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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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PvuII R-M system control region and alignment with regions upstream of C.PvuII orthologs. (A) Genetic map of the PvuII R-M system. Numbering is relative to the initiation codon of pvuIIC. The four gray rectangles represent the C-half-boxes (shaded in part A). The two transcription starts for pvuIICR are identified by rightward bent arrows: from the C-independent weak promoter (thin) and C-dependent strong promoter (thick) (43). The two pvuIIM promoters are also shown (leftward bent arrows). (B) Comparison of the regions upstream of confirmed and putative C protein genes. Genes were identified by using the Blink function of Entrez (92), and REBASE (1), with the C.PvuII amino acid sequence as seed. This set was then screened for two features: proteins with predicted recognition helix sequences (of the helix–turn–helix motif) similar to that of C.PvuII (HRTYI) (41) or of C.AhdI (DRT/SY) (52), and being part of a putative R-M system (usually indicated by an adjacent MTase gene, as REase genes are difficult to recognize). A question mark indicates an ORF not yet formally named (93). The C protein initiation codon is underlined, unless it starts farther downstream. For C.PvuII (top), the underlined nucleotides (in half-box 2B and at the C.PvuII initiation codon) indicate the previously-identified transcript starts (40,43). The shaded nucleotides indicate actual or predicted C-boxes, and the proposed symmetrical core (40,43) is shown at the bottom. (C) Sequence Logos for the two subsets of C-box/promoter regions associated with C proteins having HRTY (upper) or DRT/SY (bottom) in the recognition helix. The Logo (46) was obtained from the two subsets of sequences in panel A, generated by the server at: http://weblogo.berkeley.edu. In Logo analysis, the full height (two bits of information content) applies to completely conserved positions. The C-box elements are boxed. (D) The pvuIIC promoter region sequence showing the C-box and promoter elements. The palindromic operators each contain a pair of C-boxes, designated as boxes 1AB or OL (operator left), and 2AB or OR (operator right). Conserved elements of the stronger, C-dependent promoter are indicated by heavy rectangles, while thinner rectangles indicate the weak C-independent promoter. Transcript starts are indicated as in (A). The upper sequence is from the wild-type (WT) PvuII R-M system, while the lower sequence has one substitution each in half-boxes 1A and 2A such that they match the symmetrical cores (SC) shown at the bottom of (B).
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Figure 1: PvuII R-M system control region and alignment with regions upstream of C.PvuII orthologs. (A) Genetic map of the PvuII R-M system. Numbering is relative to the initiation codon of pvuIIC. The four gray rectangles represent the C-half-boxes (shaded in part A). The two transcription starts for pvuIICR are identified by rightward bent arrows: from the C-independent weak promoter (thin) and C-dependent strong promoter (thick) (43). The two pvuIIM promoters are also shown (leftward bent arrows). (B) Comparison of the regions upstream of confirmed and putative C protein genes. Genes were identified by using the Blink function of Entrez (92), and REBASE (1), with the C.PvuII amino acid sequence as seed. This set was then screened for two features: proteins with predicted recognition helix sequences (of the helix–turn–helix motif) similar to that of C.PvuII (HRTYI) (41) or of C.AhdI (DRT/SY) (52), and being part of a putative R-M system (usually indicated by an adjacent MTase gene, as REase genes are difficult to recognize). A question mark indicates an ORF not yet formally named (93). The C protein initiation codon is underlined, unless it starts farther downstream. For C.PvuII (top), the underlined nucleotides (in half-box 2B and at the C.PvuII initiation codon) indicate the previously-identified transcript starts (40,43). The shaded nucleotides indicate actual or predicted C-boxes, and the proposed symmetrical core (40,43) is shown at the bottom. (C) Sequence Logos for the two subsets of C-box/promoter regions associated with C proteins having HRTY (upper) or DRT/SY (bottom) in the recognition helix. The Logo (46) was obtained from the two subsets of sequences in panel A, generated by the server at: http://weblogo.berkeley.edu. In Logo analysis, the full height (two bits of information content) applies to completely conserved positions. The C-box elements are boxed. (D) The pvuIIC promoter region sequence showing the C-box and promoter elements. The palindromic operators each contain a pair of C-boxes, designated as boxes 1AB or OL (operator left), and 2AB or OR (operator right). Conserved elements of the stronger, C-dependent promoter are indicated by heavy rectangles, while thinner rectangles indicate the weak C-independent promoter. Transcript starts are indicated as in (A). The upper sequence is from the wild-type (WT) PvuII R-M system, while the lower sequence has one substitution each in half-boxes 1A and 2A such that they match the symmetrical cores (SC) shown at the bottom of (B).

Mentions: C proteins, where tested, activate their own transcription (‘autogenous’ activation), and are believed to be responsible for the delay in REase activity that is crucial when an R-M system enters a new host cell. In R-M systems having a C gene, the REase gene typically does not have its own promoter (an exception is LlaI (39)). The C and REase open reading frames usually overlap (as in the PvuII system; Figure 1A), and the REase gene is completely dependent on transcription from the upstream autogenously regulated C gene (40). Disruption of pvuIIC leads to a drastic reduction in REase expression that is restored by supplying the C gene in trans (27,41). Thus, in a new cell, REase expression should be low until C protein accumulates. The role of this activation requirement in delaying REase expression is indicated by the observation that pre-expressing C protein prevents transformation by the intact cognate R-M system, presumably due to premature REase expression and cleavage of recipient cells’ chromosomal DNA (13,40).Figure 1.


