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Regulatory circuit based on autogenous activation-repression: roles of C-boxes and spacer sequences in control of the PvuII restriction-modification system.

Mruk I, Rajesh P, Blumenthal RM - Nucleic Acids Res. (2007)

Bottom Line: In other systems, this type of circuit can result in oscillatory behavior.Mutational analysis associated the repression with O(R), which overlaps the promoter -35 hexamer but is otherwise dispensable for activation.A nonrepressing mutant exhibited poor establishment in new cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology and Immunology, University of Toledo Health Sciences Campus, Toledo, OH 43614-2598, USA.

ABSTRACT
Type II restriction-modification (R-M) systems comprise a restriction endonuclease (REase) and a protective methyltransferase (MTase). After R-M genes enter a new cell, MTase must appear before REase or the chromosome will be cleaved. PvuII and some other R-M systems achieve this delay by cotranscribing the REase gene with the gene for an autogenous transcription activator (the controlling or 'C' protein C.PvuII). This study reveals, through in vivo titration, that C.PvuII is not only an activator but also a repressor for its own gene. In other systems, this type of circuit can result in oscillatory behavior. Despite the use of identical, symmetrical C protein-binding sequences (C-boxes) in the left and right operators, C.PvuII showed higher in vitro affinity for O(L) than for O(R), implicating the spacer sequences in this difference. Mutational analysis associated the repression with O(R), which overlaps the promoter -35 hexamer but is otherwise dispensable for activation. A nonrepressing mutant exhibited poor establishment in new cells. Comparing promoter-operator regions from PvuII and 29 R-M systems controlled by C proteins revealed that the most-highly conserved sequence is the tetranucleotide spacer separating O(L) from O(R). Any changes in that spacer reduced the stability of C.PvuII-operator complexes and abolished activation.

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In vivo titration with C.PvuII and its effect on pvuIICR transcription. Cells were grown in minimal media with 0.2% glycerol and the indicated concentration of arabinose, and supplemented as described in Materials and Methods section. In addition, the cells were grown without (A) or with (B) 0.2% glucose. Expression from transcriptional fusion PpvuIICR:lacZ (pDK435) was measured as β-galactosidase specific activity, determined by linear regression of the slopes of the lines generated by plotting LacZ activity (modified Miller units) versus optical density of the culture (Platko et al., 1990). Unconnected points at the beginning of the curve (panel A) indicate values obtained in glucose with no arabinose. Closed symbols represent LacZ activity from cells with the native PvuII C-boxes (circles, pDK435) or its symmetrized variant (diamonds, pIM8), in the presence of PBAD–pvuIIC (plasmid pIM1); open circles represent LacZ fusion activity when the vector plasmid (lacking pvuIIC) is present (plasmid pBAD24). (C) Extracts from the same cultures shown in (A) were resolved on a 4–12% acrylamide SDS gel. Ten micrograms of total protein was loaded per lane, except for the MW protein markers in the far left lane and 150 ng of purified C.PvuII in the far right lane. Lanes 1–7 contained culture samples: 1-glucose, 0% arabinose; 2–0% arabinose; 3–7-increasing concentrations of arabinose, in 10-fold steps, from 0.00002 to 0.2%. The gel was then silver stained and photographed. (D) A parallel gel to that shown in (C) was electroblotted to a PVDF membrane, blocked, and probed with polyclonal rabbit anti-C.PvuII antiserum and then with horseradish peroxidase-coupled goat anti-rabbit IgG. The positions of the prestained MW markers are indicated by bars. The expected subunit size of native C.PvuII is 9.4 kDa.
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Figure 2: In vivo titration with C.PvuII and its effect on pvuIICR transcription. Cells were grown in minimal media with 0.2% glycerol and the indicated concentration of arabinose, and supplemented as described in Materials and Methods section. In addition, the cells were grown without (A) or with (B) 0.2% glucose. Expression from transcriptional fusion PpvuIICR:lacZ (pDK435) was measured as β-galactosidase specific activity, determined by linear regression of the slopes of the lines generated by plotting LacZ activity (modified Miller units) versus optical density of the culture (Platko et al., 1990). Unconnected points at the beginning of the curve (panel A) indicate values obtained in glucose with no arabinose. Closed symbols represent LacZ activity from cells with the native PvuII C-boxes (circles, pDK435) or its symmetrized variant (diamonds, pIM8), in the presence of PBAD–pvuIIC (plasmid pIM1); open circles represent LacZ fusion activity when the vector plasmid (lacking pvuIIC) is present (plasmid pBAD24). (C) Extracts from the same cultures shown in (A) were resolved on a 4–12% acrylamide SDS gel. Ten micrograms of total protein was loaded per lane, except for the MW protein markers in the far left lane and 150 ng of purified C.PvuII in the far right lane. Lanes 1–7 contained culture samples: 1-glucose, 0% arabinose; 2–0% arabinose; 3–7-increasing concentrations of arabinose, in 10-fold steps, from 0.00002 to 0.2%. The gel was then silver stained and photographed. (D) A parallel gel to that shown in (C) was electroblotted to a PVDF membrane, blocked, and probed with polyclonal rabbit anti-C.PvuII antiserum and then with horseradish peroxidase-coupled goat anti-rabbit IgG. The positions of the prestained MW markers are indicated by bars. The expected subunit size of native C.PvuII is 9.4 kDa.

