Unusual interaction of RNA polymerase with the bacteriophage Mu middle promoter P m in the absence of its activator protein Mor

The bacteriophage Mu Mor activator protein is absolutely required for transcription from the Mu middle promoter P m . However, when RNA polymerase (RNAP) was incubated with P m DNA in the absence of Mor, a band at promoter position −51 was hypersensitive to DNase I cleavage, demonstrating an interaction of RNAP with the promoter DNA. The hypersensitivity was similar at four different lengths of P m DNA assayed from −62 to +10, −62 to +46, −96 to +10, and −96 to +46. The hypersensitivity occurred equally well at 5°C, 15°C, and 30°C, indicating that it did not require open complex formation, which only occurred at 30°C. The −51 hypersensitivity at 5°C and 15°C was eliminated by the addition of heparin, consistent with the possibility that it arose by formation of unstable closed complexes of RNAP bound to P m DNA. Generation of the hypersensitive band required the complete RNAP with its α CTDs, but neither the α CTD nor intact α were sufficient for the interaction and resulting hypersensitivity. There was no correlation between the level of hypersensitivity observed in vitro and the level of P m activity in vivo , as assayed by the Mor‐dependent production of β ‐galactosidase from a P m ‐ lacZ fusion. In an “order of addition” experiment, preincubation of P m DNA with Mor followed by addition of RNAP led to the fastest open complex formation, whereas preincubation of P m DNA with RNAP gave the slowest. These results support the conclusion that Mor recruits RNAP to P m rather than reposition a prebound RNAP, as occurs for C‐dependent repositioning of RNAP at the Mu late promoter P mom .