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Transcription regulation of restriction-modification system Esp1396I.

Bogdanova E, Zakharova M, Streeter S, Taylor J, Heyduk T, Kneale G, Severinov K - Nucleic Acids Res. (2009)

Bottom Line: In contrast, a C-protein dimer binds to a single site at the M-promoter to repress the gene, with an affinity much greater than for the CR promoter.Mutational analysis of promoter binding sites reveals that the tetranucleotide inverted repeats long believed to be important for C-protein binding to DNA are less significant than previously thought.Instead, symmetry-related elements outside of these repeats appear to be critical for the interaction and are discussed in terms of the recent crystal structure of C.Esp139I bound to the CR promoter.

View Article: PubMed Central - PubMed

Affiliation: Waksman Institute for Microbiology, Department of Biochemistry and Molecular Biology, Rutgers, State University of New Jersey, Piscataway, NJ 08854, USA.

ABSTRACT
The convergently transcribed restriction (R) and methylase (M) genes of the Restriction-Modification system Esp1396I are tightly regulated by a controller (C) protein that forms part of the CR operon. We have mapped the transcriptional start sites from each promoter and examined the regulatory role of C.Esp1396I in vivo and in vitro. C-protein binding at the CR and M promoters was analyzed by DNA footprinting and a range of biophysical techniques. The distal and proximal C-protein binding sites at the CR promoter are responsible for activation and repression, respectively. In contrast, a C-protein dimer binds to a single site at the M-promoter to repress the gene, with an affinity much greater than for the CR promoter. Thus, during establishment of the system in a naïve host, the activity of the M promoter is turned off early, preventing excessive synthesis of methylase. Mutational analysis of promoter binding sites reveals that the tetranucleotide inverted repeats long believed to be important for C-protein binding to DNA are less significant than previously thought. Instead, symmetry-related elements outside of these repeats appear to be critical for the interaction and are discussed in terms of the recent crystal structure of C.Esp139I bound to the CR promoter.

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Expression of the esp1396I genes in vivo. (A) The horizontal lines on the left panel show overnight 37°C growth of E. coli cells harboring a plasmid containing the entire Esp1396I system of without such a plasmid on an LB agar plate. Cells were spotted with indicated dilutions of λ-vir phage lysate. Two panels on the right show the results of primer extension analysis carried out with RNA purified from cells harboring a plasmid containing the entire Esp1396I system with a primer specific for esp1396ICR transcription unit (middle panel) or with a esp1396IM-specific primer (right panel). Primer extension products are indicated by arrows. (B and C) Top panels shows the results of overnight 37°C growth of E. coli cells harboring esp1396ICR (B) or esp1396IM (C) promoters fused to promotorless galK in the absence (1) or presence (2) or a compatible plasmid expressing C.Esp1396I on McConkey agar plates. Bottom panels show the results of primer extension analysis of RNA purified from cells shown at the top with a galK specific primer.
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Figure 2: Expression of the esp1396I genes in vivo. (A) The horizontal lines on the left panel show overnight 37°C growth of E. coli cells harboring a plasmid containing the entire Esp1396I system of without such a plasmid on an LB agar plate. Cells were spotted with indicated dilutions of λ-vir phage lysate. Two panels on the right show the results of primer extension analysis carried out with RNA purified from cells harboring a plasmid containing the entire Esp1396I system with a primer specific for esp1396ICR transcription unit (middle panel) or with a esp1396IM-specific primer (right panel). Primer extension products are indicated by arrows. (B and C) Top panels shows the results of overnight 37°C growth of E. coli cells harboring esp1396ICR (B) or esp1396IM (C) promoters fused to promotorless galK in the absence (1) or presence (2) or a compatible plasmid expressing C.Esp1396I on McConkey agar plates. Bottom panels show the results of primer extension analysis of RNA purified from cells shown at the top with a galK specific primer.