Mentions: To examine the DNA-binding sites important for activation of the promoter for the C and REase genes (PpvuIICR) by C.PvuII, we first tested the effects of an in vivo titration in which intracellular C.PvuII protein levels were varied from undetectably low to overexpression. We used the E. coli araBAD promoter (PBAD), as its activity can be modulated over a wide range (68–70). We fused pvuIIC to PBAD, followed by strong transcription terminators, to generate plasmid pIM1 (Table 1). This plasmid also carries the gene for the bifunctional AraC protein, that represses PBAD in the absence of arabinose and activates it when arabinose is present. To reveal the effects of the C.PvuII titration, we cloned the PpvuIICR region (including wild-type C-boxes, positions −93 to +88, Figure 1A) upstream of the lacZ reporter gene (plasmid pDK435 or its variant pIM8 with the symmetrized C-boxes), generating transcriptional fusions. The host was E. coli MC1061, which lacks the lac and ara operons but carries araE for arabinose transport. Experiments were carried out in minimal media with concentrations of arabinose from 0 to 0.2% (saturating induction) (Figure 2A and B). The effects of induction on C.PvuII levels were confirmed by western blot (Figure 2D, and I.M. unpublished data), and show the strong nonlinear response to arabinose concentration characteristic of PBAD (68). Expression of the PpvuIIC-lacZ transcriptional fusion was measured by β-galactosidase assay.Figure 2.


Regulatory circuit based on autogenous activation-repression: roles of C-boxes and spacer sequences in control of the PvuII restriction-modification system.

Mruk I, Rajesh P, Blumenthal RM - Nucleic Acids Res. (2007)

In vivo titration with C.PvuII and its effect on pvuIICR transcription. Cells were grown in minimal media with 0.2% glycerol and the indicated concentration of arabinose, and supplemented as described in Materials and Methods section. In addition, the cells were grown without (A) or with (B) 0.2% glucose. Expression from transcriptional fusion PpvuIICR:lacZ (pDK435) was measured as β-galactosidase specific activity, determined by linear regression of the slopes of the lines generated by plotting LacZ activity (modified Miller units) versus optical density of the culture (Platko et al., 1990). Unconnected points at the beginning of the curve (panel A) indicate values obtained in glucose with no arabinose. Closed symbols represent LacZ activity from cells with the native PvuII C-boxes (circles, pDK435) or its symmetrized variant (diamonds, pIM8), in the presence of PBAD–pvuIIC (plasmid pIM1); open circles represent LacZ fusion activity when the vector plasmid (lacking pvuIIC) is present (plasmid pBAD24). (C) Extracts from the same cultures shown in (A) were resolved on a 4–12% acrylamide SDS gel. Ten micrograms of total protein was loaded per lane, except for the MW protein markers in the far left lane and 150 ng of purified C.PvuII in the far right lane. Lanes 1–7 contained culture samples: 1-glucose, 0% arabinose; 2–0% arabinose; 3–7-increasing concentrations of arabinose, in 10-fold steps, from 0.00002 to 0.2%. The gel was then silver stained and photographed. (D) A parallel gel to that shown in (C) was electroblotted to a PVDF membrane, blocked, and probed with polyclonal rabbit anti-C.PvuII antiserum and then with horseradish peroxidase-coupled goat anti-rabbit IgG. The positions of the prestained MW markers are indicated by bars. The expected subunit size of native C.PvuII is 9.4 kDa.
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Related In: Results  -  Collection

