Localization of the Escherichia coli RNA polymerase beta' subunit residue phosphorylated by bacteriophage T7 kinase Gp0.7

Abstract

During bacteriophage T7 infection, the Escherichia coli RNA polymerase beta' subunit is phosphorylated by the phage-encoded kinase Gp0.7. Here, we used proteolytic degradation and mutational analysis to localize the phosphorylation site to a single amino acid, Thr(1068), in the evolutionarily hypervariable segment of beta'. Using a phosphomimetic substitution of Thr(1068), we show that phosphorylation of beta' leads to increased rho-dependent transcription termination, which may help to switch from host to viral RNA polymerase transcription during phage development.

FIG. 1.

Localization of the β′ phosphorylation site by use of chemical proteases. (A) E. coli MG1655 cells were infected with bacteriophage T7 in the presence of radioactive orthophosphate as described in Materials and Methods. Cells were collected and lysed, proteins were resolved by SDS-PAGE, and phosphoproteins were revealed by autoradiography. Lane 1 shows phosphoproteins in control, mock-infected cells. The position of the E. coli RNAP β′ subunit is indicated. (B) ³²P-labeled RNAP was purified from T7-infected cells prepared as described for panel A and subjected to complete proteolysis with Cys-specific NTCBA (left panel) or limited proteolysis with Met-specific CNBr (right panel). Met residues are labeled at right (right panel). The products were resolved by SDS-PAGE and revealed by autoradiography. (C) ³²P-labeled RNAP β′ from T7-infected cells was subjected to complete acid hydrolysis, and phosphoamino acids were revealed, after thin-layer chromatography, by autoradiography. Positions of phosphoamino acid markers are indicated.

FIG. 2.

Genetic context of the Gp0.7 phosphorylation site in the E. coli RNAP β′ subunit. The thick bar at the top represents the 1,407-amino-acid-long β′ subunit of E. coli RNAP. Hatched boxes labeled A to H represent segments highly conserved in evolution (1). The E. coli hypervariable region that contains the Gp0.7 phosphorylation site and that is absent from β′ homologues from gram-positive bacteria (46) is shown by an open box, and its sequence is expanded underneath. The E. coli sequence (E. c.) is aligned with homologous sequences from Yersinia pestis (Y. p.), Kluyvera cryocrescens (K. c.), Caulobacter crescentus (C. c.), and Bacillus subtilis (B. s.). Dots indicate identity to the E. coli sequence, and dashes indicate gaps. Methionine residues that flank the CNBr fragment that contains the phosphorylation site are indicated above the E. coli sequence, threonine residues that are contained within this CNBr fragment are italicized, and two phosphomimetic substitutions used in this work are indicated. The bar above the E. coli sequence shows the deletion of the β′ downstream jaw in the E. coli JE1144 cells.

FIG. 3.

β′ Thr¹⁰⁶⁸ is the target of Gp0.7 phosphorylation. JE1134 cells harboring a chromosomal deletion of rpoC codons 1149 to 1190 were transformed with plasmids expressing the indicated alleles of rpoC. Cells were grown and infected with T7 phage as described in the legend for Fig. 1A. Proteins were resolved by SDS-PAGE, the gel was silver stained (left panel), and phosphoproteins were revealed by autoradiography (autorad) (right panel). Plasmid-free JE1134 and wild-type (wt) MG1655 cells were used as controls (lanes 1 and 1′ and 6 and 6′). Lane 7 is a marker lane (RNAP σ⁷⁰ holoenzyme).

FIG. 4.

β′ substitution T1068E leads to increased transcription termination in vivo. E. coli RL211 cells were transformed with pRL663 or its indicated derivatives and plated as shown schematically in the center of the figure. The plate on the left contained rich unselective medium, and the plate on the right contained minimal medium with 5-MAA. The results of overnight growth at 30°C are presented. T1328I is a point substitution in β′ that was previously shown to increase RL211 survival in the presence of 5-MAA due to increased transcription termination (43). IPTG, isopropyl-β-d-thiogalactopyranoside; VB, Vogel-Bonner; Amp, ampicillin; wt, wild type.

FIG.5.

β′ substitution T1068E leads to increased ρ-dependent termination in vitro. Transcription reactions were performed on a template containing the λ tR1 ρ-dependent terminator, as described in Materials and Methods, in the presence (+) or in the absence (−) of the ρ factor. Where indicated, transcription reactions were chased (+) with 1 mM NTPs. The numbers at the right indicate expected sizes of the terminated and runoff transcription products. The gel shown is representative of three independent experiments. wt, wild type.