of the one-hybrid system is deleterious to systems that detect mutations or molecules that dissociate growth. Under these conditions, dissociation ofthe interaction protein-protein or DNA-protein interactions, respectively. provides a selective growth advantage, thereby facilitating detection. These methods referred to as the "reverse two- hybrid system" and "reverse one-hybrid system" facilitate the METHODS study of the structure-function relationships and regulation Yeast Strains and Manipulations. MaV52 (MATa ura3-52 of protein-protein and DNA-protein interactions. They leu2-3, 112 trpl-901 his3A200 ade2-101 gal4A gal80A canlR should also facilitate the selection of dissociator molecules cyh2R GAL1::HIS3@LYS2 GALJ::lacZ) is a derivative of that could be used as therapeutic agents. Y153 (3) obtained by 5-fluoroorotic acid (FOA) selection to eliminate GAL1::lacZ@URA3 and subsequent canavanine Many biological processes rely upon macromolecular interac- selection. Isogenic derivatives of MaV52 containing SPO13:: tions such as protein-protein and DNA-protein interactions. URA3 fusions with various numbers (n) of GAL4 binding sites Traditionally, the tools available to identify and characterize (SPALn::URA3) are MaV95 (SPAL5::URA3), MaV96 (SPAL7:: molecular interactions have been limited to biochemistry. URA3), MaV97 (SPAL8::URA3), MaV99 (SPAL10::URA3), and However, a genetic method, the two-hybrid system, was de- MaV94 (SPALX::URA3 for which the number of Gal4 binding scribed that allows in a single step both the identification of sites has not been established). Strains MaV108 (MATa) and potential interacting proteins and the isolation of the encoding MaV103 (MATa) are segregants of a cross between MaV99 and genes (1). Two interacting proteins (X and Y) are expressed in PCY2 (8). Methods foryeast manipulationswere as described (9). Saccharomyces cerevisiae as hybrids fused to a DNA-binding Construction and Integration of SPAL::URA3 Reporter domain (DB-X) or an activation domain (AD-Y), respectively. Genes. We used a construct in which the SPO13 promoter DB-X/AD-Y interaction reconstitutes a functional transcrip- drives the expression of a protein fusion in which the first 15 tion factor that activates a reporter gene driven by a promoter aa of SP013 are fused to URA3 (pPL128; R. Strich and R. containing DB binding sites. Positive growth selections using Esposito, personal communication). The SPO13::URA3 frag- prototrophic selectable markers as reporter genes are possible ment was excised from pPL128 and cloned into a pBSK (2-4) and facilitate detection of protein-protein interactions: plasmid (Stratagene). pMV252, the resulting plasmid, contains a few growing yeast colonies expressing DB-X/AD-Y- EcoRI sites at positions -170 and -368 in the SPO13 pro- interacting proteins can conveniently be identified among moter. A fragment containing 5 GAL4 binding sites (10) was many nongrowing colonies. cloned into pMV252 in the EcoRI sites to yield pMV262-11 A one-hybrid system was also described that allows the and pMV262-12 with 5 and 15 GAL4 binding sites, respec- identification of DNA-binding proteins (DBPX) and the iso- tively. lation of their encoding genes (5, 6). In that setting, DBPX is The SPAL::URA3 alleles were integrated at the ura3-52 expressed as a hybrid with an activation domain (DBPX-AD). locus by homologous recombination of the products of a PCR. Interaction between DBPX-AD and its binding site(s) acti- The 5' primer used was JB516 that contains 40 nt of the URA3 vates a reporter gene that can also be a prototrophic selectable sequence upstream of its promoter (positions -257 to -218) marker (5, 7). fused to 20 nt of the SP013 promoter [positions -370 to -351 Subsequent to their identification, it is often crucial to (11)]: 5 '-GAAGGTTAATGTGGCTGTGGTTTCA- characterize