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Transcription regulation of the type II restriction-modification system AhdI.

Bogdanova E, Djordjevic M, Papapanagiotou I, Heyduk T, Kneale G, Severinov K - Nucleic Acids Res. (2008)

Bottom Line: We show that AhdI transcription is controlled by two independent regulatory loops that are well-optimized to ensure successful establishment in a naïve bacterial host.We develop a mathematical model that is in quantitative agreement with the experiment and indicates that RNA polymerase outcompetes C protein from the promoter-proximal-binding site.Such an unusual mechanism leads to a very inefficient activation of the R gene transcription, which presumably helps control the level of the endonuclease in the cell.

View Article: PubMed Central - PubMed

Affiliation: Waksman Institute, Piscataway, NJ 08854, USA.

ABSTRACT
The Restriction-modification system AhdI contains two convergent transcription units, one with genes encoding methyltransferase subunits M and S and another with genes encoding the controller (C) protein and the restriction endonuclease (R). We show that AhdI transcription is controlled by two independent regulatory loops that are well-optimized to ensure successful establishment in a naïve bacterial host. Transcription from the strong MS promoter is attenuated by methylation of an AhdI site overlapping the -10 element of the promoter. Transcription from the weak CR promoter is regulated by the C protein interaction with two DNA-binding sites. The interaction with the promoter-distal high-affinity site activates transcription, while interaction with the weaker promoter-proximal site represses it. Because of high levels of cooperativity, both C protein-binding sites are always occupied in the absence of RNA polymerase, raising a question how activated transcription is achieved. We develop a mathematical model that is in quantitative agreement with the experiment and indicates that RNA polymerase outcompetes C protein from the promoter-proximal-binding site. Such an unusual mechanism leads to a very inefficient activation of the R gene transcription, which presumably helps control the level of the endonuclease in the cell.

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Regulation the PahdIMS activity in vitro and in vivo. (A) A single-round transcription in vitro by E.coli RNAP σ70 holoenzyme (100 nM) from regulatory region DNA fragment (13 nM) containing methylated and unmethylated promoter region were performed and products were separated by gel electrophoreses. Autoradiographs of 8% denaturing polyacrylamide gels are shown. (B) Effect of methylation on the expression of ahdIM gene. Plasmid pPM containing transcriptional fusion PahdIM’::’lacZ were co-transformed with the pAhdIMRinC plasmid (only methylase gene is active), bacterial cultures were grown until OD600 0.5 and β-galactosidase activity was measured in three independent sets of experiments.
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Figure 8: Regulation the PahdIMS activity in vitro and in vivo. (A) A single-round transcription in vitro by E.coli RNAP σ70 holoenzyme (100 nM) from regulatory region DNA fragment (13 nM) containing methylated and unmethylated promoter region were performed and products were separated by gel electrophoreses. Autoradiographs of 8% denaturing polyacrylamide gels are shown. (B) Effect of methylation on the expression of ahdIM gene. Plasmid pPM containing transcriptional fusion PahdIM’::’lacZ were co-transformed with the pAhdIMRinC plasmid (only methylase gene is active), bacterial cultures were grown until OD600 0.5 and β-galactosidase activity was measured in three independent sets of experiments.

Mentions: Inspection of the PahdIMS sequence revealed the presence of an AhdI site that partially overlaps with the −10 element of the promoter (Figure 1). The subunit composition and the sequence of the AhdI methyltransferase are highly similar to enzymes from Type I R-M systems, which invariably methylate the sixth position of an adenine in the target sequence (30,31). Based on this similarity, it is highly likely that the AhdI methylase also methylates an adenine. In the case of the AhdI site embedded in PahdIMS, a non-template strand A between the extended −10 TG motif and the CATACT −10 element and the template strand A two bases downstream of the −10 element should be methylated (Figure 1). To determine whether methylation of the AhdI site affects transcription from PahdIMS, two PahdIMS-containing fragments, identical except for the methylation state of the AhdI site, were prepared and used in in vitro transcription (Figure 8A). The results of multiple experiments consistently showed that the methylated template was utilized slightly less (∼50%) efficiently than the unmodified template. The effect was highly reproducible with different preparations of PahdIMS DNA. In vitro transcription from both fragments was independent of the presence of C.AhdI and initiated from the same start site as that determined in vivo (data not shown).Figure 8.


