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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 operator spacer library and selected variants. (A) Sequencing trace of the pooled randomized spacer plasmid library before selection. The randomized spacer lies between C-half-boxes 1B and 2A. The promoter region library is upstream of a promoterless cat gene (chloramphenicol acetyltransferase). (B) The plasmid library was used to transform an E. coli strain that already carried a compatible plasmid producing C.PvuII at physiological levels, and transformants were plated onto agar with a high Cml concentration (120 µg/ml) to select functional C.PvuII-activated variants, followed by a screen for reduced resistance in the absence of C.PvuII. The sequencing result is shown, along with the WT sequence for comparison. (C) Non-activated variants, that grew at a low Cml concentration (20 µg/ml) but not at 120 µg/ml. Sequences are shown, with matches to the WT sequence in bold, and alterations in outline. (D) Logo analysis of the Cml-sensitive variants shown in (C). (E) Promoter activity for selected variants in the presence of physiological steady-state levels of C.PvuII (pvuIIC under native control from plasmid pDK200). CAT levels were determined in triplicate via immunoassay as described in Material and Methods section. Error bars indicate the SD. CAT levels are expressed as nanograms of protein per microgram of total protein and then normalized to the WT PpvuIICR-cat level (TGTA). (F) EMSA performed for WT (TGTA) and three representative variants. The experiments were carried out as in Figure 3B. The stars and Roman numerals denote positions of unbound DNA and C.PvuII–DNA complexes, respectively.
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Figure 4: Randomized operator spacer library and selected variants. (A) Sequencing trace of the pooled randomized spacer plasmid library before selection. The randomized spacer lies between C-half-boxes 1B and 2A. The promoter region library is upstream of a promoterless cat gene (chloramphenicol acetyltransferase). (B) The plasmid library was used to transform an E. coli strain that already carried a compatible plasmid producing C.PvuII at physiological levels, and transformants were plated onto agar with a high Cml concentration (120 µg/ml) to select functional C.PvuII-activated variants, followed by a screen for reduced resistance in the absence of C.PvuII. The sequencing result is shown, along with the WT sequence for comparison. (C) Non-activated variants, that grew at a low Cml concentration (20 µg/ml) but not at 120 µg/ml. Sequences are shown, with matches to the WT sequence in bold, and alterations in outline. (D) Logo analysis of the Cml-sensitive variants shown in (C). (E) Promoter activity for selected variants in the presence of physiological steady-state levels of C.PvuII (pvuIIC under native control from plasmid pDK200). CAT levels were determined in triplicate via immunoassay as described in Material and Methods section. Error bars indicate the SD. CAT levels are expressed as nanograms of protein per microgram of total protein and then normalized to the WT PpvuIICR-cat level (TGTA). (F) EMSA performed for WT (TGTA) and three representative variants. The experiments were carried out as in Figure 3B. The stars and Roman numerals denote positions of unbound DNA and C.PvuII–DNA complexes, respectively.

Mentions: To test the role of the operator spacer, we generated a plasmid library in which the 4 nt spacer was replaced with a randomized sequence, and the promoter region including this modification was cloned in front of a promotorless cat (chloramphenicol acetyltransferase) reporter gene (details in Materials and Methods section, and in Table S1 and Figure S1). Electroporation into E. coli produced about 104 transformants, giving a theoretical ∼40-fold coverage of the 256-possibility library. The pooled library was purified and sequence confirmed, consistent with randomization where expected and with no other changes (Figure 4A). This confirmed library was then electroporated into E. coli cells containing pDK200, a compatible plasmid that supplies intact pvuIIC in trans. The objective was to determine which spacer sequences conferred high-level resistance to Cml, due to increased cat expression, in the presence, but not the absence of C.PvuII.Figure 4.


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 operator spacer library and selected variants. (A) Sequencing trace of the pooled randomized spacer plasmid library before selection. The randomized spacer lies between C-half-boxes 1B and 2A. The promoter region library is upstream of a promoterless cat gene (chloramphenicol acetyltransferase). (B) The plasmid library was used to transform an E. coli strain that already carried a compatible plasmid producing C.PvuII at physiological levels, and transformants were plated onto agar with a high Cml concentration (120 µg/ml) to select functional C.PvuII-activated variants, followed by a screen for reduced resistance in the absence of C.PvuII. The sequencing result is shown, along with the WT sequence for comparison. (C) Non-activated variants, that grew at a low Cml concentration (20 µg/ml) but not at 120 µg/ml. Sequences are shown, with matches to the WT sequence in bold, and alterations in outline. (D) Logo analysis of the Cml-sensitive variants shown in (C). (E) Promoter activity for selected variants in the presence of physiological steady-state levels of C.PvuII (pvuIIC under native control from plasmid pDK200). CAT levels were determined in triplicate via immunoassay as described in Material and Methods section. Error bars indicate the SD. CAT levels are expressed as nanograms of protein per microgram of total protein and then normalized to the WT PpvuIICR-cat level (TGTA). (F) EMSA performed for WT (TGTA) and three representative variants. The experiments were carried out as in Figure 3B. The stars and Roman numerals denote positions of unbound DNA and C.PvuII–DNA complexes, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2175313&req=5

Figure 4: Randomized operator spacer library and selected variants. (A) Sequencing trace of the pooled randomized spacer plasmid library before selection. The randomized spacer lies between C-half-boxes 1B and 2A. The promoter region library is upstream of a promoterless cat gene (chloramphenicol acetyltransferase). (B) The plasmid library was used to transform an E. coli strain that already carried a compatible plasmid producing C.PvuII at physiological levels, and transformants were plated onto agar with a high Cml concentration (120 µg/ml) to select functional C.PvuII-activated variants, followed by a screen for reduced resistance in the absence of C.PvuII. The sequencing result is shown, along with the WT sequence for comparison. (C) Non-activated variants, that grew at a low Cml concentration (20 µg/ml) but not at 120 µg/ml. Sequences are shown, with matches to the WT sequence in bold, and alterations in outline. (D) Logo analysis of the Cml-sensitive variants shown in (C). (E) Promoter activity for selected variants in the presence of physiological steady-state levels of C.PvuII (pvuIIC under native control from plasmid pDK200). CAT levels were determined in triplicate via immunoassay as described in Material and Methods section. Error bars indicate the SD. CAT levels are expressed as nanograms of protein per microgram of total protein and then normalized to the WT PpvuIICR-cat level (TGTA). (F) EMSA performed for WT (TGTA) and three representative variants. The experiments were carried out as in Figure 3B. The stars and Roman numerals denote positions of unbound DNA and C.PvuII–DNA complexes, respectively.
Mentions: To test the role of the operator spacer, we generated a plasmid library in which the 4 nt spacer was replaced with a randomized sequence, and the promoter region including this modification was cloned in front of a promotorless cat (chloramphenicol acetyltransferase) reporter gene (details in Materials and Methods section, and in Table S1 and Figure S1). Electroporation into E. coli produced about 104 transformants, giving a theoretical ∼40-fold coverage of the 256-possibility library. The pooled library was purified and sequence confirmed, consistent with randomization where expected and with no other changes (Figure 4A). This confirmed library was then electroporated into E. coli cells containing pDK200, a compatible plasmid that supplies intact pvuIIC in trans. The objective was to determine which spacer sequences conferred high-level resistance to Cml, due to increased cat expression, in the presence, but not the absence of C.PvuII.Figure 4.

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