Identifying N60D mutation in ω subunit of Escherichia coli RNA polymerase by bottom-up proteomic approach.
Escherichia coli RNA polymerase is a multi-subunit enzyme containing α(2)ββ'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.
Sabareesh
Varatharajan
V
Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bengaluru, 560064, India. sabareesh@jncasr.ac.in
Sarkar
Paramita
P
Sardesai
Abhijit A
AA
Chatterji
Dipankar
D
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D056148:Chromatography, Reverse-Phase / Q000379:methods; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D040901:Proteomics / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D014357:Trypsin / Q000378:metabolism
D029968:Escherichia coli Proteins; D010455:Peptides; D021122:Protein Subunits; C111232:RNA polymerase omega subunit; D012321:DNA-Directed RNA Polymerases; D014357:Trypsin