Limits...
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.

Show MeSH

Related in: MedlinePlus

The effects of altered OR on activation and repression by C.PvuII. Quantitative evaluation of selected C.PvuII-activatable variants from the WWNW and WWNR libraries, upstream of a promoterless cat gene, was determined with C.PvuII produced from PBAD-pvuIIC (pIM4; black bars) or without pvuIIC (white bars). Furthermore, experiments were carried out at two levels of C.PvuII that, for the WT C-boxes, are respectively associated with activation (A, upper panel) and repression (B, lower panel). In panel A, cultures were grown in the absence of arabinose, leading to lower C.PvuII levels, while for panel B, cultures were grown in 0.2% arabinose, associated with high levels of C.PvuII (see Figure 2A). CAT levels were determined in triplicate immunoassays as described in Material and Methods section. For comparison, variants having inactive OR (WWRR) or OL (RRWW) were also analyzed. pKK represents vector (pKK232-8) control. The error bars indicate SDs. For each variant, the sequence of C-half-box 2A is shown (WT = GACT); the horizontal black bar indicates those variants that were selected from the library on the basis of nonrepressibility. (C) Logo analysis of the nonrepressing variants from the WWNR library, showing the interoperator spacer and C-half-box 2A.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2175313&req=5

Figure 6: The effects of altered OR on activation and repression by C.PvuII. Quantitative evaluation of selected C.PvuII-activatable variants from the WWNW and WWNR libraries, upstream of a promoterless cat gene, was determined with C.PvuII produced from PBAD-pvuIIC (pIM4; black bars) or without pvuIIC (white bars). Furthermore, experiments were carried out at two levels of C.PvuII that, for the WT C-boxes, are respectively associated with activation (A, upper panel) and repression (B, lower panel). In panel A, cultures were grown in the absence of arabinose, leading to lower C.PvuII levels, while for panel B, cultures were grown in 0.2% arabinose, associated with high levels of C.PvuII (see Figure 2A). CAT levels were determined in triplicate immunoassays as described in Material and Methods section. For comparison, variants having inactive OR (WWRR) or OL (RRWW) were also analyzed. pKK represents vector (pKK232-8) control. The error bars indicate SDs. For each variant, the sequence of C-half-box 2A is shown (WT = GACT); the horizontal black bar indicates those variants that were selected from the library on the basis of nonrepressibility. (C) Logo analysis of the nonrepressing variants from the WWNR library, showing the interoperator spacer and C-half-box 2A.

Mentions: CAT production was determined quantitatively for selected WWNR variants and, for comparison, some previously obtained C-dependent variants from the WWNW library (selected under lower C.PvuII expression levels; Figure 5G). The measurements were carried out under two conditions, chosen based on the in vivo titrations (Figure 2A). The first condition favors activation and maximum cat expression (0% arabinose; Figure 6A). The second condition favors repression (0.2% arabinose; Figure 6B). The results for all tested WWNW variants reveal similar levels of cat expression in low levels of C.PvuII (Figure 6A and B; white bars). When the C.PvuII level was increased by induction of its PBAD promoter, cat expression was significantly reduced in all cases.Figure 6.


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)

The effects of altered OR on activation and repression by C.PvuII. Quantitative evaluation of selected C.PvuII-activatable variants from the WWNW and WWNR libraries, upstream of a promoterless cat gene, was determined with C.PvuII produced from PBAD-pvuIIC (pIM4; black bars) or without pvuIIC (white bars). Furthermore, experiments were carried out at two levels of C.PvuII that, for the WT C-boxes, are respectively associated with activation (A, upper panel) and repression (B, lower panel). In panel A, cultures were grown in the absence of arabinose, leading to lower C.PvuII levels, while for panel B, cultures were grown in 0.2% arabinose, associated with high levels of C.PvuII (see Figure 2A). CAT levels were determined in triplicate immunoassays as described in Material and Methods section. For comparison, variants having inactive OR (WWRR) or OL (RRWW) were also analyzed. pKK represents vector (pKK232-8) control. The error bars indicate SDs. For each variant, the sequence of C-half-box 2A is shown (WT = GACT); the horizontal black bar indicates those variants that were selected from the library on the basis of nonrepressibility. (C) Logo analysis of the nonrepressing variants from the WWNR library, showing the interoperator spacer and C-half-box 2A.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175313&req=5

Figure 6: The effects of altered OR on activation and repression by C.PvuII. Quantitative evaluation of selected C.PvuII-activatable variants from the WWNW and WWNR libraries, upstream of a promoterless cat gene, was determined with C.PvuII produced from PBAD-pvuIIC (pIM4; black bars) or without pvuIIC (white bars). Furthermore, experiments were carried out at two levels of C.PvuII that, for the WT C-boxes, are respectively associated with activation (A, upper panel) and repression (B, lower panel). In panel A, cultures were grown in the absence of arabinose, leading to lower C.PvuII levels, while for panel B, cultures were grown in 0.2% arabinose, associated with high levels of C.PvuII (see Figure 2A). CAT levels were determined in triplicate immunoassays as described in Material and Methods section. For comparison, variants having inactive OR (WWRR) or OL (RRWW) were also analyzed. pKK represents vector (pKK232-8) control. The error bars indicate SDs. For each variant, the sequence of C-half-box 2A is shown (WT = GACT); the horizontal black bar indicates those variants that were selected from the library on the basis of nonrepressibility. (C) Logo analysis of the nonrepressing variants from the WWNR library, showing the interoperator spacer and C-half-box 2A.
Mentions: CAT production was determined quantitatively for selected WWNR variants and, for comparison, some previously obtained C-dependent variants from the WWNW library (selected under lower C.PvuII expression levels; Figure 5G). The measurements were carried out under two conditions, chosen based on the in vivo titrations (Figure 2A). The first condition favors activation and maximum cat expression (0% arabinose; Figure 6A). The second condition favors repression (0.2% arabinose; Figure 6B). The results for all tested WWNW variants reveal similar levels of cat expression in low levels of C.PvuII (Figure 6A and B; white bars). When the C.PvuII level was increased by induction of its PBAD promoter, cat expression was significantly reduced in all cases.Figure 6.

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