Transcription regulation of the type II restriction-modification system AhdI.

Bogdanova E, Djordjevic M, Papapanagiotou I, Heyduk T, Kneale G, Severinov K - Nucleic Acids Res. (2008)

Regulation the PahdIMS activity in vitro and in vivo. (A) A single-round transcription in vitro by E.coli RNAP σ70 holoenzyme (100 nM) from regulatory region DNA fragment (13 nM) containing methylated and unmethylated promoter region were performed and products were separated by gel electrophoreses. Autoradiographs of 8% denaturing polyacrylamide gels are shown. (B) Effect of methylation on the expression of ahdIM gene. Plasmid pPM containing transcriptional fusion PahdIM’::’lacZ were co-transformed with the pAhdIMRinC plasmid (only methylase gene is active), bacterial cultures were grown until OD600 0.5 and β-galactosidase activity was measured in three independent sets of experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 8: Regulation the PahdIMS activity in vitro and in vivo. (A) A single-round transcription in vitro by E.coli RNAP σ70 holoenzyme (100 nM) from regulatory region DNA fragment (13 nM) containing methylated and unmethylated promoter region were performed and products were separated by gel electrophoreses. Autoradiographs of 8% denaturing polyacrylamide gels are shown. (B) Effect of methylation on the expression of ahdIM gene. Plasmid pPM containing transcriptional fusion PahdIM’::’lacZ were co-transformed with the pAhdIMRinC plasmid (only methylase gene is active), bacterial cultures were grown until OD600 0.5 and β-galactosidase activity was measured in three independent sets of experiments.
Mentions: Inspection of the PahdIMS sequence revealed the presence of an AhdI site that partially overlaps with the −10 element of the promoter (Figure 1). The subunit composition and the sequence of the AhdI methyltransferase are highly similar to enzymes from Type I R-M systems, which invariably methylate the sixth position of an adenine in the target sequence (30,31). Based on this similarity, it is highly likely that the AhdI methylase also methylates an adenine. In the case of the AhdI site embedded in PahdIMS, a non-template strand A between the extended −10 TG motif and the CATACT −10 element and the template strand A two bases downstream of the −10 element should be methylated (Figure 1). To determine whether methylation of the AhdI site affects transcription from PahdIMS, two PahdIMS-containing fragments, identical except for the methylation state of the AhdI site, were prepared and used in in vitro transcription (Figure 8A). The results of multiple experiments consistently showed that the methylated template was utilized slightly less (∼50%) efficiently than the unmodified template. The effect was highly reproducible with different preparations of PahdIMS DNA. In vitro transcription from both fragments was independent of the presence of C.AhdI and initiated from the same start site as that determined in vivo (data not shown).Figure 8.

Bottom Line: We show that AhdI transcription is controlled by two independent regulatory loops that are well-optimized to ensure successful establishment in a naïve bacterial host.We develop a mathematical model that is in quantitative agreement with the experiment and indicates that RNA polymerase outcompetes C protein from the promoter-proximal-binding site.Such an unusual mechanism leads to a very inefficient activation of the R gene transcription, which presumably helps control the level of the endonuclease in the cell.

View Article: PubMed Central - PubMed

Affiliation: Waksman Institute, Piscataway, NJ 08854, USA.

ABSTRACT
The Restriction-modification system AhdI contains two convergent transcription units, one with genes encoding methyltransferase subunits M and S and another with genes encoding the controller (C) protein and the restriction endonuclease (R). We show that AhdI transcription is controlled by two independent regulatory loops that are well-optimized to ensure successful establishment in a naïve bacterial host. Transcription from the strong MS promoter is attenuated by methylation of an AhdI site overlapping the -10 element of the promoter. Transcription from the weak CR promoter is regulated by the C protein interaction with two DNA-binding sites. The interaction with the promoter-distal high-affinity site activates transcription, while interaction with the weaker promoter-proximal site represses it. Because of high levels of cooperativity, both C protein-binding sites are always occupied in the absence of RNA polymerase, raising a question how activated transcription is achieved. We develop a mathematical model that is in quantitative agreement with the experiment and indicates that RNA polymerase outcompetes C protein from the promoter-proximal-binding site. Such an unusual mechanism leads to a very inefficient activation of the R gene transcription, which presumably helps control the level of the endonuclease in the cell.

Show MeSH
Related in: MedlinePlus