Mentions: To better understand the regulation of Esp1396I, we identified transcription start points of Esp1396I genes by performing primer extension reactions with total RNA prepared from E. coli cells harboring a plasmid containing complete Esp1396I R–M system (8). This plasmid is stably maintained in E. coli and cells harboring this plasmid restrict the growth of phage λ (Figure 2A, left panel) indicating that both Esp1396I methyltransferase and restriction endonuclease are expressed. We wished to use RNA from cells harboring an Esp1396I R–M plasmid containing a lesion in the esp1396IC gene to reveal C.Esp1396I-dependent transcription start sites. However, no such plasmid could be constructed, indicating that intact esp1396IC is necessary for establishment and/or maintenance of esp1396I genes in the cell. Similar observations were made previously and were explained by postulating that expression of the esp1396IM gene in the absence of C.Esp1396I is toxic to the cell (8). Extension of primers annealing to various places within the esp1396ICR genes revealed a single primer extension product (Figure 2A, center panel and data not shown), supporting the notion that the esp1396ICR gene pair is transcribed from a single promoter (8). The primer extension product mapped to an adenine located 27-bp upstream of the initiating ATG codon of esp1396IC. Thus, unlike several other studied CR transcripts that were shown to be leaderless (7,17,28), the esp1396ICR transcript is translated in a conventional way (indeed, there is a recognizable Shine–Dalgarno sequence at an appropriate distance from the initiating ATG codon of esp1396I.C, Figure 1). The esp1396ICR transcription start site is preceded by a CATTAT sequence that is appropriately positioned to serve as a −10 promoter element (consensus sequence TATAAT). The place where the −35 promoter element (consensus sequence TTGACA) should be located contains a GTGTGA sequence. Thus, the similarity of the esp1396ICR promoter (Pesp1396ICR) elements to consensus elements is low, suggesting that basal activity level of this promoter is also low. Primer extension reactions with a primer designed to reveal transcription from the Esp1396I methylase promoter (Pesp1396IM) revealed a single product whose 5′-end corresponded to a thymidine located 20-bp upstream of the initiating ATG of esp1396IM (Figure 2A, right panel). This residue is preceded by an appropriately positioned TAGAAT sequence that is highly similar to the consensus −10 promoter element sequence TATAAT; at an appropriate distance upstream, a TTGAAA sequence that is similar to the −35 promoter consensus element sequence TTGACA is located (Figure 1).Figure 2.


Transcription regulation of restriction-modification system Esp1396I.

Bogdanova E, Zakharova M, Streeter S, Taylor J, Heyduk T, Kneale G, Severinov K - Nucleic Acids Res. (2009)

Expression of the esp1396I genes in vivo. (A) The horizontal lines on the left panel show overnight 37°C growth of E. coli cells harboring a plasmid containing the entire Esp1396I system of without such a plasmid on an LB agar plate. Cells were spotted with indicated dilutions of λ-vir phage lysate. Two panels on the right show the results of primer extension analysis carried out with RNA purified from cells harboring a plasmid containing the entire Esp1396I system with a primer specific for esp1396ICR transcription unit (middle panel) or with a esp1396IM-specific primer (right panel). Primer extension products are indicated by arrows. (B and C) Top panels shows the results of overnight 37°C growth of E. coli cells harboring esp1396ICR (B) or esp1396IM (C) promoters fused to promotorless galK in the absence (1) or presence (2) or a compatible plasmid expressing C.Esp1396I on McConkey agar plates. Bottom panels show the results of primer extension analysis of RNA purified from cells shown at the top with a galK specific primer.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: Expression of the esp1396I genes in vivo. (A) The horizontal lines on the left panel show overnight 37°C growth of E. coli cells harboring a plasmid containing the entire Esp1396I system of without such a plasmid on an LB agar plate. Cells were spotted with indicated dilutions of λ-vir phage lysate. Two panels on the right show the results of primer extension analysis carried out with RNA purified from cells harboring a plasmid containing the entire Esp1396I system with a primer specific for esp1396ICR transcription unit (middle panel) or with a esp1396IM-specific primer (right panel). Primer extension products are indicated by arrows. (B and C) Top panels shows the results of overnight 37°C growth of E. coli cells harboring esp1396ICR (B) or esp1396IM (C) promoters fused to promotorless galK in the absence (1) or presence (2) or a compatible plasmid expressing C.Esp1396I on McConkey agar plates. Bottom panels show the results of primer extension analysis of RNA purified from cells shown at the top with a galK specific primer.
Mentions: To better understand the regulation of Esp1396I, we identified transcription start points of Esp1396I genes by performing primer extension reactions with total RNA prepared from E. coli cells harboring a plasmid containing complete Esp1396I R–M system (8). This plasmid is stably maintained in E. coli and cells harboring this plasmid restrict the growth of phage λ (Figure 2A, left panel) indicating that both Esp1396I methyltransferase and restriction endonuclease are expressed. We wished to use RNA from cells harboring an Esp1396I R–M plasmid containing a lesion in the esp1396IC gene to reveal C.Esp1396I-dependent transcription start sites. However, no such plasmid could be constructed, indicating that intact esp1396IC is necessary for establishment and/or maintenance of esp1396I genes in the cell. Similar observations were made previously and were explained by postulating that expression of the esp1396IM gene in the absence of C.Esp1396I is toxic to the cell (8). Extension of primers annealing to various places within the esp1396ICR genes revealed a single primer extension product (Figure 2A, center panel and data not shown), supporting the notion that the esp1396ICR gene pair is transcribed from a single promoter (8). The primer extension product mapped to an adenine located 27-bp upstream of the initiating ATG codon of esp1396IC. Thus, unlike several other studied CR transcripts that were shown to be leaderless (7,17,28), the esp1396ICR transcript is translated in a conventional way (indeed, there is a recognizable Shine–Dalgarno sequence at an appropriate distance from the initiating ATG codon of esp1396I.C, Figure 1). The esp1396ICR transcription start site is preceded by a CATTAT sequence that is appropriately positioned to serve as a −10 promoter element (consensus sequence TATAAT). The place where the −35 promoter element (consensus sequence TTGACA) should be located contains a GTGTGA sequence. Thus, the similarity of the esp1396ICR promoter (Pesp1396ICR) elements to consensus elements is low, suggesting that basal activity level of this promoter is also low. Primer extension reactions with a primer designed to reveal transcription from the Esp1396I methylase promoter (Pesp1396IM) revealed a single product whose 5′-end corresponded to a thymidine located 20-bp upstream of the initiating ATG of esp1396IM (Figure 2A, right panel). This residue is preceded by an appropriately positioned TAGAAT sequence that is highly similar to the consensus −10 promoter element sequence TATAAT; at an appropriate distance upstream, a TTGAAA sequence that is similar to the −35 promoter consensus element sequence TTGACA is located (Figure 1).Figure 2.

Bottom Line: In contrast, a C-protein dimer binds to a single site at the M-promoter to repress the gene, with an affinity much greater than for the CR promoter.Mutational analysis of promoter binding sites reveals that the tetranucleotide inverted repeats long believed to be important for C-protein binding to DNA are less significant than previously thought.Instead, symmetry-related elements outside of these repeats appear to be critical for the interaction and are discussed in terms of the recent crystal structure of C.Esp139I bound to the CR promoter.

View Article: PubMed Central - PubMed

Affiliation: Waksman Institute for Microbiology, Department of Biochemistry and Molecular Biology, Rutgers, State University of New Jersey, Piscataway, NJ 08854, USA.

ABSTRACT
The convergently transcribed restriction (R) and methylase (M) genes of the Restriction-Modification system Esp1396I are tightly regulated by a controller (C) protein that forms part of the CR operon. We have mapped the transcriptional start sites from each promoter and examined the regulatory role of C.Esp1396I in vivo and in vitro. C-protein binding at the CR and M promoters was analyzed by DNA footprinting and a range of biophysical techniques. The distal and proximal C-protein binding sites at the CR promoter are responsible for activation and repression, respectively. In contrast, a C-protein dimer binds to a single site at the M-promoter to repress the gene, with an affinity much greater than for the CR promoter. Thus, during establishment of the system in a naïve host, the activity of the M promoter is turned off early, preventing excessive synthesis of methylase. Mutational analysis of promoter binding sites reveals that the tetranucleotide inverted repeats long believed to be important for C-protein binding to DNA are less significant than previously thought. Instead, symmetry-related elements outside of these repeats appear to be critical for the interaction and are discussed in terms of the recent crystal structure of C.Esp139I bound to the CR promoter.

Show MeSH
Related in: MedlinePlus