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Autoregulation of the Escherichia coli melR promoter: repression involves four molecules of MelR.

Samarasinghe S, El-Robh MS, Grainger DC, Zhang W, Soultanas P, Busby SJ - Nucleic Acids Res. (2008)

Bottom Line: Optimal repression requires MelR binding to a site that overlaps the melR transcription start point and to upstream sites.In this work, we have investigated the different determinants needed for optimal repression and their spatial requirements.We show that repression requires a complex involving four DNA-bound MelR molecules, and that the global CRP regulator plays little or no role.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.

ABSTRACT
The Escherichia coli MelR protein is a transcription activator that autoregulates its own promoter by repressing transcription initiation. Optimal repression requires MelR binding to a site that overlaps the melR transcription start point and to upstream sites. In this work, we have investigated the different determinants needed for optimal repression and their spatial requirements. We show that repression requires a complex involving four DNA-bound MelR molecules, and that the global CRP regulator plays little or no role.

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Binding of Fe-BABE-labelled MelR to melR promoter DNA. (A) The figure shows an autoradiogram of a segment of a sequencing gel that analyses DNA cleavage at MelR-binding site R, resulting from hydroxyl radicals generated by Fe-BABE attached to residue 269 of purified MelR. For this experiment, 0.75 μM of MelR labelled with Fe-BABE was incubated with 10-nM DNA fragment, either without (lane 1) or with (lane 2) 10 mM melibiose (see Materials and methods section). The DNA fragment was purified from a restriction digest of plasmid pSR carrying the TB10 melR promoter fragment and end-labelled at the HindIII site downstream of the melR promoter. The gel was calibrated with a Maxam–Gilbert G+A reaction (lane M). The location of MelR-binding site R is shown by vertical box and calibrations correspond to positions with respect to the melR transcript start point. (B) The figure shows the base sequence around the melR transcript start point (+1). The locations of DNA cleavage, resulting from hydroxyl radicals generated by Fe-ABE attached to residue 269 of MelR are indicated by stars. The 18-bp MelR-binding site R is enclosed by a box.
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Figure 4: Binding of Fe-BABE-labelled MelR to melR promoter DNA. (A) The figure shows an autoradiogram of a segment of a sequencing gel that analyses DNA cleavage at MelR-binding site R, resulting from hydroxyl radicals generated by Fe-BABE attached to residue 269 of purified MelR. For this experiment, 0.75 μM of MelR labelled with Fe-BABE was incubated with 10-nM DNA fragment, either without (lane 1) or with (lane 2) 10 mM melibiose (see Materials and methods section). The DNA fragment was purified from a restriction digest of plasmid pSR carrying the TB10 melR promoter fragment and end-labelled at the HindIII site downstream of the melR promoter. The gel was calibrated with a Maxam–Gilbert G+A reaction (lane M). The location of MelR-binding site R is shown by vertical box and calibrations correspond to positions with respect to the melR transcript start point. (B) The figure shows the base sequence around the melR transcript start point (+1). The locations of DNA cleavage, resulting from hydroxyl radicals generated by Fe-ABE attached to residue 269 of MelR are indicated by stars. The 18-bp MelR-binding site R is enclosed by a box.

