Dimeric association of Escherichia coli RNA polymerase α subunits, studied by cleavage of single-cysteine α subunits conjugated to iron-(S)-1- [p-(bromoacetamido)benzyl]ethylenediaminetetraacetate

Reiko Miyake, Katsuhiko Murakami, Jeffrey T. Owens, Douglas P. Greiner, Olga N. Ozoline, Akira Ishihama, Claude F. Meares

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

Proximity relationships between the two associated monomers of the Escherichia coli RNA polymerase α subunit were studied using a set of four mutant α subunits, each with a single Cys residue at one of the naturally occurring positions (54, 131, 176, and 269). These mutant α subunits were conjugated with the cutting reagent iron-(S)-1-[p- (bromoacetamido)benzyl]ethylenediaminetetraacetate (Fe-BABE), and the peptide backbone was cleaved at locations near the modified Cys. Analysis of the cleavage sites identified segments within ≃12 Å of the conjugation site. These results show that, for intermolecular cutting, segments of the subunit assembly domain (N-terminal domain) of one subunit and the linker region between N- and C-terminal domains of the other subunit are near each other, and the N-terminal domains of both subunits are in close proximity to one another. Intramolecular cutting however, was observed only within an individual N- or C-terminal domain.

Original languageEnglish (US)
Pages (from-to)1344-1349
Number of pages6
JournalBiochemistry
Volume37
Issue number5
DOIs
StatePublished - Feb 3 1998

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DNA-Directed RNA Polymerases
Escherichia coli
Cysteine
Iron
Peptides
Monomers
1-(4-bromoacetamidobenzyl)EDTA

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Miyake, Reiko ; Murakami, Katsuhiko ; Owens, Jeffrey T. ; Greiner, Douglas P. ; Ozoline, Olga N. ; Ishihama, Akira ; Meares, Claude F. / Dimeric association of Escherichia coli RNA polymerase α subunits, studied by cleavage of single-cysteine α subunits conjugated to iron-(S)-1- [p-(bromoacetamido)benzyl]ethylenediaminetetraacetate. In: Biochemistry. 1998 ; Vol. 37, No. 5. pp. 1344-1349.
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abstract = "Proximity relationships between the two associated monomers of the Escherichia coli RNA polymerase α subunit were studied using a set of four mutant α subunits, each with a single Cys residue at one of the naturally occurring positions (54, 131, 176, and 269). These mutant α subunits were conjugated with the cutting reagent iron-(S)-1-[p- (bromoacetamido)benzyl]ethylenediaminetetraacetate (Fe-BABE), and the peptide backbone was cleaved at locations near the modified Cys. Analysis of the cleavage sites identified segments within ≃12 {\AA} of the conjugation site. These results show that, for intermolecular cutting, segments of the subunit assembly domain (N-terminal domain) of one subunit and the linker region between N- and C-terminal domains of the other subunit are near each other, and the N-terminal domains of both subunits are in close proximity to one another. Intramolecular cutting however, was observed only within an individual N- or C-terminal domain.",
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Dimeric association of Escherichia coli RNA polymerase α subunits, studied by cleavage of single-cysteine α subunits conjugated to iron-(S)-1- [p-(bromoacetamido)benzyl]ethylenediaminetetraacetate. / Miyake, Reiko; Murakami, Katsuhiko; Owens, Jeffrey T.; Greiner, Douglas P.; Ozoline, Olga N.; Ishihama, Akira; Meares, Claude F.

In: Biochemistry, Vol. 37, No. 5, 03.02.1998, p. 1344-1349.

Research output: Contribution to journalArticle

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AU - Miyake, Reiko

AU - Murakami, Katsuhiko

AU - Owens, Jeffrey T.

AU - Greiner, Douglas P.

AU - Ozoline, Olga N.

AU - Ishihama, Akira

AU - Meares, Claude F.

PY - 1998/2/3

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AB - Proximity relationships between the two associated monomers of the Escherichia coli RNA polymerase α subunit were studied using a set of four mutant α subunits, each with a single Cys residue at one of the naturally occurring positions (54, 131, 176, and 269). These mutant α subunits were conjugated with the cutting reagent iron-(S)-1-[p- (bromoacetamido)benzyl]ethylenediaminetetraacetate (Fe-BABE), and the peptide backbone was cleaved at locations near the modified Cys. Analysis of the cleavage sites identified segments within ≃12 Å of the conjugation site. These results show that, for intermolecular cutting, segments of the subunit assembly domain (N-terminal domain) of one subunit and the linker region between N- and C-terminal domains of the other subunit are near each other, and the N-terminal domains of both subunits are in close proximity to one another. Intramolecular cutting however, was observed only within an individual N- or C-terminal domain.

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