Systematic structure-activity analysis of microcin J25.
Microcin J25 (MccJ25) is a 21-residue plasmid-encoded ribosomally synthesized lariat-protoknot antibacterial peptide that targets bacterial RNA polymerase. MccJ25 consists of an 8-residue cycle followed by a 13-residue tail that loops back and threads through the cycle. We have performed systematic mutational scanning of MccJ25, constructing and analyzing more than 380 singly substituted derivatives of MccJ25. The results define residues important for production of MccJ25 (comprising synthesis of MccJ25 precursor, processing of MccJ25 precursor, export of mature MccJ25, and stability of mature MccJ25), inhibition of RNA polymerase, and inhibition of bacterial growth. The results show that only a small number of residues (three in the cycle and one in the threaded segment of the tail) are important for MccJ25 production. The results further show that only a small number of additional residues (two in the cycle and four in the threaded segment of the tail) are important for inhibition of transcription. The results open the way for design and construction of more potent MccJ25-based inhibitors of bacterial growth.
Pavlova
Olga
O
Waksman Institute of Microbiology, Piscataway, New Jersey 08844; Institute of Gene Biology, Russian Academy of Sciences, Moscow 119334, Russia.
Mukhopadhyay
Jayanta
J
Waksman Institute of Microbiology, Piscataway, New Jersey 08844; Howard Hughes Medical Institute, Piscataway, New Jersey 08904.
Sineva
Elena
E
Waksman Institute of Microbiology, Piscataway, New Jersey 08844; Howard Hughes Medical Institute, Piscataway, New Jersey 08904.
Ebright
Richard H
RH
Waksman Institute of Microbiology, Piscataway, New Jersey 08844; Howard Hughes Medical Institute, Piscataway, New Jersey 08904; Department of Chemistry, Piscataway, New Jersey 08904. Electronic address: ebright@waksman.rutgers.edu.
Severinov
Konstantin
K
Waksman Institute of Microbiology, Piscataway, New Jersey 08844; Institute of Gene Biology, Russian Academy of Sciences, Moscow 119334, Russia; Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08904. Electronic address: severik@waksman.rutgers.edu.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001430:Bacteriocins / Q000737:chemistry*; D004252:DNA Mutational Analysis; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D008954:Models, Biological; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic
D000596:Amino Acids; D001430:Bacteriocins; C020411:microcin; D012321:DNA-Directed RNA Polymerases