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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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Related in: MedlinePlus

Regional mutagenesis of Pm sequence upstream of the Mor binding site. The Pm promoter sequence is shown from −103 to +11 with respect to the transcription initiation site as +1. Dots above the sequence indicate 10-bp intervals that are numbered by their position in the promoter with respect to +1. The −10 sequence is in a box and +1 is indicated by the bent arrow. The bold lines above the sequence indicate the positions of the DNase I footprints for Mor and for the αCTD. The short inverted arrows above the sequence identify the locations of the 5-bp inverted repeats of the Mor binding site. The long thin arrows below the sequence show the Pm bases present in the primers used for mutagenesis. The bold lines below the sequence show the regions I-V that were mutagenized.
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fig3: Regional mutagenesis of Pm sequence upstream of the Mor binding site. The Pm promoter sequence is shown from −103 to +11 with respect to the transcription initiation site as +1. Dots above the sequence indicate 10-bp intervals that are numbered by their position in the promoter with respect to +1. The −10 sequence is in a box and +1 is indicated by the bent arrow. The bold lines above the sequence indicate the positions of the DNase I footprints for Mor and for the αCTD. The short inverted arrows above the sequence identify the locations of the 5-bp inverted repeats of the Mor binding site. The long thin arrows below the sequence show the Pm bases present in the primers used for mutagenesis. The bold lines below the sequence show the regions I-V that were mutagenized.

Mentions: The goal of this study was to determine whether Pm contains an UP element. Our approach was to isolate and characterize Pm mutants with base changes in the region upstream of the Mor binding site. The mutagenesis strategy was to divide the Pm sequence upstream of the Mor binding site into multiple regions based on their GC-content, helix face, and proximity to the Mor binding site (Figure 3). Mutagenesis of each region was performed separately using degenerate oligonucleotide primers (Table 1). For regions I (−88 to −79) and II (−78 to −69), the primers were designed to change the targeted region to be more A/T- or G/C-rich. For regions III (−69 to −62), IV (−63 to −57), and V (−57 to −52), a random mixture of all four bases was used at targeted positions. PCRs were performed first with the degenerate oligonucleotide primers and an opposing wild-type primer (MLK7) to produce a template for a second PCR with overlapping wild-type primers (MEG4 and MLK7). This second PCR completed the promoter sequence and provided the EcoRI and BamHI restriction sites for cloning. The PCR products were cloned between the EcoRI and BamHI sites in the promoter-less lacZ fusion vector pIA12 (Figure 2) just upstream of the lacZ gene. The ligation mix was transformed into strain MH13335 containing the activator plasmid pKM78, which expresses Mor when induced by IPTG (Figure 2). The transcription activities of the mutant promoters were examined by color development of each transformant on MacConkey lactose plates with different concentrations of IPTG, and scored as Up, Down, or wild-type relative to the color developed by the control strain (MH 15001) containing a plasmid with wild-type Pm on the same plate.


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

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

Regional mutagenesis of Pm sequence upstream of the Mor binding site. The Pm promoter sequence is shown from −103 to +11 with respect to the transcription initiation site as +1. Dots above the sequence indicate 10-bp intervals that are numbered by their position in the promoter with respect to +1. The −10 sequence is in a box and +1 is indicated by the bent arrow. The bold lines above the sequence indicate the positions of the DNase I footprints for Mor and for the αCTD. The short inverted arrows above the sequence identify the locations of the 5-bp inverted repeats of the Mor binding site. The long thin arrows below the sequence show the Pm bases present in the primers used for mutagenesis. The bold lines below the sequence show the regions I-V that were mutagenized.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Regional mutagenesis of Pm sequence upstream of the Mor binding site. The Pm promoter sequence is shown from −103 to +11 with respect to the transcription initiation site as +1. Dots above the sequence indicate 10-bp intervals that are numbered by their position in the promoter with respect to +1. The −10 sequence is in a box and +1 is indicated by the bent arrow. The bold lines above the sequence indicate the positions of the DNase I footprints for Mor and for the αCTD. The short inverted arrows above the sequence identify the locations of the 5-bp inverted repeats of the Mor binding site. The long thin arrows below the sequence show the Pm bases present in the primers used for mutagenesis. The bold lines below the sequence show the regions I-V that were mutagenized.
Mentions: The goal of this study was to determine whether Pm contains an UP element. Our approach was to isolate and characterize Pm mutants with base changes in the region upstream of the Mor binding site. The mutagenesis strategy was to divide the Pm sequence upstream of the Mor binding site into multiple regions based on their GC-content, helix face, and proximity to the Mor binding site (Figure 3). Mutagenesis of each region was performed separately using degenerate oligonucleotide primers (Table 1). For regions I (−88 to −79) and II (−78 to −69), the primers were designed to change the targeted region to be more A/T- or G/C-rich. For regions III (−69 to −62), IV (−63 to −57), and V (−57 to −52), a random mixture of all four bases was used at targeted positions. PCRs were performed first with the degenerate oligonucleotide primers and an opposing wild-type primer (MLK7) to produce a template for a second PCR with overlapping wild-type primers (MEG4 and MLK7). This second PCR completed the promoter sequence and provided the EcoRI and BamHI restriction sites for cloning. The PCR products were cloned between the EcoRI and BamHI sites in the promoter-less lacZ fusion vector pIA12 (Figure 2) just upstream of the lacZ gene. The ligation mix was transformed into strain MH13335 containing the activator plasmid pKM78, which expresses Mor when induced by IPTG (Figure 2). The transcription activities of the mutant promoters were examined by color development of each transformant on MacConkey lactose plates with different concentrations of IPTG, and scored as Up, Down, or wild-type relative to the color developed by the control strain (MH 15001) containing a plasmid with wild-type Pm on the same plate.

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