the structure-function relationships and the reg- GGGTCCATAAAGCTTGTCCTGGAAGTCT- ulation of protein-protein or DNA-protein interactions. In CATGGAG-3'. The 3' primer used was 3'URA3 [URA3 both cases, rare events that dissociate the interaction must be sequence of positions +656 to +632 (12)]: 5'-TCAGGATC- recognized. (i) The detailed characterization of the structure- Abbreviations: FOA, 5-fluoroorotic acid; Foas, FOA sensitive; FoaR, The publication costs of this article were defrayed in part by page charge FOA resistant; pRB, retinoblastoma gene product. payment. This article must therefore be hereby marked "advertisement" in tTo whom reprint requests should be addressed. e-mail: Vidal@ accordance with 18 U.S.C. §1734 solely to indicate this fact. Helix.MGH.Harvard.edu. 10315 Downloaded by guest on September 24, 2021 10316 Genetics: Vidal et al. Proc. Natl. Acad. Sci. USA 93 (1996) CCTAGGTTCCTTTGTTACTTCTTCCG-3'. The PCR Protein Steady-State Levels by Western Blot Analysis. Yeast products were transformed (13) directly into MaV82 [MaV52 cells were grown to midlogarithmic phase, harvested, washed, transformed with pCL1 (1)]. Integration at ura3 was confirmed and resuspended in 100 mM Tris HCl, pH 8.0/20% glycerol/i and the number of GAL4 binding sites was estimated by a PCR mM EDTA/0.1% Triton X-100/5 mM MgCl2/10 mM 2-mer- using genomic DNA as template, JB536 (URA3 sequence from captoethanol (2ME)/1 mM phenylmethylsulfonyl fluoride positions -298 to -276, 5'-GCGAGGCATATTTATGGT- (PMSF)/leupeptin (1 ,g/ml)/pepstatin (1 [kg/ml) for pRB/ GAAGG-3') as 5' primer, and 13-5 [SPO13 antisense se- E2F1-related experiments or 50 mM Tris-HCl, pH 7.5/1% quence from positions -124 to -145, 5'-CATTTCCGTG- SDS/5 mM EDTA, pH 8.0/14 mM 2ME/1 mM PMSF for the CAAGGTACTAAC-3' (11)] as 3' primer. p53-related experiment at a cell density of about 109 cells per Construction and Integration of UAS53::URA3 Reporter ml. Cells were disrupted by vortex mixing in the presence of Genes. For construction of the lcUAS53::URA3 reporter gene, glass beads for 10 min at 4°C. Debris was pelleted by centrif- oligonucleotides corresponding to the p53 consensus DNA ugation at 12,000 rpm in a microcentrifuge for 15 min at 4°C. binding site [JB820, 5'-AATTTAGGCATGTCTAGGCAT- Approximately 50,tg of proteins was subjected to Western blot GTCTA-3', and JB821, 5'-AATTTAGACATGCCTAGA- analysis (21). CATGCCTA-3' (14)] were annealed, phosphorylated, and ligated into EcoRI-digested pMV252. RESULTS The 1cUAS53::URA3 fusion was PCR-amplified as de- scribed for the SPAL::URA3 fusions and transformed into Yeast strains were generated in which expression of DB-X/ BY385 (MATa ura3-52 his3A200 trplA63 leu2Al1 lys2A202) AD-Y or DBPX hybrid proteins is toxic under particular containing pRB16. About 20% of the transformants selected conditions (negative selection). Under these conditions, dis- on Sc-URA had a plasmid-dependent Ural phenotype. sociation of an interaction should provide a selective advan- Plasmid Constructions. The cFos and cJun fusions (DB- tage thereby facilitating detection: a few growing yeast colo- nies in which DB-X/AD-Y (or DBPX/binding site) fail to cFos, pPC76; DB-Jun, pPC75; AD-Jun, pPC79) are gifts from interact should be identified among many nongrowing colonies P. Chevray and D. Nathans (8). The remaining two-hybrid containing interacting DB-X/AD-Y (or DBPX/binding site) fusions were generated by cloning PCR products in-frame into (Fig. 1). This concept was demonstrated using well- plasmids pPC97 (DB) (pPC97 is pPC62 containing the pPC86 characterized protein-protein and DNA-protein dissociation polylinker) or pPC86 (AD) (8). DB-pRB, aa 302-928 of the events. retinoblastoma gene product (pRB); DB-pRBA22, aa 281-894 URA3 Counterselectable Marker. The reverse two-hybrid of mutant pRB