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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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Analysis of C.Esp1396I complexes with wild-type and mutant esp1396I promoters using electrophoretic mobility shift assay. (A) The sequences of the wild-type esp1396ICR C-boxes with symmetrical elements highlighted is presented at the top of the figure. Numbers below refer to positions of nucleotides in the C-box. The gels below show the results of native gel separation of complexes formed in the absence or in the presence of increasing concentrations of C.Esp1396I using wild-type esp1396ICR and various mutants. Note on nomenclature, every mutant fragment (‘mut’) is labeled to indicate positions of the boxes substituted (i.e. 1,2 in the second gel) and the bases introduced by the mutations (GT in the second gel). (B) As in (A), but using wild-type or mutant esp1396IM DNA fragments.
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Figure 7: Analysis of C.Esp1396I complexes with wild-type and mutant esp1396I promoters using electrophoretic mobility shift assay. (A) The sequences of the wild-type esp1396ICR C-boxes with symmetrical elements highlighted is presented at the top of the figure. Numbers below refer to positions of nucleotides in the C-box. The gels below show the results of native gel separation of complexes formed in the absence or in the presence of increasing concentrations of C.Esp1396I using wild-type esp1396ICR and various mutants. Note on nomenclature, every mutant fragment (‘mut’) is labeled to indicate positions of the boxes substituted (i.e. 1,2 in the second gel) and the bases introduced by the mutations (GT in the second gel). (B) As in (A), but using wild-type or mutant esp1396IM DNA fragments.

Mentions: To investigate further the interactions that contribute to site recognition by C.Esp1396I, EMSA experiments were performed with double-stranded DNA fragments carrying mutations in a 33 bp Pesp1396ICR fragment containing both C.Esp1396I binding sites. We specifically sought to determine the importance of ‘outer’ dinucleotides TG and CA in positions 1, 2 and 32, 33, respectively, and what the consequences would be of introducing the ‘missing’ internal symmetry-related dinucleotide on C.Esp1396I binding to each site. Based on structural analysis, we expected that the former substitutions would decrease the binding, while introduction of symmetry related dinucleotides between the individual binding sites would increase the binding of a C.Esp1396I dimer to a site, while decreasing the cooperativity of the binding. We also introduced double substitutions in the tetranucleotide repeats, to check their importance for the binding, despite the apparent lack of visible specific interaction and the fact that these repeats are not symmetry related in the complex. The results are presented in Figure 7A. For the wild-type fragment, a shifted band corresponding to a bound C.Esp1396I tetramer was observed, in agreement with the analyses above. A minor band corresponding to a dimer was seen only transiently at low concentrations of C.Esp1396I.Figure 7.


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)

Analysis of C.Esp1396I complexes with wild-type and mutant esp1396I promoters using electrophoretic mobility shift assay. (A) The sequences of the wild-type esp1396ICR C-boxes with symmetrical elements highlighted is presented at the top of the figure. Numbers below refer to positions of nucleotides in the C-box. The gels below show the results of native gel separation of complexes formed in the absence or in the presence of increasing concentrations of C.Esp1396I using wild-type esp1396ICR and various mutants. Note on nomenclature, every mutant fragment (‘mut’) is labeled to indicate positions of the boxes substituted (i.e. 1,2 in the second gel) and the bases introduced by the mutations (GT in the second gel). (B) As in (A), but using wild-type or mutant esp1396IM DNA fragments.
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Related In: Results  -  Collection

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Figure 7: Analysis of C.Esp1396I complexes with wild-type and mutant esp1396I promoters using electrophoretic mobility shift assay. (A) The sequences of the wild-type esp1396ICR C-boxes with symmetrical elements highlighted is presented at the top of the figure. Numbers below refer to positions of nucleotides in the C-box. The gels below show the results of native gel separation of complexes formed in the absence or in the presence of increasing concentrations of C.Esp1396I using wild-type esp1396ICR and various mutants. Note on nomenclature, every mutant fragment (‘mut’) is labeled to indicate positions of the boxes substituted (i.e. 1,2 in the second gel) and the bases introduced by the mutations (GT in the second gel). (B) As in (A), but using wild-type or mutant esp1396IM DNA fragments.
Mentions: To investigate further the interactions that contribute to site recognition by C.Esp1396I, EMSA experiments were performed with double-stranded DNA fragments carrying mutations in a 33 bp Pesp1396ICR fragment containing both C.Esp1396I binding sites. We specifically sought to determine the importance of ‘outer’ dinucleotides TG and CA in positions 1, 2 and 32, 33, respectively, and what the consequences would be of introducing the ‘missing’ internal symmetry-related dinucleotide on C.Esp1396I binding to each site. Based on structural analysis, we expected that the former substitutions would decrease the binding, while introduction of symmetry related dinucleotides between the individual binding sites would increase the binding of a C.Esp1396I dimer to a site, while decreasing the cooperativity of the binding. We also introduced double substitutions in the tetranucleotide repeats, to check their importance for the binding, despite the apparent lack of visible specific interaction and the fact that these repeats are not symmetry related in the complex. The results are presented in Figure 7A. For the wild-type fragment, a shifted band corresponding to a bound C.Esp1396I tetramer was observed, in agreement with the analyses above. A minor band corresponding to a dimer was seen only transiently at low concentrations of C.Esp1396I.Figure 7.

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