Streptolydigin-resistant Mutants in an Evolutionarily Conserved Region of the β′ Subunit of Escherichia coli RNA Polymerase

Abstract

Mutations conferring streptolydigin resistance ontoEscherichia coli RNA polymerase have been found exclusively in the ␤ subunit (Heisler, L. M., Suzuki, H., Landick, R., and Gross, C. A. (1993) J. Biol. Chem. 268, 25369 -25375). We report here the isolation of a streptolydigin-resistant mutation in the E. coli rpoC gene, encoding the ␤ subunit. The mutation is the Phe 793 3 Ser substitution, which occurred in an evolutionarily conserved segment of the ␤ subunit. The homologous segment in the eukaryotic RNA polymerase II largest subunit harbors mutations conferring ␣-amanitin resistance. Both streptolydigin and ␣-amanitin are inhibitors of transcription elongation. Thus, the two antibiotics may inhibit transcription in their respective systems by a similar mechanism, despite their very different chemical nature. Streptolydigin (Stl)1 is a 3-acyltetramic acid antibiotic (1), which specifically inhibits bacterial DNA-dependent RNA polymerase (2-6). Stl interacts with RNAP in ternary transcription complexes and inhibits growth of nascent RNA chains during transcription initiation and elongation (4, 6). The binding of RNAP to template DNA is not affected by Stl (2). Thus, the likely target of Stl is either the binding of incoming NTP in the substrate binding site of RNAP or the catalysis of phosphodiester bond formation (4, 6).Transcription by RNAP purified from mutant cells that acquired resistance to the drug is resistant to Stl (3). In Escherichia coli, all Stl-resistant RNAPs studied to date have an altered ␤ subunit (7,8), and all known mutations leading to Stl R RNAP map to the rpoB gene, which codes for the ␤ subunit (8). Substitutions of amino acids in ␤ between 540 and 546 lead to Stl resistance (9 -11). The highest resistance levels in vivo and in vitro were found in the case of substitutions at positions 544 and 545 (10 -11). In the absence of more direct data it has been assumed that these ␤ amino acids participate in Stl binding to RNA polymerase.Since Stl inhibits elongation of nascent RNA, mutations changing RNAP Stl-binding site are likely to change the catalytic properties of the enzyme. However, RNAP with the known ␤ subunit Stl R mutations has unaltered transcription elongation and transcription termination properties in vivo and in vitro (11). Moreover, the site of Stl resistance in the ␤ subunit is dispensable for RNAP function, since mutant RNAPs with deletions spanning amino acids 534 -545 are functional both in vivo and in vitro (10). This apparent discrepancy led Heisler et al. (11) to hypothesize that other site(s) in RNAP may be involved in Stl binding. This hypothesis was substantiated by a report (12) that in RNAP reconstituted in vitro, the ␤Ј subunit from a mutant strain of Bacillus subtilis was responsible for Stl resistance. However, no ␤Ј subunit Stl R mutants have ever been reported in E. coli. Here, we report an isolation and localization of such a mutation. The mutation leads to an amino acid substitution in an evolutionarily conserved region of the ␤Ј subunit. In the ␤Ј homolo...

Genetic Changes That Correlate with Reduced Susceptibility to Daptomycin in Staphylococcus aureus

“…The amino acid changes found in association with increases in the daptomycin MIC have not been described previously. They do not lie within the regions of rpoB known to confer resistance to the RNA polymerase inhibitor rifampin, nor do they overlap with mutations in rpoB and rpoC that confer resistance to other antibiotics, e.g., zwittermicin, streptolydigin, and microcin J25 (6,35,39,42,44,45). Mutations in rpoB and rpoC could reflect direct interaction between daptomycin and the polymerase or could alter global gene expression in ways that alter susceptibility.…”

Escherichia coli RNA Polymerase Is the Target of the Cyclopeptide Antibiotic Microcin J25

“…These are rifampin (10), which inhibits initiation of RNA synthesis, and streptolydigin (28), which blocks transcription upon addition to elongating, as well as initiating, RNAP. All known mutations leading to rifampin resistance map to the rpoB gene (reference 14 and references cited therein), while mutations causing streptolydigin resistance have been found in both ␤ and ␤Ј subunits (11,26,27). Recently, rpoB and rpoC mutants of E. coli resistant to a novel aminopolyol antibiotic, zwittermicin A, have been described (32).…”

Isolation, purification, and in vitro characterization of recessive-lethal-mutant RNA polymerases from Escherichia coli

“…Four distinct regions of the ␤ polypeptide give rise to Rif r mutants, suggesting that the binding site for rifampin is composed of distinct segments of the polypeptide (14,20). Streptolydigin appears to contact both ␤ and ␤Ј (34). The contacts for streptolydigin in ␤ appear to consist of a single set of contiguous amino acids (residues 543 to 546) located between two of the domains (residues 507 to 533 and residues 563 to 572) implicated in binding rifampin (11,35).…”