Limits...
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.

Show MeSH

Related in: MedlinePlus

Interaction of C. Esp1396I with esp1396I binding sites measured by fluorescence polarization. (A) DNA binding of C.Esp1396I analyzed by fluorescence polarization of fluorescein-labeled Pesp1396ICR (left) and Pesp1396IM (right) DNA fragments. Solid lines show best fits of the models to experimental data. The best fit parameters for Pesp1936ICR were Kp = 322 nM, Kd = 6.9 nM and cooperativity 219. The best fit equilibrium constant for Pesp1936IM DNA was K = 0.3 nM. (B) Left panel: binding of C.Esp1396I to labeled Pesp1396ICR DNA in the absence (gray symbols) and presence (black symbols) of unlabeled Pesp1396IM DNA. Right panel: binding of C.Esp1396I to labeled Pesp1396IM DNA in the absence (gray symbols) and presence (black symbols) of unlabeled Pesp1396ICR DNA. Both labeled and competing unlabeled DNA (when present) were at 50 nM. Dashed lines depict predicted simulated binding using equilibrium constants derived from the analysis of the data illustrated in (A).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2691842&req=5

Figure 6: Interaction of C. Esp1396I with esp1396I binding sites measured by fluorescence polarization. (A) DNA binding of C.Esp1396I analyzed by fluorescence polarization of fluorescein-labeled Pesp1396ICR (left) and Pesp1396IM (right) DNA fragments. Solid lines show best fits of the models to experimental data. The best fit parameters for Pesp1936ICR were Kp = 322 nM, Kd = 6.9 nM and cooperativity 219. The best fit equilibrium constant for Pesp1936IM DNA was K = 0.3 nM. (B) Left panel: binding of C.Esp1396I to labeled Pesp1396ICR DNA in the absence (gray symbols) and presence (black symbols) of unlabeled Pesp1396IM DNA. Right panel: binding of C.Esp1396I to labeled Pesp1396IM DNA in the absence (gray symbols) and presence (black symbols) of unlabeled Pesp1396ICR DNA. Both labeled and competing unlabeled DNA (when present) were at 50 nM. Dashed lines depict predicted simulated binding using equilibrium constants derived from the analysis of the data illustrated in (A).

Mentions: To derive equilibrium binding constants of C.Esp1396I interactions with its binding sites, we performed solution-based fluorescence polarization assays. Fluorescence polarization does not involve steps that could potentially perturb the binding equilibria being measured (such as physical separation of free and bound species) and therefore allows true equilibrium determinations of macromolecular interactions (29,30). Promoter fragments were labeled at the 5′-end with fluorescein and fluorescence polarization of DNA fragments was measured as a function of C.Esp1396I concentration (Figure 6A). Equilibrium constants describing the binding of C.Esp1396I were obtained by nonlinear regression analysis and fitting the data to a model involving binding of a single dimer of C.Esp1396I to Pesp1396IM-containing fragment and cooperative binding of two C.Esp1396I dimers to Pesp1396ICR-containing fragment. While satisfactory fits of polarization data for both Pesp1396IM and Pesp1396ICR sites could be obtained using either of these models, only the specific models described above were considered since they were consistent with data described above. Also, the models were consistent with higher total fluorescence polarization changes observed with Pesp1396ICR DNA indicating larger complexes formed with this DNA (Figure 6A, compare panels on the left and on the right).Figure 6.


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)

Interaction of C. Esp1396I with esp1396I binding sites measured by fluorescence polarization. (A) DNA binding of C.Esp1396I analyzed by fluorescence polarization of fluorescein-labeled Pesp1396ICR (left) and Pesp1396IM (right) DNA fragments. Solid lines show best fits of the models to experimental data. The best fit parameters for Pesp1936ICR were Kp = 322 nM, Kd = 6.9 nM and cooperativity 219. The best fit equilibrium constant for Pesp1936IM DNA was K = 0.3 nM. (B) Left panel: binding of C.Esp1396I to labeled Pesp1396ICR DNA in the absence (gray symbols) and presence (black symbols) of unlabeled Pesp1396IM DNA. Right panel: binding of C.Esp1396I to labeled Pesp1396IM DNA in the absence (gray symbols) and presence (black symbols) of unlabeled Pesp1396ICR DNA. Both labeled and competing unlabeled DNA (when present) were at 50 nM. Dashed lines depict predicted simulated binding using equilibrium constants derived from the analysis of the data illustrated in (A).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2691842&req=5

Figure 6: Interaction of C. Esp1396I with esp1396I binding sites measured by fluorescence polarization. (A) DNA binding of C.Esp1396I analyzed by fluorescence polarization of fluorescein-labeled Pesp1396ICR (left) and Pesp1396IM (right) DNA fragments. Solid lines show best fits of the models to experimental data. The best fit parameters for Pesp1936ICR were Kp = 322 nM, Kd = 6.9 nM and cooperativity 219. The best fit equilibrium constant for Pesp1936IM DNA was K = 0.3 nM. (B) Left panel: binding of C.Esp1396I to labeled Pesp1396ICR DNA in the absence (gray symbols) and presence (black symbols) of unlabeled Pesp1396IM DNA. Right panel: binding of C.Esp1396I to labeled Pesp1396IM DNA in the absence (gray symbols) and presence (black symbols) of unlabeled Pesp1396ICR DNA. Both labeled and competing unlabeled DNA (when present) were at 50 nM. Dashed lines depict predicted simulated binding using equilibrium constants derived from the analysis of the data illustrated in (A).
Mentions: To derive equilibrium binding constants of C.Esp1396I interactions with its binding sites, we performed solution-based fluorescence polarization assays. Fluorescence polarization does not involve steps that could potentially perturb the binding equilibria being measured (such as physical separation of free and bound species) and therefore allows true equilibrium determinations of macromolecular interactions (29,30). Promoter fragments were labeled at the 5′-end with fluorescein and fluorescence polarization of DNA fragments was measured as a function of C.Esp1396I concentration (Figure 6A). Equilibrium constants describing the binding of C.Esp1396I were obtained by nonlinear regression analysis and fitting the data to a model involving binding of a single dimer of C.Esp1396I to Pesp1396IM-containing fragment and cooperative binding of two C.Esp1396I dimers to Pesp1396ICR-containing fragment. While satisfactory fits of polarization data for both Pesp1396IM and Pesp1396ICR sites could be obtained using either of these models, only the specific models described above were considered since they were consistent with data described above. Also, the models were consistent with higher total fluorescence polarization changes observed with Pesp1396ICR DNA indicating larger complexes formed with this DNA (Figure 6A, compare panels on the left and on the right).Figure 6.

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