The Phage Mu Middle Promoter Pm Contains a Partial UP Element

Abstract

Abstract There are three phases of transcription during lytic development of bacteriophage Mu: early, middle, and late. Transcription from the middle phase promoter Pm requires the activator protein Mor. In the presence of Mor, transcription from Pm is carried out by the Escherichia coli RNA polymerase holoenzyme containing σ70. A Mor dimer binds to two 5-bp inverted repeats within a 16-bp element centered at −43.5 in Pm, replacing the normal −35 element contacted by RNA polymerase (RNAP). In this study random and targeted mutagenesis of the sequence upstream (−88 to −52) of the Mor binding site was performed to determine whether Pm also contains an UP element for binding of the RNAP α subunit, thereby stimulating transcription. The results demonstrated that mutations upstream of −57 had no effect on Pm activity in vivo, assayed by expression of lacZ fused downstream of a wild-type or mutant Pm. Mutations at positions −57 through −52 led to decreased transcription from Pm, consistent with the presence of an UP element. In DNase I footprinting and gel mobility shift assays, paired mutations at positions −55 and −54 did not affect Mor binding but decreased the synergistic binding of Mor with histidine tagged α (His-α), indicating that His-α binds to Pm in a sequence- and/or structure-specific manner. Taken together, these results demonstrate that Pm has a strong proximal UP element subsite, but lacks a distal subsite.