One- plus two-hybrid system, a novel yeast genetic selection for specific missense mutations disrupting protein/protein interactions.
To facilitate analysis of protein/protein interaction interfaces, we devised a novel yeast genetic screening method, named the "one- plus two-hybrid system," for the efficient selection of missense mutations that specifically disrupt known protein/protein interactions. This system modifies the standard yeast two-hybrid system to allow the operation of dual reporter systems within the same cell. The one-hybrid system is first used to select the intact interacting partner (prey), resulting in the positive selection of informative missense mutants from a large library of randomly generated mutant alleles. Then in a second screening step, interaction-defective prey mutants for a given protein are selected using the two-hybrid reporter system among the isolated missense mutants. We used this method to characterize the interactions between unliganded nuclear receptors (NRs) and the conserved motif within the bipartite NR interaction domains (IDs) of the NR corepressor (N-CoR) and identified the specific residues of N-CoR-IDs required either generally for optimal NR binding or to interact with a particular NR. This efficient and rapid method should allow us to quickly analyze a large number of interaction interfaces.
Kim
Ji Young
JY
Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, South Korea.
Park
Ok Gu
OG
Lee
Jae Woon
JW
Lee
Young Chul
YC
D000595:Amino Acid Sequence; D002460:Cell Line; D002999:Clone Cells; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D050505:Mutant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020125:Mutation, Missense / Q000235:genetics*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D047488:Retinoid X Receptors / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D012641:Selection, Genetic; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques
D050505:Mutant Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D018168:Receptors, Retinoic Acid; D012097:Repressor Proteins; D047488:Retinoid X Receptors; D029701:Saccharomyces cerevisiae Proteins; D005982:Glutathione Transferase