Streptolydigin Resistance Can Be Conferred by Alterations to Either the β or β′ Subunits of Bacillus subtilis RNA Polymerase

Abstract

Rifampicin and streptolydigin are antibiotics which inhibit prokaryotic RNA polymerase at the initiation and elongation steps, respectively. In Escherichia coli, resistance to each antibiotic results from alterations in the ␤ subunit of the core enzyme. However, in Bacillus subtilis, reconstitution studies found rifampicin resistance (Rif R ) associated with the ␤ subunit and streptolydigin resistance (Stl R ) with ␤. To understand the basis of bacterial Stl R , we isolated the B. subtilis rpoC gene, which encodes a 1,199-residue product that is 53% identical to E. coli ␤. Two spontaneous Stl R mutants carried the same D796G substitution in rpoC, and this substitution alone was sufficient to confer Stl R in vivo. D796 falls within Region F, which is conserved among the largest subunits of prokaryotic and eukaryotic RNA polymerases. Among eukaryotes, alterations in Region F promote resistance to ␣-amanitin, a toxin which inhibits transcription elongation; among prokaryotes, alterations in Region F cause aberrant termination. To determine whether alterations in the ␤ subunit of B. subtilis could also confer Stl R , we made three Stl R substitutions (A499V, G500R, and E502V) in the rif region of rpoB. Together these results suggest that ␤ and ␤ interact to form an Stl binding site, and that this site is important for transcription elongation.The RNA polymerase core enzyme of eubacteria has a multisubunit structure containing one ␤, one ␤Ј, and two ␣ subunits (see Ref. 1 for a review). Most of the catalytic functions of the enzyme are thought to reside in the two largest subunits, ␤ and ␤Ј. Because the ␤ and ␤Ј subunits share colinear blocks of conserved sequence with the two largest subunits of eukaryotic RNA polymerases, genetic and biochemical analysis of the eubacterial enzyme can contribute to an understanding of the structure-function relationships among RNA polymerases of all organisms (1).One way to explore these structure-function relationships in eubacteria is to study the action of two antibiotics that specifically target RNA polymerase, rifampicin (Rif) 1 and streptolydigin (Stl), each of which has a different mechanism of inhibition. Rif arrests transcriptional initiation at the promoter by locking RNA polymerase in an abortive initiation complex capable of synthesizing only short oligonucleotides, but this antibiotic has no effect once the transcription complex has elongated past the promoter (2). In contrast, Stl blocks both transcriptional initiation and elongation by inhibiting the translocation step, thereby reducing the rate of chain formation. This translocation inhibition has been suggested to result from interference with either the nucleotide triphosphate binding site or the ability to form the phosphodiester bond between the incoming triphosphate and the nascent RNA chain (3-5).In the Gram-negative bacterium Escherichia coli, mutations that confer either Rif resistance (Rif R ) or Stl resistance (Stl R ) have been mapped to rpoB, the gene encoding the ␤ subunit (6 -10). Likewise, reconstituti...

Does RNA polymerase help drive chromosome segregation in bacteria?

“…We interpret this to indicate that replication had resumed at the permissive temperature, and that the newly duplicated origin regions had moved apart. Inhibition of RNA polymerase was achieved by the use of streptolydigin, which prevents transcription initiation and elongation through interaction with the ␤Ј-subunit of RNA polymerase (24). Streptolydigin was added after the temperature shift down to investigate the dependence of DNA separation on transcription.…”

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.…”

A zinc-binding site in the largest subunit of DNA-dependent RNA polymerase is involved in enzyme assembly

“…Thus, the site II cleavage occurred carboxy terminal to the ␤Ј conserved segment F (Fig. 2B) involved in transcription elongation and termination (Waelbaecher et al 1994), the binding of streptolydigin in bacterial RNAP Yang and Price Cold Spring Harbor Laboratory Press on May 11, 2018 -Published by genesdev.cshlp.org Downloaded from 1995), and the binding of ␣-amanitin in eukaryotic RNAP (Bartolomei and Corden 1987). Similar to site I, there is a cysteine residue at or very close to cleavage site II, Cys-814, that is strictly conserved within the eubacterial lineage but is absent from archeal and eukaryotic ␤Ј homologs.…”