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. 2008 Mar;7(3):1244-50.
doi: 10.1021/pr070451j. Epub 2008 Feb 14.

Rapid isolation and identification of bacteriophage T4-encoded modifications of Escherichia coli RNA polymerase: a generic method to study bacteriophage/host interactions

Lars F Westblade  1 , Leonid Minakhin, Konstantin Kuznedelov, Alan J Tackett, Emmanuel J Chang, Rachel A Mooney, Irina Vvedenskaya, Qing Jun Wang, David Fenyö, Michael P Rout, Robert Landick, Brian T Chait, Konstantin Severinov, Seth A Darst
Affiliations

Rapid isolation and identification of bacteriophage T4-encoded modifications of Escherichia coli RNA polymerase: a generic method to study bacteriophage/host interactions

Lars F Westblade et al. J Proteome Res. 2008 Mar.

Abstract

Bacteriophages are bacterial viruses that infect bacterial cells, and they have developed ingenious mechanisms to modify the bacterial RNA polymerase. Using a rapid, specific, single-step affinity isolation procedure to purify Escherichia coli RNA polymerase from bacteriophage T4-infected cells, we have identified bacteriophage T4-dependent modifications of the host RNA polymerase. We suggest that this methodology is broadly applicable for the identification of bacteriophage-dependent alterations of the host synthesis machinery.

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Figures

Figure 1
Figure 1
Immunoisolation of β′-2PrA and co-isolating proteins from T4-infected Ec cells. Complexes were isolated via a PrA tag under conditions that co-isolated interacting proteins. Proteins were resolved by denaturing SDS-PAGE, visualized by Coomassie blue staining, and analyzed by MALDI MS. Lanes are loaded as follows: lane 1, Immunoisolated proteins from Ec wild-type cells infected with T4; lane 2, immunoisolated proteins from EcrpoC::2PrA cells infected with T4. RNAP subunits in the 2PrA-tagged sample (lane 2) are labeled.
Figure 2
Figure 2
Non-denaturing PAGE and denaturing SDS-PAGE were used to probe the associations of Alc with the core enzyme and the σ70-holoenzyme. (A) Different combinations of Alc, core, and σ70-holoenzyme were incubated together and resolved under non-denaturing conditions. (B) Bands due to the proteins separated by non-denaturing PAGE, labeled in (A), were excised and their composition revealed by denaturing SDS-PAGE.
Figure 3
Figure 3
Identification of T4-dependent ADP-ribosylation of RNAP α subunit Arg 265. Bands due to the RNAP α subunit isolated from uninfected and T4-infected cells were excised, digested with trypsin, and analysed by MALDI MS. The resulting mass spectra were aligned and scanned for peptides present in the T4 infected sample, but absent from the uninfected sample. (A) Mass spectrum of tryptic peptides due to the RNAP α subunit isolated from uninfected Ec rpoC::2PrA cells (top panel) and Ec rpoC::2PrA cells infected with T4 (bottom panel). The appearance of a peak at 3838.0 m/z, with a mass that corresponds to a singly protonated ADP-ribsoylated peptide derived from α residues 244 to 271 in the spectrum of the infected phage that is absent in the uninfected sample spectrum suggests that this peptide is ADP-ribosylated in vivo in a T4 dependent manner. (B) The mass spectrum of the MS2 analysis of the species with an m/z of 3838.0 described in (A) illustrates neutral loss of AMP, ADP and ADP-ribose, together with various water/ammonia neutral losses. This fragmentation is consistent with the interpretation that the peptide ion at 3838.0 m/z is indeed an ADP-ribsoylated species. (C) MS3 analysis of the ions in the MS2 spectrum with a nominal m/z of 3237.0 (corresponding to the neutral loss of ADP-Ribose +HCN2 +water/ammonia) yielded backbone fragmentation providing partial sequence of the peptide. The fragmentation pattern is consistent with the interpretation that the original peptide ion at 3838.0 m/z is indeed derived from α residues 244 to 271. Observed are fragments corresponding to the preferential cleavage of the singly-protonated peptide ion carboxy-terminal to acidic residues and amino-terminal to Pro residues. Together, the MS, MS2, and MS3 spectra in panels (A), (B) and (C) provide strong evidence that α is ADP-ribosylated on an internal Arg residue within the tryptic peptide encoding residues 244-271.
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