title: A genetic system for studying the activity of a proteolytic enzyme.
abstract: We describe a genetic system for monitoring the activity of a specific proteolytic enzyme by taking advantage of the properties of the yeast transcriptional activator GAL4. The GAL4 protein contains two separable and functionally essential domains: the amino-terminal DNA binding domain and the carboxyl-terminal transcriptional activating domain. We constructed two hybrid proteins by inserting between the DNA binding domain and the activation domain of GAL4 either (i) a self-cleaving protease (3C protease of a picornavirus, coxsackievirus B3) or (ii) a mutant form of the protease that is unable to cleave. We show that, although the hybrid protein containing the mutant protease activates transcription of GAL1-lacZ reporter gene, the hybrid protein bearing the wild-type protease is proteolytically cleaved and fails to activate transcription. Our approach to monitor the proteolytic activity could be used to develop simple genetic systems to study other proteases.
authors.affiliation: Antiviral Chemotherapy, Schering-Plough Research Institute, Bloomfield, NJ 07003.
authors.forename: B
authors.initials: B
authors.lastname: Dasmahapatra
authors.forename: B
authors.initials: B
authors.lastname: DiDomenico
authors.forename: S
authors.initials: S
authors.lastname: Dwyer
authors.forename: J
authors.initials: J
authors.lastname: Ma
authors.forename: I
authors.initials: I
authors.lastname: Sadowski
authors.forename: J
authors.initials: J
authors.lastname: Schwartz
mesh_terms: D000595:Amino Acid Sequence; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors