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The phage Mu middle promoter Pm contains a partial UP element.

Ma J, Howe MM - G3 (Bethesda) (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology & Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee 38163.

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Middle promoter two-plasmid in vivo assay system for promoter activity. DNA fragments with promoters, in this case Pm sequence −98 to +10, are cloned between the EcoRI and BamHI sites in pIA12 to generate the Pm-lacZ fusions. Plasmid pKM78 contains a WT mor gene under the control of the PlacUV5 promoter. When cells contain both plasmids, the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) leads to induction of Mor expression from pKM78. Mor then binds and activates transcription from the WT or mutant Pm promoter cloned in pIA12, leading to synthesis of β-galactosidase at levels proportional to Pm activity. This figure was published previously by Artsimovitch and Howe (1996).
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fig2: Middle promoter two-plasmid in vivo assay system for promoter activity. DNA fragments with promoters, in this case Pm sequence −98 to +10, are cloned between the EcoRI and BamHI sites in pIA12 to generate the Pm-lacZ fusions. Plasmid pKM78 contains a WT mor gene under the control of the PlacUV5 promoter. When cells contain both plasmids, the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) leads to induction of Mor expression from pKM78. Mor then binds and activates transcription from the WT or mutant Pm promoter cloned in pIA12, leading to synthesis of β-galactosidase at levels proportional to Pm activity. This figure was published previously by Artsimovitch and Howe (1996).

Mentions: The promoter cloning plasmid, pIA12 (Figure 2), contains a HindIII-EcoRI-BamHI linker upstream from a promoter-less lacZ gene (Chiang and Howe 1993; Artsimovitch and Howe 1996). Plasmid pMM1 contains Pm sequence from −98 to +10 cloned into the EcoRI and BamHI sites in pIA12 (Chiang et al. 1993). Plasmid pKM78 (Figure 2) expresses Mor under the control of PlacUV5 (Mathee and Howe 1990). Plasmid pKM90 contains the mor gene downstream of the strong T7 promoter and ribosome binding site in pT7-7 (Tabor and Richardson 1985; Mathee and Howe 1993; Ma and Howe 2004). Plasmid pHTT7f1-NHα contains an rpoA gene encoding the N-terminal His6-tagged α subunit of E. coli RNA polymerase (Tang et al. 1994).


The phage Mu middle promoter Pm contains a partial UP element.

Ma J, Howe MM - G3 (Bethesda) (2015)

Middle promoter two-plasmid in vivo assay system for promoter activity. DNA fragments with promoters, in this case Pm sequence −98 to +10, are cloned between the EcoRI and BamHI sites in pIA12 to generate the Pm-lacZ fusions. Plasmid pKM78 contains a WT mor gene under the control of the PlacUV5 promoter. When cells contain both plasmids, the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) leads to induction of Mor expression from pKM78. Mor then binds and activates transcription from the WT or mutant Pm promoter cloned in pIA12, leading to synthesis of β-galactosidase at levels proportional to Pm activity. This figure was published previously by Artsimovitch and Howe (1996).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390567&req=5

fig2: Middle promoter two-plasmid in vivo assay system for promoter activity. DNA fragments with promoters, in this case Pm sequence −98 to +10, are cloned between the EcoRI and BamHI sites in pIA12 to generate the Pm-lacZ fusions. Plasmid pKM78 contains a WT mor gene under the control of the PlacUV5 promoter. When cells contain both plasmids, the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) leads to induction of Mor expression from pKM78. Mor then binds and activates transcription from the WT or mutant Pm promoter cloned in pIA12, leading to synthesis of β-galactosidase at levels proportional to Pm activity. This figure was published previously by Artsimovitch and Howe (1996).
Mentions: The promoter cloning plasmid, pIA12 (Figure 2), contains a HindIII-EcoRI-BamHI linker upstream from a promoter-less lacZ gene (Chiang and Howe 1993; Artsimovitch and Howe 1996). Plasmid pMM1 contains Pm sequence from −98 to +10 cloned into the EcoRI and BamHI sites in pIA12 (Chiang et al. 1993). Plasmid pKM78 (Figure 2) expresses Mor under the control of PlacUV5 (Mathee and Howe 1990). Plasmid pKM90 contains the mor gene downstream of the strong T7 promoter and ribosome binding site in pT7-7 (Tabor and Richardson 1985; Mathee and Howe 1993; Ma and Howe 2004). Plasmid pHTT7f1-NHα contains an rpoA gene encoding the N-terminal His6-tagged α subunit of E. coli RNA polymerase (Tang et al. 1994).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology & Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee 38163.

Show MeSH
Related in: MedlinePlus