A genetic screen for isolating "lariat" Peptide inhibitors of protein function.
Functional genomic analyses provide information that allows hypotheses to be formulated on protein function. These hypotheses, however, need to be validated using reverse genetic approaches, which are difficult to perform on a large scale and in diploid organisms. We developed a genetic screen for isolating "lariat" peptides that function as trans dominant inhibitors of protein function. A lariat consists of a lactone-cyclized peptide with a covalently attached transcription activation domain, which allows combinatorial lariat libraries to be screened for protein interactions using the yeast two-hybrid assay. We isolated lariats against the bacterial repressor protein LexA. LexA regulates bacterial SOS response and LexA mutants that cannot undergo autoproteolysis make bacteria more sensitive to, and inhibit resistance against, cytotoxic reagents. We showed that an anti-LexA lariat blocked LexA autoproteolysis and potentiated the antimicrobial activity of mitomycin C.
Barreto
Kris
K
University of Saskatchewan, Saskatoon, Canada.
Bharathikumar
V M
VM
Ricardo
Alonso
A
DeCoteau
John F
JF
Luo
Yu
Y
Geyer
C Ronald
CR
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D020650:Combinatorial Chemistry Techniques; D017930:Genes, Reporter; D007783:Lactones / Q000737:chemistry* / Q000494:pharmacology; D019151:Peptide Library; D010456:Peptides, Cyclic / Q000737:chemistry* / Q000494:pharmacology; D011485:Protein Binding; D013014:SOS Response, Genetics; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance; D015533:Transcriptional Activation; D020798:Two-Hybrid System Techniques; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D007783:Lactones; D019151:Peptide Library; D010456:Peptides, Cyclic; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein