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Regulation of gene expression in restriction-modification system Eco29kI.

Nagornykh M, Zakharova M, Protsenko A, Bogdanova E, Solonin AS, Severinov K - Nucleic Acids Res. (2011)

Bottom Line: We further show that transcription from two intragenic antisense promoters strongly decreases the levels of eco29kIR gene transcripts.The antisense transcripts act by preventing translation initiation from the bicistronic eco29kIR-eco29kIM mRNA and causing its degradation.Both eco29kIM and antisense promoters are necessary for Eco29kI genes establishment and/or stable maintenance, indicating that they jointly contribute to coordinated expression of Eco29kI genes.

View Article: PubMed Central - PubMed

Affiliation: Waksman Institute of Microbiology, 190 Frelinghuysen Road, Piscataway, NJ 08854, USA.

ABSTRACT
The Eco29kI restriction-modification (R-M) system consists of two partially overlapping genes, eco29kIR, encoding a restriction endonuclease and eco29kIM, encoding methyltransferase. The two genes are thought to form an operon with the eco29kIR gene preceding the eco29kIM gene. Such an organization is expected to complicate establishment of plasmids containing this R-M system in naive hosts, since common logic dictates that methyltransferase should be synthesized first to protect the DNA from cleavage by the endonuclease. Here, we characterize the Eco29kI gene transcription. We show that a separate promoter located within the eco29kIR gene is sufficient to synthesize enough methyltransferase to completely modify host DNA. We further show that transcription from two intragenic antisense promoters strongly decreases the levels of eco29kIR gene transcripts. The antisense transcripts act by preventing translation initiation from the bicistronic eco29kIR-eco29kIM mRNA and causing its degradation. Both eco29kIM and antisense promoters are necessary for Eco29kI genes establishment and/or stable maintenance, indicating that they jointly contribute to coordinated expression of Eco29kI genes.

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Expression of eco29kIR is negatively affected by the activity of antisense-promoters. (A) β-Galactosidase activity in cells harboring plasmids with translational fusions of the lacZ reporter with the following fragments of eco29kIR or their mutational derivatives: pR-lacZ—a DNA fragment (from −44 to +168) of Eco29kI system fused to lacZ; pRmut1—pR-lacZ with inactive Res_P1 promoter; pRmut2—pR-lacZ with inactive Res_P2 promoter; pRAS-lacZ—a DNA fragment (from −44 to +282) of Eco29kI system fused to lacZ; pRASM-lacZ—a DNA fragment (from −44 to +464) of Eco29kI system fused to lacZ; pRASmut3-lacZ—pRAS-lacZ with inactive AS promoters; pM-lacZ—a DNA fragment (from +364 to +806) of Eco29kI system fused to lacZ. (B) Relative lacZ transcript abundance measured with qRT–PCR. The amount of cat transcript revealed with appropriate primers was used to normalize lacZ transcript abundance in each reaction mix.
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Figure 6: Expression of eco29kIR is negatively affected by the activity of antisense-promoters. (A) β-Galactosidase activity in cells harboring plasmids with translational fusions of the lacZ reporter with the following fragments of eco29kIR or their mutational derivatives: pR-lacZ—a DNA fragment (from −44 to +168) of Eco29kI system fused to lacZ; pRmut1—pR-lacZ with inactive Res_P1 promoter; pRmut2—pR-lacZ with inactive Res_P2 promoter; pRAS-lacZ—a DNA fragment (from −44 to +282) of Eco29kI system fused to lacZ; pRASM-lacZ—a DNA fragment (from −44 to +464) of Eco29kI system fused to lacZ; pRASmut3-lacZ—pRAS-lacZ with inactive AS promoters; pM-lacZ—a DNA fragment (from +364 to +806) of Eco29kI system fused to lacZ. (B) Relative lacZ transcript abundance measured with qRT–PCR. The amount of cat transcript revealed with appropriate primers was used to normalize lacZ transcript abundance in each reaction mix.

Mentions: A series of translational fusions of eco29kIR promoters to the lacZ gene with intervening Eco29kI DNA of various lengths was prepared. The resulting plasmids were introduced in lacZ E. coli cells and the levels of LacZ activity and the amounts of lacZ RNA were determined. The results are summarized in Figure 6. As can be seen, inclusion of Eco29kI DNA fragments containing AS promoters had a strong negative effect on LacZ activity levels (Figure 6A) and the steady-state levels of lacZ transcripts (Figure 6B) synthesized from eco29kIR promoters. The inclusion of additional Eco29kI sequences between eco29kIR promoters and lacZ—up to and downstream of Met_P—had no further negative effect on LacZ activity or lacZ RNA abundance. AS promoters activity was responsible for decrease in LacZ synthesis and steady-state levels of eco29kIR promoter-initiated transcripts since inactivation of both AS promoters increased the levels of LacZ activity and lacZ transcripts to those seen in cells that contained plasmids with the shortest eco29kIR::lacZ fusion from which both AS promoters were absent (note that down mutations in AS promoters did not affect the sequence of the R.Eco29kI-LacZ fusion protein). Analysis of cells harboring plasmids with individually inactivated AS promoters indicated that the more active AS_P2 was primarily responsible for inhibition of lacZ expression from eco29kIR promoters (data not shown).Figure 6.


