The primary structure ot<i>E. coli</i>RNA porymerase. Nucleotide sequence of the rpoC gene and amino acid sequence of the β′-sabunit

Ovchinnikov, Yu.A.; Monastyrskaya, G.S.; Gubanov, V.V.; Guryev, S.O.; Саломатина, И С; Shuvaeva, T. M.; Липкин, В. М.; Свердлов, Е. Д.

doi:10.1093/nar/10.13.4035

Cited by 129 publications

(23 citation statements)

References 12 publications

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“…Comparison of the sequences revealed a single nucleotide difference between pMKa201 and the mutant plasmids. Both mutant plasmids have a TTC at codon position 793, while pMKa201 has a CTC at that codon position, as does the published sequence (14). As a result of this change, the ␤Ј subunit expressed from the mutant plasmids has a phenylalanine at amino acid position 793 instead of serine, as found in the wild-type protein (Fig.…”

Section: Resultsmentioning

confidence: 77%

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

Severinov

Markov

Severinova

et al. 1995

Journal of Biological Chemistry

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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...

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Section: Resultsmentioning

confidence: 77%

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

Severinov

Markov

Severinova

et al. 1995

Journal of Biological Chemistry

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“…Section 1734 solely to indicate this fact. AGCAAGAGAAGCTTTNGTRATNCC), designed by comparing the conserved 3Ј ends of the rpoC genes from E. coli (17) and Staphylococcus aureus (18). This PCR probe was used to screen a random library of B. subtilis chromosomal DNA carried in gt11 (15).…”

Section: Methodsmentioning

confidence: 99%

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

Yang

Price

1995

Journal of Biological Chemistry

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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...

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“…In addition, untethered iron-EDTA has been used to study the accessibility of sites on the surface of a small DNA-binding protein (20 (24) was used to produce rabbit polyclonal antibodies. The C-terminal 14-mer peptides Leu-Ala-Glu-Leu-Leu-Asn-Ala-Gly-Leu-Gly-Gly-SerAsp-Asn and Leu-Ala-Ser-Arg-Gly-Leu-Ser-Leu-Gly-MetArg-Leu-Glu-Asn, corresponding to residues 1393-1406 of the 1407-residue ,l3' subunit (25) and to residues 307-320 of the 329-residue a subunit (26), were synthesized on an 8-branch Fmoc-MAP resin with a model 430A peptide synthesizer (Applied Biosystems). After problems were encountered with antisera to the X3 subunit C terminus, the N-terminal 14-mer peptide Met-Val-Tyr-Ser-Tyr-Thr-Glu-Lys-Lys-Arg-Ile-ArgLys-Asp of the 1342-residue ,B subunit (27) was synthesized by Phoenix Pharmaceuticals (Mountain View, CA).…”

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confidence: 99%

Binding of the sigma 70 protein to the core subunits of Escherichia coli RNA polymerase, studied by iron-EDTA protein footprinting.

Greiner

Hughes

Gunasekera

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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We have used a nonspecific protein cleaving reagent to map the interactions between subunits of the multisubunit enzyme RNA polymerase (Escherichia coli). We developed suitable conditions for using an untethered Fe-EDTA reagent, which does not bind significantly to proteins. Comparison of the cleaved fragments of the subunits from the core enzyme (a!2313') and the holoenzyme (core + .70) shows that absence of the Or70 subunit is associated with the appearance of several cleavage sites on the subunits fJ (within 10 residues of sequence positions 745, 764, 795, and 812) and 3' (within 10 residues of sequence positions 581, 613, and 728). A cleavage site near 13 residue 604 is present in the holoenzyme but absent in the core, demonstrating that a conformational change occurs when cr70 binds. No differences are observed for the a subunit.Gene transcription in living cells is a complex process, with large numbers of protein factors involved in selecting the correct initiation site on DNA and initiating and carrying out RNA synthesis to the appropriate end. The proteins responsible for transcription in both prokaryotic and eukaryotic cells are actively being identified and their functions explored (1-8).Bacterial RNA polymerase is a well-studied example. It is composed of five protein subunits (Ct2f1'o), of which one (o-) is a member of a group of related proteins that convey different DNA-binding specificities. The most common a subunit in Escherichia coli has a mass of 70 kDa and is designated a70.It has long been known that oa70 binds reversibly to the at23' core enzyme (9-11), but the actual binding site has been difficult to identify. This problem has been addressed by chemical cross-linking (12-14) and by molecular genetics (15), but identification of the particular amino acid residues on the core subunits involved in oJ7() binding remains a challenge.

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The primary structure otE. coliRNA porymerase. Nucleotide sequence of the rpoC gene and amino acid sequence of the β′-sabunit

Cited by 129 publications

References 12 publications

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

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

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

Binding of the sigma 70 protein to the core subunits of Escherichia coli RNA polymerase, studied by iron-EDTA protein footprinting.

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