Structural Studies of a potent Escherichia coli RNAP inhibitor T7 Gp2 and its interaction with RNAP β' Subunit - An early step towards devising new antibiotics

Abstract

Infection of Escherichia coli by the T7 bacteriophage leads to the rapid and selective inhibition of the host RNA polymerase (RNAP) - a multisubunit enzyme responsible for gene transcription - by a small (7 kDa) phage-encoded protein called Gp2 (Gene 2 protein). Gp2 is also a potent inhibitor of E. coli RNAP in vitro. Here, we provide the first structural insight into Gp2. The structure of Gp2 revealed a distinct separation of positive and negative surface charges on different sides of the molecule. The two highly exposed arginines are also in agreement with the mutagenesis studies, which suggested that they have an important role on the Gp2-RNAP interaction and Gp2's function. We have also provided the structural insight into a small domain of the RNAP β' subunit, the β' jaw domain, which is known from previous mutagenesis studies to possess the site for Gp2 binding. Evidence for an interaction between Gp2 and β' jaw domain were provided by Nuclear Magnetic Resonances (NMR) titration experiments. The Gp2-Jaw complex structure solved by NMR spectroscopy has largely facilitated the elucidation of Gp2's inhibition mechanism by allowing the construction of a Gp2-RNAP complex model, which also suggested other possible interaction sites between RNAP and Gp2. Furthermore, the new selective methyl labelling methods may provide experimental data for interactions between Gp2 and RNAP by NMR. RNAP has been used as an important target for broad-spectrum antibacterial therapy and for anti-tuberculosis therapy. According to the structural model of the Gp2-RNAP complex, the binding site of Gp2 is distal to the catalytic cleft, which does not overlap with the binding site of the antibiotic Rifamycin. Understanding the inhibition mechanism of Gp2 may thus open up a new route for the development of RNAP targeted antibiotics.