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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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Randomized C-half-box 2A libraries and selected variants. (A) Sequencing trace of the pooled randomized C-half-box 2A plasmid library before selection. All other C-half-boxes were WT (WWNW, where ‘N’ indicates the randomization), and the promoter region library is upstream of a promoterless cat gene. (B) Sequencing trace as in (A), except that in this library C-half-box 2B is replaced by the reversed complement (AGTC→GACT; WWNR). (C) The selection for C.PvuII-activated variants was as described in Figure 4B. The resulting sequences from the WWNW library, number of variants showing each recovered sequence, and Logo analysis are shown. (D) Sequences that could be activated detectably as in (C), but for the WWNR library. (E) The isolation of variants that are not detectably activated by C.PvuII was as described in Figure 4C. The resulting sequences from the WWNW library and Logo analysis are shown. (F) Sequences that are not detectably activated as in (E), but for the WWNR library. (G) Quantitative evaluation of C.PvuII-activated variants from WWNW (panel C) or WWNR (panel D) libraries. CAT levels were measured either in the presence of physiological steady-state levels of C.PvuII (pvuIIC under native control on plasmid pDK200; black bars), or without pvuIIC (white bars). CAT levels were determined via triplicate immunoassays as described in Material and Methods section. For comparison, mutants with inactive OR (WWRR) or OL (RRWW) were also analyzed. pKK represents vector (pKK232-8) control. The error bars indicate SDs.
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Figure 5: Randomized C-half-box 2A libraries and selected variants. (A) Sequencing trace of the pooled randomized C-half-box 2A plasmid library before selection. All other C-half-boxes were WT (WWNW, where ‘N’ indicates the randomization), and the promoter region library is upstream of a promoterless cat gene. (B) Sequencing trace as in (A), except that in this library C-half-box 2B is replaced by the reversed complement (AGTC→GACT; WWNR). (C) The selection for C.PvuII-activated variants was as described in Figure 4B. The resulting sequences from the WWNW library, number of variants showing each recovered sequence, and Logo analysis are shown. (D) Sequences that could be activated detectably as in (C), but for the WWNR library. (E) The isolation of variants that are not detectably activated by C.PvuII was as described in Figure 4C. The resulting sequences from the WWNW library and Logo analysis are shown. (F) Sequences that are not detectably activated as in (E), but for the WWNR library. (G) Quantitative evaluation of C.PvuII-activated variants from WWNW (panel C) or WWNR (panel D) libraries. CAT levels were measured either in the presence of physiological steady-state levels of C.PvuII (pvuIIC under native control on plasmid pDK200; black bars), or without pvuIIC (white bars). CAT levels were determined via triplicate immunoassays as described in Material and Methods section. For comparison, mutants with inactive OR (WWRR) or OL (RRWW) were also analyzed. pKK represents vector (pKK232-8) control. The error bars indicate SDs.

Mentions: If C.PvuII binding to OR is associated exclusively with repression, then altering that site should not reduce activation by C.PvuII. To test this prediction, we again prepared a randomized plasmid library for selection on plates and CAT reporter assays. We generated two separate libraries, each of them containing a randomized half-box 2A in OR. In one library, designated WWNW (where ‘N’ indicates the randomized 4 nt sequence), the background sequence of the other three half-boxes and the TGTA spacer remain intact (Figure 5A). In contrast, the second library, labeled WWNR, includes a reversed complement of half-box 2B (Figure 5B).Figure 5.


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)

Randomized C-half-box 2A libraries and selected variants. (A) Sequencing trace of the pooled randomized C-half-box 2A plasmid library before selection. All other C-half-boxes were WT (WWNW, where ‘N’ indicates the randomization), and the promoter region library is upstream of a promoterless cat gene. (B) Sequencing trace as in (A), except that in this library C-half-box 2B is replaced by the reversed complement (AGTC→GACT; WWNR). (C) The selection for C.PvuII-activated variants was as described in Figure 4B. The resulting sequences from the WWNW library, number of variants showing each recovered sequence, and Logo analysis are shown. (D) Sequences that could be activated detectably as in (C), but for the WWNR library. (E) The isolation of variants that are not detectably activated by C.PvuII was as described in Figure 4C. The resulting sequences from the WWNW library and Logo analysis are shown. (F) Sequences that are not detectably activated as in (E), but for the WWNR library. (G) Quantitative evaluation of C.PvuII-activated variants from WWNW (panel C) or WWNR (panel D) libraries. CAT levels were measured either in the presence of physiological steady-state levels of C.PvuII (pvuIIC under native control on plasmid pDK200; black bars), or without pvuIIC (white bars). CAT levels were determined via triplicate immunoassays as described in Material and Methods section. For comparison, mutants with inactive OR (WWRR) or OL (RRWW) were also analyzed. pKK represents vector (pKK232-8) control. The error bars indicate SDs.
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Related In: Results  -  Collection

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Figure 5: Randomized C-half-box 2A libraries and selected variants. (A) Sequencing trace of the pooled randomized C-half-box 2A plasmid library before selection. All other C-half-boxes were WT (WWNW, where ‘N’ indicates the randomization), and the promoter region library is upstream of a promoterless cat gene. (B) Sequencing trace as in (A), except that in this library C-half-box 2B is replaced by the reversed complement (AGTC→GACT; WWNR). (C) The selection for C.PvuII-activated variants was as described in Figure 4B. The resulting sequences from the WWNW library, number of variants showing each recovered sequence, and Logo analysis are shown. (D) Sequences that could be activated detectably as in (C), but for the WWNR library. (E) The isolation of variants that are not detectably activated by C.PvuII was as described in Figure 4C. The resulting sequences from the WWNW library and Logo analysis are shown. (F) Sequences that are not detectably activated as in (E), but for the WWNR library. (G) Quantitative evaluation of C.PvuII-activated variants from WWNW (panel C) or WWNR (panel D) libraries. CAT levels were measured either in the presence of physiological steady-state levels of C.PvuII (pvuIIC under native control on plasmid pDK200; black bars), or without pvuIIC (white bars). CAT levels were determined via triplicate immunoassays as described in Material and Methods section. For comparison, mutants with inactive OR (WWRR) or OL (RRWW) were also analyzed. pKK represents vector (pKK232-8) control. The error bars indicate SDs.
Mentions: If C.PvuII binding to OR is associated exclusively with repression, then altering that site should not reduce activation by C.PvuII. To test this prediction, we again prepared a randomized plasmid library for selection on plates and CAT reporter assays. We generated two separate libraries, each of them containing a randomized half-box 2A in OR. In one library, designated WWNW (where ‘N’ indicates the randomized 4 nt sequence), the background sequence of the other three half-boxes and the TGTA spacer remain intact (Figure 5A). In contrast, the second library, labeled WWNR, includes a reversed complement of half-box 2B (Figure 5B).Figure 5.

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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