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)

PvuII R-M system control region and alignment with regions upstream of C.PvuII orthologs. (A) Genetic map of the PvuII R-M system. Numbering is relative to the initiation codon of pvuIIC. The four gray rectangles represent the C-half-boxes (shaded in part A). The two transcription starts for pvuIICR are identified by rightward bent arrows: from the C-independent weak promoter (thin) and C-dependent strong promoter (thick) (43). The two pvuIIM promoters are also shown (leftward bent arrows). (B) Comparison of the regions upstream of confirmed and putative C protein genes. Genes were identified by using the Blink function of Entrez (92), and REBASE (1), with the C.PvuII amino acid sequence as seed. This set was then screened for two features: proteins with predicted recognition helix sequences (of the helix–turn–helix motif) similar to that of C.PvuII (HRTYI) (41) or of C.AhdI (DRT/SY) (52), and being part of a putative R-M system (usually indicated by an adjacent MTase gene, as REase genes are difficult to recognize). A question mark indicates an ORF not yet formally named (93). The C protein initiation codon is underlined, unless it starts farther downstream. For C.PvuII (top), the underlined nucleotides (in half-box 2B and at the C.PvuII initiation codon) indicate the previously-identified transcript starts (40,43). The shaded nucleotides indicate actual or predicted C-boxes, and the proposed symmetrical core (40,43) is shown at the bottom. (C) Sequence Logos for the two subsets of C-box/promoter regions associated with C proteins having HRTY (upper) or DRT/SY (bottom) in the recognition helix. The Logo (46) was obtained from the two subsets of sequences in panel A, generated by the server at: http://weblogo.berkeley.edu. In Logo analysis, the full height (two bits of information content) applies to completely conserved positions. The C-box elements are boxed. (D) The pvuIIC promoter region sequence showing the C-box and promoter elements. The palindromic operators each contain a pair of C-boxes, designated as boxes 1AB or OL (operator left), and 2AB or OR (operator right). Conserved elements of the stronger, C-dependent promoter are indicated by heavy rectangles, while thinner rectangles indicate the weak C-independent promoter. Transcript starts are indicated as in (A). The upper sequence is from the wild-type (WT) PvuII R-M system, while the lower sequence has one substitution each in half-boxes 1A and 2A such that they match the symmetrical cores (SC) shown at the bottom of (B).
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Related In: Results  -  Collection

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Figure 1: PvuII R-M system control region and alignment with regions upstream of C.PvuII orthologs. (A) Genetic map of the PvuII R-M system. Numbering is relative to the initiation codon of pvuIIC. The four gray rectangles represent the C-half-boxes (shaded in part A). The two transcription starts for pvuIICR are identified by rightward bent arrows: from the C-independent weak promoter (thin) and C-dependent strong promoter (thick) (43). The two pvuIIM promoters are also shown (leftward bent arrows). (B) Comparison of the regions upstream of confirmed and putative C protein genes. Genes were identified by using the Blink function of Entrez (92), and REBASE (1), with the C.PvuII amino acid sequence as seed. This set was then screened for two features: proteins with predicted recognition helix sequences (of the helix–turn–helix motif) similar to that of C.PvuII (HRTYI) (41) or of C.AhdI (DRT/SY) (52), and being part of a putative R-M system (usually indicated by an adjacent MTase gene, as REase genes are difficult to recognize). A question mark indicates an ORF not yet formally named (93). The C protein initiation codon is underlined, unless it starts farther downstream. For C.PvuII (top), the underlined nucleotides (in half-box 2B and at the C.PvuII initiation codon) indicate the previously-identified transcript starts (40,43). The shaded nucleotides indicate actual or predicted C-boxes, and the proposed symmetrical core (40,43) is shown at the bottom. (C) Sequence Logos for the two subsets of C-box/promoter regions associated with C proteins having HRTY (upper) or DRT/SY (bottom) in the recognition helix. The Logo (46) was obtained from the two subsets of sequences in panel A, generated by the server at: http://weblogo.berkeley.edu. In Logo analysis, the full height (two bits of information content) applies to completely conserved positions. The C-box elements are boxed. (D) The pvuIIC promoter region sequence showing the C-box and promoter elements. The palindromic operators each contain a pair of C-boxes, designated as boxes 1AB or OL (operator left), and 2AB or OR (operator right). Conserved elements of the stronger, C-dependent promoter are indicated by heavy rectangles, while thinner rectangles indicate the weak C-independent promoter. Transcript starts are indicated as in (A). The upper sequence is from the wild-type (WT) PvuII R-M system, while the lower sequence has one substitution each in half-boxes 1A and 2A such that they match the symmetrical cores (SC) shown at the bottom of (B).
Mentions: C proteins, where tested, activate their own transcription (‘autogenous’ activation), and are believed to be responsible for the delay in REase activity that is crucial when an R-M system enters a new host cell. In R-M systems having a C gene, the REase gene typically does not have its own promoter (an exception is LlaI (39)). The C and REase open reading frames usually overlap (as in the PvuII system; Figure 1A), and the REase gene is completely dependent on transcription from the upstream autogenously regulated C gene (40). Disruption of pvuIIC leads to a drastic reduction in REase expression that is restored by supplying the C gene in trans (27,41). Thus, in a new cell, REase expression should be low until C protein accumulates. The role of this activation requirement in delaying REase expression is indicated by the observation that pre-expressing C protein prevents transformation by the intact cognate R-M system, presumably due to premature REase expression and cleavage of recipient cells’ chromosomal DNA (13,40).Figure 1.

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