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Systematic structure-activity analysis of microcin J25.

Pavlova O, Mukhopadhyay J, Sineva E, Ebright RH, Severinov K - J. Biol. Chem. (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Waksman Institute of Microbiology, Piscataway, NJ 08844, USA.

ABSTRACT
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.

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Effects of single-amino acid substitutions in MccJ25 on inhibition of RNAP. Data are presented for 242 single-amino acid substitution derivatives of MccJ25, comprising all substitutions shown to be competent for production/maturation/export/stability (see Fig. 1). The sequence of MccJ25 is shown at the bottom. The heights of bars indicate the percentage of tested amino acid substitutions that do not prevent inhibition of RNAP by MccJ25. Letters within bars list amino acid substitutions that do not prevent inhibition of RNAP by MccJ25. (Positions 1, 2, and 8 (gray bars) were not analyzed, since no single-amino acid substitution derivatives at these positions were shown to be competent for production/maturation/export/stability (see Fig. 1).)
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fig2: Effects of single-amino acid substitutions in MccJ25 on inhibition of RNAP. Data are presented for 242 single-amino acid substitution derivatives of MccJ25, comprising all substitutions shown to be competent for production/maturation/export/stability (see Fig. 1). The sequence of MccJ25 is shown at the bottom. The heights of bars indicate the percentage of tested amino acid substitutions that do not prevent inhibition of RNAP by MccJ25. Letters within bars list amino acid substitutions that do not prevent inhibition of RNAP by MccJ25. (Positions 1, 2, and 8 (gray bars) were not analyzed, since no single-amino acid substitution derivatives at these positions were shown to be competent for production/maturation/export/stability (see Fig. 1).)

Mentions: Determinants of MccJ25 for Inhibition of RNAP—To assess the effects of substitutions on inhibition of RNAP by MccJ25, we prepared culture supernatants of cells harboring substituted mcjABCD biosynthetic gene clusters, and we assayed culture supernatants for the ability to inhibit RNAP in vitro. We tested 242 single-amino acid substitutions of MccJ25, comprising all single-amino acid substitutions found to be compatible with production/maturation/export/stability of MccJ25 (see above). The results indicate that 155 of the 242 substituted MccJ25 derivatives competent for production/maturation/export/stability also are competent for inhibition of RNAP (64%; Fig. 2).


Systematic structure-activity analysis of microcin J25.

Pavlova O, Mukhopadhyay J, Sineva E, Ebright RH, Severinov K - J. Biol. Chem. (2008)

Effects of single-amino acid substitutions in MccJ25 on inhibition of RNAP. Data are presented for 242 single-amino acid substitution derivatives of MccJ25, comprising all substitutions shown to be competent for production/maturation/export/stability (see Fig. 1). The sequence of MccJ25 is shown at the bottom. The heights of bars indicate the percentage of tested amino acid substitutions that do not prevent inhibition of RNAP by MccJ25. Letters within bars list amino acid substitutions that do not prevent inhibition of RNAP by MccJ25. (Positions 1, 2, and 8 (gray bars) were not analyzed, since no single-amino acid substitution derivatives at these positions were shown to be competent for production/maturation/export/stability (see Fig. 1).)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2533079&req=5

fig2: Effects of single-amino acid substitutions in MccJ25 on inhibition of RNAP. Data are presented for 242 single-amino acid substitution derivatives of MccJ25, comprising all substitutions shown to be competent for production/maturation/export/stability (see Fig. 1). The sequence of MccJ25 is shown at the bottom. The heights of bars indicate the percentage of tested amino acid substitutions that do not prevent inhibition of RNAP by MccJ25. Letters within bars list amino acid substitutions that do not prevent inhibition of RNAP by MccJ25. (Positions 1, 2, and 8 (gray bars) were not analyzed, since no single-amino acid substitution derivatives at these positions were shown to be competent for production/maturation/export/stability (see Fig. 1).)
Mentions: Determinants of MccJ25 for Inhibition of RNAP—To assess the effects of substitutions on inhibition of RNAP by MccJ25, we prepared culture supernatants of cells harboring substituted mcjABCD biosynthetic gene clusters, and we assayed culture supernatants for the ability to inhibit RNAP in vitro. We tested 242 single-amino acid substitutions of MccJ25, comprising all single-amino acid substitutions found to be compatible with production/maturation/export/stability of MccJ25 (see above). The results indicate that 155 of the 242 substituted MccJ25 derivatives competent for production/maturation/export/stability also are competent for inhibition of RNAP (64%; Fig. 2).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Waksman Institute of Microbiology, Piscataway, NJ 08844, USA.

ABSTRACT
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.

Show MeSH