Phage Mu enlists the β-sliding clamp for late gene transcription.

Abstract

The phage Mu C protein (MuC), along with core RNA polymerase (RNAP) and σ⁷⁰, is required for transcription of phage late genes. We found that overexpression of MuC was lethal in <i><i>Escherichia coli</i></i> and observed that host replication was overinitiating under these conditions. Suppressors of MuC lethality mapped to <i>dnaA</i>, <i>diaA</i>, and <i>dnaX</i>. DnaA initiates replication at <i>oriC</i>, assisted by DiaA. Reinitiation is prevented by hydrolysis of ATP-DnaA to ADP-DnaA by Hda and DnaN (β-sliding clamp or Clamp). DnaX, the tau/gamma subunit of Pol III, is part of the Clamp loader complex. Coexpression of <i>hda</i> and <i>dnaN</i> rescued MuC lethality. This result suggested that MuC was interfering with either Hda or DnaN. We noticed that MuC contains two near-consensus Clamp-binding motifs (CBM), one at the N terminus and one at the C-terminus. Changing the consensus residues of either CBM abolished MuC lethality, abrogated MuC-dependent transcription, and reduced plaque-forming units. Inactivation of a <i>ts</i> Clamp through temperature shift specifically inhibited MuC-dependent transcription but not σ⁷⁰-dependent transcription. We show that MuC interacts with the Clamp to activate late gene transcription both in vivo and in vitro. This study demonstrates the involvement of the <i><i>E. coli</i></i> Clamp, a processivity factor essential for DNA replication, for transcription by <i><i>E. coli</i></i> RNAP. We observed that members of the Mor/MuC family of transcription factors all possess at least one CBM, suggesting that engaging the Clamp for transcription is likely to be more prevalent than hitherto recognized.