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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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Genetic organization of restriction-modification system Eco29kI. The eco29kI genes are schematically shown by colored arrows, with arrow direction matching the direction of transcription. The DNA sequences of parts of the eco29kIR gene that include eco29kIR and AS promoters, and the eco29kIM promoter are expanded, respectively, above and below the eco29kI genes schematics (both DNA strands are shown). The initiating codons of both eco29kI genes are highlighted. The Res_P1, Res_P2 and Meth_P start sites are indicated by rightward arrows above the sequence. The AS_P1 and AS_P2 start sites are indicated by leftward arrows below the sequence. Experimentally defined −10-boxes are labeled and over- or underlined. Nucleotide positions substituted in engineered down-mutations discussed in the text are marked with ‘asterisks’.
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Figure 1: Genetic organization of restriction-modification system Eco29kI. The eco29kI genes are schematically shown by colored arrows, with arrow direction matching the direction of transcription. The DNA sequences of parts of the eco29kIR gene that include eco29kIR and AS promoters, and the eco29kIM promoter are expanded, respectively, above and below the eco29kI genes schematics (both DNA strands are shown). The initiating codons of both eco29kI genes are highlighted. The Res_P1, Res_P2 and Meth_P start sites are indicated by rightward arrows above the sequence. The AS_P1 and AS_P2 start sites are indicated by leftward arrows below the sequence. Experimentally defined −10-boxes are labeled and over- or underlined. Nucleotide positions substituted in engineered down-mutations discussed in the text are marked with ‘asterisks’.

Mentions: The p29 plasmid, which contains the entire Eco29kI R-M system, is stably maintained in E. coli cells and provides resistance to phages. Eco29kI promoters located upstream of the eco29kir gene were mapped by primer extension using total RNA prepared from cells harboring p29. Two primer extension products were observed (Figure 2A). Inspection of sequences upstream of primer extension end points revealed plausible promoter elements (Figure 1). In vitro transcription (Supplementary Figure S3) and KMnO4 probing (Supplementary Figure S1A) experiments confirmed that both primer extension products observed in vivo were produced from distinct E. coli RNAP σ70 holoenzyme promoters. We refer to upstream promoter as Res_P2. Approximately 70% of eco29kIR mRNA molecules are initiated from this promoter. The downstream promoter is referred to as Res_P1; it accounts for the remaining ∼30% of eco29kIR transcripts. Cells carrying a plasmid with transcriptional fusion of Eco29kI DNA upstream of the eco29kIR gene with promoterless galK contained high levels of GalK activity as judged by deep red color of cell colonies on McConkey plates (Figure 2B). This activity was unchanged when p29, a compatible plasmid carrying the entire Eco29kI R-M system, was introduced in the cells (Supplementary Figure S2). We therefore conclude that transcription from eco29kIR promoters is not affected by the presence of the Eco29kI gene products.Figure 1.


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)

Genetic organization of restriction-modification system Eco29kI. The eco29kI genes are schematically shown by colored arrows, with arrow direction matching the direction of transcription. The DNA sequences of parts of the eco29kIR gene that include eco29kIR and AS promoters, and the eco29kIM promoter are expanded, respectively, above and below the eco29kI genes schematics (both DNA strands are shown). The initiating codons of both eco29kI genes are highlighted. The Res_P1, Res_P2 and Meth_P start sites are indicated by rightward arrows above the sequence. The AS_P1 and AS_P2 start sites are indicated by leftward arrows below the sequence. Experimentally defined −10-boxes are labeled and over- or underlined. Nucleotide positions substituted in engineered down-mutations discussed in the text are marked with ‘asterisks’.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113576&req=5

Figure 1: Genetic organization of restriction-modification system Eco29kI. The eco29kI genes are schematically shown by colored arrows, with arrow direction matching the direction of transcription. The DNA sequences of parts of the eco29kIR gene that include eco29kIR and AS promoters, and the eco29kIM promoter are expanded, respectively, above and below the eco29kI genes schematics (both DNA strands are shown). The initiating codons of both eco29kI genes are highlighted. The Res_P1, Res_P2 and Meth_P start sites are indicated by rightward arrows above the sequence. The AS_P1 and AS_P2 start sites are indicated by leftward arrows below the sequence. Experimentally defined −10-boxes are labeled and over- or underlined. Nucleotide positions substituted in engineered down-mutations discussed in the text are marked with ‘asterisks’.
Mentions: The p29 plasmid, which contains the entire Eco29kI R-M system, is stably maintained in E. coli cells and provides resistance to phages. Eco29kI promoters located upstream of the eco29kir gene were mapped by primer extension using total RNA prepared from cells harboring p29. Two primer extension products were observed (Figure 2A). Inspection of sequences upstream of primer extension end points revealed plausible promoter elements (Figure 1). In vitro transcription (Supplementary Figure S3) and KMnO4 probing (Supplementary Figure S1A) experiments confirmed that both primer extension products observed in vivo were produced from distinct E. coli RNAP σ70 holoenzyme promoters. We refer to upstream promoter as Res_P2. Approximately 70% of eco29kIR mRNA molecules are initiated from this promoter. The downstream promoter is referred to as Res_P1; it accounts for the remaining ∼30% of eco29kIR transcripts. Cells carrying a plasmid with transcriptional fusion of Eco29kI DNA upstream of the eco29kIR gene with promoterless galK contained high levels of GalK activity as judged by deep red color of cell colonies on McConkey plates (Figure 2B). This activity was unchanged when p29, a compatible plasmid carrying the entire Eco29kI R-M system, was introduced in the cells (Supplementary Figure S2). We therefore conclude that transcription from eco29kIR promoters is not affected by the presence of the Eco29kI gene products.Figure 1.

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.

Show MeSH