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.
*These authors contributed equally to this work.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kouzarides T (2007) Chromatin modifications and their function. Cell 128:693–705
Kim JA, Hsu JY, Smith MM, Allis CD (2012) Mutagenesis of pairwise combinations of histone amino-terminal tails reveals functional redundancy in budding yeast. Proc Natl Acad Sci U S A 109:5779–5784
Zhang W, Bone JR, Edmondson DG, Turner BM, Roth SY (1998) Essential and redundant functions of histone acetylation revealed by mutation of target lysines and loss of the Gcn5p acetyltransferase. EMBO J 17:3155–3167
Ma XJ, Lu Q, Grunstein M (1996) A search for proteins that interact genetically with histone H3 and H4 amino termini uncovers novel regulators of the Swe1 kinase in Saccharomyces cerevisiae. Genes Dev 10:1327–1340
Gietz RD, Schiestl RH (2007) High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2:31–34
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Millau, JF., Guillemette, B., Gaudreau, L. (2017). A Method for Large-Scale Screening of Random Sequence Libraries to Determine the Function of Unstructured Regions from Essential Proteins. In: Guillemette, B., Gaudreau, L. (eds) Histones. Methods in Molecular Biology, vol 1528. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6630-1_16
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6630-1_16
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6628-8
Online ISBN: 978-1-4939-6630-1
eBook Packages: Springer Protocols