Transcription regulation of the type II restriction-modification system AhdI.
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.
Affiliation: Waksman Institute, Piscataway, NJ 08854, USA.
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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Mentions: Quantification of the amount of PahdICR transcripts produced in this single-round in vitro transcription experiments at optimal C.AhdI concentrations indicated that less than 2.5% of templates were transcriptionally active (data not shown), even though the amount of RNAP used in the experiment exceeded the amount of promoter DNA several-fold and so RNAP was not limiting. Indeed, DNase I footprinting of reactions containing both C.AhdI and RNAP holoenzyme revealed no extra protection compared to reactions containing C.AhdI only (Figure 6A, compare lanes 2 and 4). The only difference was the appearance of two DNase I sensitive bands at the downstream boundary of promoter-proximal C.AhdI-binding site. These bands, located at positions −25/−26 with respect to transcription start point likely correspond to DNase I hypersensitive bands commonly seen in open promoter complexes footprints. However, since no protection around the −10 promoter element and the transcription start site is seen in the presence of C.AhdI and RNAP, the amount of open complexes must be very low. On the other hand, the results of KMnO4 probing, which reports the presence of unpaired thymines in transcription-competent open promoter complex, revealed that thymines in the −10 element of PahdICR reacted poorly with KMnO4 in the presence of RNAP alone (Figure 6B, lane 3), but became strongly reactive when both RNAP and C.AhdI were present together (Figure 6B, lane 4).Figure 6.