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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.

Show MeSH
Comparison of operator sequences in Esp1396I. C.Esp1396I binding sites at the CR promoter (proximal, OR; distal, OL) and the M promoter (OM) are aligned. Indicated in yellow are the sites where at least 2/3 of the sequences are identical, seven of which (red lettering) are found in all three. A symmetrical consensus sequence is shown below, closely matching the OM site. Bases identified from the crystal structure as being involved in DNA–protein interactions are indicated in blue; other symmetrical bases likely to interact with helix 3 of the protein are shown in magenta.
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Figure 8: Comparison of operator sequences in Esp1396I. C.Esp1396I binding sites at the CR promoter (proximal, OR; distal, OL) and the M promoter (OM) are aligned. Indicated in yellow are the sites where at least 2/3 of the sequences are identical, seven of which (red lettering) are found in all three. A symmetrical consensus sequence is shown below, closely matching the OM site. Bases identified from the crystal structure as being involved in DNA–protein interactions are indicated in blue; other symmetrical bases likely to interact with helix 3 of the protein are shown in magenta.

Mentions: All three operators (Figure 8) have in common the central TATA sequence that allows compression of the minor groove of the DNA, forcing DNA bending at the centre of the dimer binding site in the complex (19). The strongest binding site, OM, in addition contains the symmetry related TG and CA dinucleotide sequences that interact strongly with Arg35 of the controller protein (19), whereas OL and OR have just one of these dinucleotides (clearly, both TG and CA are not possible simultaneously as the binding sites overlap in this region). It is notable that the sequence of the highest affinity site, OM, is almost perfectly palindromic, with 16 of the 18 bases being related by dyad symmetry (Figure 8). Indeed, at least two of the three operators have identical bases at these 16 sites, of which 7 consecutive bases (TATAGTC) are identical in all three operators.Figure 8.


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)

Comparison of operator sequences in Esp1396I. C.Esp1396I binding sites at the CR promoter (proximal, OR; distal, OL) and the M promoter (OM) are aligned. Indicated in yellow are the sites where at least 2/3 of the sequences are identical, seven of which (red lettering) are found in all three. A symmetrical consensus sequence is shown below, closely matching the OM site. Bases identified from the crystal structure as being involved in DNA–protein interactions are indicated in blue; other symmetrical bases likely to interact with helix 3 of the protein are shown in magenta.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 8: Comparison of operator sequences in Esp1396I. C.Esp1396I binding sites at the CR promoter (proximal, OR; distal, OL) and the M promoter (OM) are aligned. Indicated in yellow are the sites where at least 2/3 of the sequences are identical, seven of which (red lettering) are found in all three. A symmetrical consensus sequence is shown below, closely matching the OM site. Bases identified from the crystal structure as being involved in DNA–protein interactions are indicated in blue; other symmetrical bases likely to interact with helix 3 of the protein are shown in magenta.
Mentions: All three operators (Figure 8) have in common the central TATA sequence that allows compression of the minor groove of the DNA, forcing DNA bending at the centre of the dimer binding site in the complex (19). The strongest binding site, OM, in addition contains the symmetry related TG and CA dinucleotide sequences that interact strongly with Arg35 of the controller protein (19), whereas OL and OR have just one of these dinucleotides (clearly, both TG and CA are not possible simultaneously as the binding sites overlap in this region). It is notable that the sequence of the highest affinity site, OM, is almost perfectly palindromic, with 16 of the 18 bases being related by dyad symmetry (Figure 8). Indeed, at least two of the three operators have identical bases at these 16 sites, of which 7 consecutive bases (TATAGTC) are identical in all three operators.Figure 8.

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