title: RNA-ID, a Powerful Tool for Identifying and Characterizing Regulatory Sequences.
abstract: The identification and analysis of sequences that regulate gene expression is critical because regulated gene expression underlies biology. RNA-ID is an efficient and sensitive method to discover and investigate regulatory sequences in the yeast Saccharomyces cerevisiae, using fluorescence-based assays to detect green fluorescent protein (GFP) relative to a red fluorescent protein (RFP) control in individual cells. Putative regulatory sequences can be inserted either in-frame or upstream of a superfolder GFP fusion protein whose expression, like that of RFP, is driven by the bidirectional GAL1,10 promoter. In this chapter, we describe the methodology to identify and study cis-regulatory sequences in the RNA-ID system, explaining features and variations of the RNA-ID reporter, as well as some applications of this system. We describe in detail the methods to analyze a single regulatory sequence, from construction of a single GFP variant to assay of variants by flow cytometry, as well as modifications required to screen libraries of different strains simultaneously. We also describe subsequent analyses of regulatory sequences.
authors.affiliation: Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY, United States.
authors.forename: C E
authors.initials: CE
authors.lastname: Brule
authors.affiliation: Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY, United States.
authors.forename: K M
authors.initials: KM
authors.lastname: Dean
authors.affiliation: Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY, United States. Electronic address: elizabeth_grayhack@urmc.rochester.edu.
authors.forename: E J
authors.initials: EJ
authors.lastname: Grayhack
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D005434:Flow Cytometry; D005453:Fluorescence; D005686:Galactokinase / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012331:RNA, Fungal / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics; D000097573:Red Fluorescent Protein
chemical_list: D005686:Galactokinase; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D000097573:Red Fluorescent Protein; C518355:GAL10 protein, S cerevisiae; C524219:GAL1 protein, S cerevisiae
keywords: Flow cytometry; RNA-ID; Translation; Yeast