S1 nuclease mapping of the phage Mu mom gene promoter: a model for the regulation of mom expression

Abstract

The mom gene of bacteriophage Mu encodes a DNA modification function. Expression of this modification requires the host Escherichia coli Dam (DNA-adenine methylase) function and the transacting phage Mu Dad function. The mom gene was subcloned into a variety of sites on plasmid pBR322. Insertions were made into the Hindi and PvuI sites within the amp gene and into the ClaI site of the tet gene promoter. The only clones found were those in which the orientation of the mom gene prevents its transcription from the vector promoter(s), suggesting that constitutive expression of mom from a foreign promoter can occur independently of Dad function but is lethal for the cell. Employing S1 nuclease mapping, we have identified two Mu mRNA transcripts: (1) the gin transcript extends into the gin-mom intercistronic divide and terminates downstream from the BelI site; and (2) the mom transcript appears to initiate about 74 bp upstream from the BelI site, 12 bp downstream from a promoter-like sequence. Production of the mom transcript is dependent on the host Dam activity and on Dad transactivation. In contrast, the gin transcript is produced independently of Dam and Dad functions; the gin transcript may extend into the mom gene, but it appears to be either degraded at the 3' end or differentially terminated. We propose that regulation of mom gene transcription involves both positive and negative regulatory proteins, and that binding of the Dad protein (a “late” Mu protein) is required for transcription initiation by the host RNA polymerase. However, Dad protein action may be inhibited by prior binding of a represser to the mom operator, located farther upstream. We propose that this repressor (encoded by a phage or host gene) binds to the operator only when there is no active Dam enzyme present i.e., when there is no methylation of (or methylase binding to) the G-A-T-C sites within the mom operator.