Identifying N60D mutation in ω subunit of Escherichia coli RNA polymerase by bottom-up proteomic approach.
Analyst
; 135(10): 2723-9, 2010 Oct.
Article
em En
| MEDLINE
| ID: mdl-20730136
ABSTRACT
Escherichia coli RNA polymerase is a multi-subunit enzyme containing α(2)ßß'ωσ, which transcribes DNA template to intermediate RNA product in a sequence specific manner. Although most of the subunits are essential for its function, the smallest subunit ω (average molecular mass â¼ 10,105 Da) can be deleted without affecting bacterial growth. Creating a mutant of the ω subunit can aid in improving the understanding of its role. Sequencing of rpoZ gene that codes for ω subunit from a mutant variant suggested a substitution mutation at position 60 of the protein asparagine (N) â aspartic acid (D). This mutation was verified at the protein level by following a typical mass spectrometry (MS) based bottom-up proteomic approach. Characterization of in-gel trypsin digested samples by reverse phase liquid chromatography (LC) coupled to electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) enabled in ascertaining this mutation. Electron transfer dissociation (ETD) of triply charged [(M + 3H)(3+)] tryptic peptides (residues [53-67]), EIEEGLINNQILDVR from wild-type and EIEEGLIDNQILDVR from mutant, facilitated in unambiguously determining the site of mutation at residue 60.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
RNA Polimerases Dirigidas por DNA
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Proteínas de Escherichia coli
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Proteômica
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Escherichia coli
Idioma:
En
Ano de publicação:
2010
Tipo de documento:
Article