Regulation of gene expression in restriction-modification system Eco29kI.

Nagornykh M, Zakharova M, Protsenko A, Bogdanova E, Solonin AS, Severinov K - Nucleic Acids Res. (2011)

Expression of eco29kIR is negatively affected by the activity of antisense-promoters. (A) β-Galactosidase activity in cells harboring plasmids with translational fusions of the lacZ reporter with the following fragments of eco29kIR or their mutational derivatives: pR-lacZ—a DNA fragment (from −44 to +168) of Eco29kI system fused to lacZ; pRmut1—pR-lacZ with inactive Res_P1 promoter; pRmut2—pR-lacZ with inactive Res_P2 promoter; pRAS-lacZ—a DNA fragment (from −44 to +282) of Eco29kI system fused to lacZ; pRASM-lacZ—a DNA fragment (from −44 to +464) of Eco29kI system fused to lacZ; pRASmut3-lacZ—pRAS-lacZ with inactive AS promoters; pM-lacZ—a DNA fragment (from +364 to +806) of Eco29kI system fused to lacZ. (B) Relative lacZ transcript abundance measured with qRT–PCR. The amount of cat transcript revealed with appropriate primers was used to normalize lacZ transcript abundance in each reaction mix.
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Figure 6: Expression of eco29kIR is negatively affected by the activity of antisense-promoters. (A) β-Galactosidase activity in cells harboring plasmids with translational fusions of the lacZ reporter with the following fragments of eco29kIR or their mutational derivatives: pR-lacZ—a DNA fragment (from −44 to +168) of Eco29kI system fused to lacZ; pRmut1—pR-lacZ with inactive Res_P1 promoter; pRmut2—pR-lacZ with inactive Res_P2 promoter; pRAS-lacZ—a DNA fragment (from −44 to +282) of Eco29kI system fused to lacZ; pRASM-lacZ—a DNA fragment (from −44 to +464) of Eco29kI system fused to lacZ; pRASmut3-lacZ—pRAS-lacZ with inactive AS promoters; pM-lacZ—a DNA fragment (from +364 to +806) of Eco29kI system fused to lacZ. (B) Relative lacZ transcript abundance measured with qRT–PCR. The amount of cat transcript revealed with appropriate primers was used to normalize lacZ transcript abundance in each reaction mix.
Mentions: A series of translational fusions of eco29kIR promoters to the lacZ gene with intervening Eco29kI DNA of various lengths was prepared. The resulting plasmids were introduced in lacZ E. coli cells and the levels of LacZ activity and the amounts of lacZ RNA were determined. The results are summarized in Figure 6. As can be seen, inclusion of Eco29kI DNA fragments containing AS promoters had a strong negative effect on LacZ activity levels (Figure 6A) and the steady-state levels of lacZ transcripts (Figure 6B) synthesized from eco29kIR promoters. The inclusion of additional Eco29kI sequences between eco29kIR promoters and lacZ—up to and downstream of Met_P—had no further negative effect on LacZ activity or lacZ RNA abundance. AS promoters activity was responsible for decrease in LacZ synthesis and steady-state levels of eco29kIR promoter-initiated transcripts since inactivation of both AS promoters increased the levels of LacZ activity and lacZ transcripts to those seen in cells that contained plasmids with the shortest eco29kIR::lacZ fusion from which both AS promoters were absent (note that down mutations in AS promoters did not affect the sequence of the R.Eco29kI-LacZ fusion protein). Analysis of cells harboring plasmids with individually inactivated AS promoters indicated that the more active AS_P2 was primarily responsible for inhibition of lacZ expression from eco29kIR promoters (data not shown).Figure 6.

Bottom Line: We further show that transcription from two intragenic antisense promoters strongly decreases the levels of eco29kIR gene transcripts.The antisense transcripts act by preventing translation initiation from the bicistronic eco29kIR-eco29kIM mRNA and causing its degradation.Both eco29kIM and antisense promoters are necessary for Eco29kI genes establishment and/or stable maintenance, indicating that they jointly contribute to coordinated expression of Eco29kI genes.

View Article: PubMed Central - PubMed

Affiliation: Waksman Institute of Microbiology, 190 Frelinghuysen Road, Piscataway, NJ 08854, USA.

ABSTRACT
The Eco29kI restriction-modification (R-M) system consists of two partially overlapping genes, eco29kIR, encoding a restriction endonuclease and eco29kIM, encoding methyltransferase. The two genes are thought to form an operon with the eco29kIR gene preceding the eco29kIM gene. Such an organization is expected to complicate establishment of plasmids containing this R-M system in naive hosts, since common logic dictates that methyltransferase should be synthesized first to protect the DNA from cleavage by the endonuclease. Here, we characterize the Eco29kI gene transcription. We show that a separate promoter located within the eco29kIR gene is sufficient to synthesize enough methyltransferase to completely modify host DNA. We further show that transcription from two intragenic antisense promoters strongly decreases the levels of eco29kIR gene transcripts. The antisense transcripts act by preventing translation initiation from the bicistronic eco29kIR-eco29kIM mRNA and causing its degradation. Both eco29kIM and antisense promoters are necessary for Eco29kI genes establishment and/or stable maintenance, indicating that they jointly contribute to coordinated expression of Eco29kI genes.

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