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

Abstract

The bacteriophage Mu Mor activator protein is absolutely required for transcription from the Mu middle promoter Pm. However, when RNA polymerase (RNAP) was incubated with Pm 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 Pm 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 Pm DNA. Generation of the hypersensitive band required the complete RNAP with its αCTDs, but neither the αCTD 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 Pm activity in vivo, as assayed by the Mor-dependent production of β-galactosidase from a Pm-lacZ fusion. In an “order of addition” experiment, preincubation of Pm DNA with Mor followed by addition of RNAP led to the fastest open complex formation, whereas preincubation of Pm DNA with RNAP gave the slowest. These results support the conclusion that Mor recruits RNAP to Pm rather than reposition a prebound RNAP, as occurs for C-dependent repositioning of RNAP at the Mu late promoter Pmom.

The Phage Mu Middle Promoter Pm Contains a Partial UP Element

“…The footprinting patterns of Mor alone with wild-type or mutant DNA probes ( Figure 5 ) were the same as observed previously ( Artsimovitch et al 1996 ; Ma and Howe 2004 ). Reactions with RNA polymerase alone produced little or no protection but did exhibit hypersensitive bands at positions −51 and −12, demonstrating the presence of unstable or weak interactions between RNAP and P m DNA, as observed previously by Mo and Howe (2014) . The upstream mutations had no effect on the −12 hypersensitivity, presumably because it reflects interaction of RNAP with the −10 hexamer.…”

“…Media used included M9 minimal medium with 0.2% casamino acids, Luria broth (LB), and MacConkey lactose plates with only half the normal amount of lactose. Sources for ampicillin (Ap), chloramphenicol (Cm), isopropyl β-D-1-thiogalactopyranoside (IPTG), o -nitrophenyl β-galactoside, agarose, and radiolabeled [γ- 32 P]ATP (3000 Ci/mmol) are in the article by Mo and Howe (2014) . Sources of enzymes used are in that same paper.…”