title: A Method for Large-Scale Screening of Random Sequence Libraries to Determine the Function of Unstructured Regions from Essential Proteins.
abstract: In this chapter we present a method allowing the screening of random sequences to discover essential aspects of unstructured protein regions in yeast. The approach can be applied to any protein with unstructured peptide sequences for which functions are difficult to decipher, for example the N-terminal tails of histones. The protocol first describes the building and preparation of a large library of random peptides in fusion with a protein of interest. Recent technical advances in oligonucleotide synthesis allow the construction of long random sequences up to 35 residues long. The protocol details the screening of the library in yeast for sequences that can functionally replace an unstructured domain in an essential protein in vivo. Our method typically identifies sequences that, while being totally different from the wild type, retain essential features allowing yeast to live. This collection of proteins with functional synthetic sequences can subsequently be used in phenotypic tests or genetic screens in order to discover genetic interaction.
authors.affiliation: Département de biologie, Faculté des sciences, Université de Sherbrooke, Sherbrooke, QC, Canada, J1K 2R1.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Millau
authors.affiliation: Department of Biology, Université de Sherbrooke, Sherbrooke, QC, Canada, J1K 2R1.
authors.forename: Benoit
authors.initials: B
authors.lastname: Guillemette
authors.affiliation: Department of Biology, Université de Sherbrooke, Sherbrooke, QC, Canada, J1K 2R1. luc.gaudreau@usherbrooke.ca.
authors.forename: Luc
authors.initials: L
authors.lastname: Gaudreau
mesh_terms: D002843:Chromatin / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D006657:Histones / Q000378:metabolism*; D009707:Nucleosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D058615:Synthetic Biology
chemical_list: D002843:Chromatin; D006657:Histones; D009707:Nucleosomes; D029701:Saccharomyces cerevisiae Proteins
keywords: Chromatin; Genetic screen; H3; Histone modification; Histone tails; Nucleosome; Synthetic biology; Yeast