Issue 10, 2010

Identifying N60D mutation in ω subunit of Escherichia coliRNA polymerase by bottom-up proteomic approach

Abstract

Escherichia coli RNA polymerase is a multi-subunit enzyme containing α2ββ′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 ∼ 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) → 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.

Graphical abstract: Identifying N60D mutation in ω subunit of Escherichia coli RNA polymerase by bottom-up proteomic approach

Supplementary files

Article information

Article type
Paper
Submitted
10 Mar 2010
Accepted
30 Jun 2010
First published
20 Aug 2010

Analyst, 2010,135, 2723-2729

Identifying N60D mutation in ω subunit of Escherichia coli RNA polymerase by bottom-up proteomic approach

V. Sabareesh, P. Sarkar, A. A. Sardesai and D. Chatterji, Analyst, 2010, 135, 2723 DOI: 10.1039/C0AN00130A

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