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Figure 2: In vivo titration with C.PvuII and its effect on pvuIICR transcription. Cells were grown in minimal media with 0.2% glycerol and the indicated concentration of arabinose, and supplemented as described in Materials and Methods section. In addition, the cells were grown without (A) or with (B) 0.2% glucose. Expression from transcriptional fusion PpvuIICR:lacZ (pDK435) was measured as β-galactosidase specific activity, determined by linear regression of the slopes of the lines generated by plotting LacZ activity (modified Miller units) versus optical density of the culture (Platko et al., 1990). Unconnected points at the beginning of the curve (panel A) indicate values obtained in glucose with no arabinose. Closed symbols represent LacZ activity from cells with the native PvuII C-boxes (circles, pDK435) or its symmetrized variant (diamonds, pIM8), in the presence of PBAD–pvuIIC (plasmid pIM1); open circles represent LacZ fusion activity when the vector plasmid (lacking pvuIIC) is present (plasmid pBAD24). (C) Extracts from the same cultures shown in (A) were resolved on a 4–12% acrylamide SDS gel. Ten micrograms of total protein was loaded per lane, except for the MW protein markers in the far left lane and 150 ng of purified C.PvuII in the far right lane. Lanes 1–7 contained culture samples: 1-glucose, 0% arabinose; 2–0% arabinose; 3–7-increasing concentrations of arabinose, in 10-fold steps, from 0.00002 to 0.2%. The gel was then silver stained and photographed. (D) A parallel gel to that shown in (C) was electroblotted to a PVDF membrane, blocked, and probed with polyclonal rabbit anti-C.PvuII antiserum and then with horseradish peroxidase-coupled goat anti-rabbit IgG. The positions of the prestained MW markers are indicated by bars. The expected subunit size of native C.PvuII is 9.4 kDa.
Mentions: To examine the DNA-binding sites important for activation of the promoter for the C and REase genes (PpvuIICR) by C.PvuII, we first tested the effects of an in vivo titration in which intracellular C.PvuII protein levels were varied from undetectably low to overexpression. We used the E. coli araBAD promoter (PBAD), as its activity can be modulated over a wide range (68–70). We fused pvuIIC to PBAD, followed by strong transcription terminators, to generate plasmid pIM1 (Table 1). This plasmid also carries the gene for the bifunctional AraC protein, that represses PBAD in the absence of arabinose and activates it when arabinose is present. To reveal the effects of the C.PvuII titration, we cloned the PpvuIICR region (including wild-type C-boxes, positions −93 to +88, Figure 1A) upstream of the lacZ reporter gene (plasmid pDK435 or its variant pIM8 with the symmetrized C-boxes), generating transcriptional fusions. The host was E. coli MC1061, which lacks the lac and ara operons but carries araE for arabinose transport. Experiments were carried out in minimal media with concentrations of arabinose from 0 to 0.2% (saturating induction) (Figure 2A and B). The effects of induction on C.PvuII levels were confirmed by western blot (Figure 2D, and I.M. unpublished data), and show the strong nonlinear response to arabinose concentration characteristic of PBAD (68). Expression of the PpvuIIC-lacZ transcriptional fusion was measured by β-galactosidase assay.Figure 2.

Bottom Line: In other systems, this type of circuit can result in oscillatory behavior.Mutational analysis associated the repression with O(R), which overlaps the promoter -35 hexamer but is otherwise dispensable for activation.A nonrepressing mutant exhibited poor establishment in new cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology and Immunology, University of Toledo Health Sciences Campus, Toledo, OH 43614-2598, USA.

ABSTRACT
Type II restriction-modification (R-M) systems comprise a restriction endonuclease (REase) and a protective methyltransferase (MTase). After R-M genes enter a new cell, MTase must appear before REase or the chromosome will be cleaved. PvuII and some other R-M systems achieve this delay by cotranscribing the REase gene with the gene for an autogenous transcription activator (the controlling or 'C' protein C.PvuII). This study reveals, through in vivo titration, that C.PvuII is not only an activator but also a repressor for its own gene. In other systems, this type of circuit can result in oscillatory behavior. Despite the use of identical, symmetrical C protein-binding sequences (C-boxes) in the left and right operators, C.PvuII showed higher in vitro affinity for O(L) than for O(R), implicating the spacer sequences in this difference. Mutational analysis associated the repression with O(R), which overlaps the promoter -35 hexamer but is otherwise dispensable for activation. A nonrepressing mutant exhibited poor establishment in new cells. Comparing promoter-operator regions from PvuII and 29 R-M systems controlled by C proteins revealed that the most-highly conserved sequence is the tetranucleotide spacer separating O(L) from O(R). Any changes in that spacer reduced the stability of C.PvuII-operator complexes and abolished activation.

Show MeSH
Related in: MedlinePlus