Phage-Encoded Inhibitor of Staphylococcus aureus Transcription Exerts Context-Dependent Effects on Promoter Function in a Modified Escherichia coli-Based Transcription System

Abstract

Promoter recognition in bacteria is mediated primarily by the sigma subunit of RNA polymerase (RNAP), which makes sequence-specific contacts with the promoter -10 and -35 elements in the context of the RNAP holoenzyme. However, the RNAP alpha subunit can also contribute to promoter recognition by making sequence-specific contacts with upstream (UP) elements that are associated with a subset of promoters, including the rRNA promoters. In Escherichia coli, these interactions between the RNAP alpha subunit (its C-terminal domain [ CTD], in particular) and UP element DNA result in significant stimulation of rRNA transcription. Among the many cellular and bacteriophage-encoded regulators of transcription initiation that have been functionally dissected, most exert their effects via a direct interaction with either the sigma or the alpha subunit. An unusual example is provided by a phage-encoded inhibitor of RNA synthesis in Staphylococcus aureus. This protein, phage G1 gp67, which binds tightly to sigma in the context of the S. aureus RNAP holoenzyme, has recently been shown to exert selective effects on transcription by inhibiting the function of the alpha subunit CTD (alpha CTD). Here we report the development of a gp67-responsive E. coli-based transcription system. We examine transcription in vitro from promoters that do or do not carry the UP element associated with a well-characterized E. coli rRNA promoter. Our findings indicate that the alpha CTD can increase promoter activity significantly even in the absence of an UP element. We also find that gp67 can exert alpha CTD-dependent or alpha CTD-independent effects on transcription depending on the particular promoter, indicating that the mechanism of gp67 action is context dependent.