Mentions: To determine experimentally the orientation of MelR bound at site R we used complementary genetic and biochemical approaches. First, we exploited the observation that the RV273 substitution in helix–turn–helix 2, permits MelR to recognize binding targets with a T at position 13 (20). Results in Figure 3C show that Val273 MelR gives a small enhancement in repression of the melR promoter carrying the 13T substitution in site R, compared to wild-type MelR. In contrast, repression of the 5C derivative is reduced with Val273 MelR. These data are consistent with a model in which helix–turn–helix 2 of MelR contacts the downstream part of site R, with residue 273 interacting with the base pair at position 13. To confirm this, we used a preparation of purified MelR that had been labelled with an inorganic DNA cleavage reagent at residue 269, adjacent to helix–turn–helix 2 (16). The reagent, p-bromoacetamidobenzyl-EDTA-Fe (Fe-BABE), generates a pulse of hydroxyl radicals that can be used to find the location of DNA binding of a protein and its orientation (26–28), and previously we exploited this to determine the orientation of MelR binding at sites 1′, 1, 2 and 2′ (16). Figure 4A shows the pattern of DNA cleavage due to Fe-BABE covalently attached at residue 269 of purified MelR, bound at site R. As expected, two sets of bands are observed. These result from cleavage at minor grooves on either side of the site where helix–turn–helix 2 is bound, which occurs as a pulse of hydroxyl radicals encounters the neighbouring DNA. Figure 4B shows the location of these sites of cleavage in the context of MelR-binding site R. The results confirm that helix—turn–helix 2 of MelR binds to the downstream half of site R and hence MelR must bind in opposite orientations at site R and site 2 (Figure 1A).Figure 4.


Autoregulation of the Escherichia coli melR promoter: repression involves four molecules of MelR.

Samarasinghe S, El-Robh MS, Grainger DC, Zhang W, Soultanas P, Busby SJ - Nucleic Acids Res. (2008)

Binding of Fe-BABE-labelled MelR to melR promoter DNA. (A) The figure shows an autoradiogram of a segment of a sequencing gel that analyses DNA cleavage at MelR-binding site R, resulting from hydroxyl radicals generated by Fe-BABE attached to residue 269 of purified MelR. For this experiment, 0.75 μM of MelR labelled with Fe-BABE was incubated with 10-nM DNA fragment, either without (lane 1) or with (lane 2) 10 mM melibiose (see Materials and methods section). The DNA fragment was purified from a restriction digest of plasmid pSR carrying the TB10 melR promoter fragment and end-labelled at the HindIII site downstream of the melR promoter. The gel was calibrated with a Maxam–Gilbert G+A reaction (lane M). The location of MelR-binding site R is shown by vertical box and calibrations correspond to positions with respect to the melR transcript start point. (B) The figure shows the base sequence around the melR transcript start point (+1). The locations of DNA cleavage, resulting from hydroxyl radicals generated by Fe-ABE attached to residue 269 of MelR are indicated by stars. The 18-bp MelR-binding site R is enclosed by a box.
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Related In: Results  -  Collection

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Show All Figures
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Figure 4: Binding of Fe-BABE-labelled MelR to melR promoter DNA. (A) The figure shows an autoradiogram of a segment of a sequencing gel that analyses DNA cleavage at MelR-binding site R, resulting from hydroxyl radicals generated by Fe-BABE attached to residue 269 of purified MelR. For this experiment, 0.75 μM of MelR labelled with Fe-BABE was incubated with 10-nM DNA fragment, either without (lane 1) or with (lane 2) 10 mM melibiose (see Materials and methods section). The DNA fragment was purified from a restriction digest of plasmid pSR carrying the TB10 melR promoter fragment and end-labelled at the HindIII site downstream of the melR promoter. The gel was calibrated with a Maxam–Gilbert G+A reaction (lane M). The location of MelR-binding site R is shown by vertical box and calibrations correspond to positions with respect to the melR transcript start point. (B) The figure shows the base sequence around the melR transcript start point (+1). The locations of DNA cleavage, resulting from hydroxyl radicals generated by Fe-ABE attached to residue 269 of MelR are indicated by stars. The 18-bp MelR-binding site R is enclosed by a box.
Mentions: To determine experimentally the orientation of MelR bound at site R we used complementary genetic and biochemical approaches. First, we exploited the observation that the RV273 substitution in helix–turn–helix 2, permits MelR to recognize binding targets with a T at position 13 (20). Results in Figure 3C show that Val273 MelR gives a small enhancement in repression of the melR promoter carrying the 13T substitution in site R, compared to wild-type MelR. In contrast, repression of the 5C derivative is reduced with Val273 MelR. These data are consistent with a model in which helix–turn–helix 2 of MelR contacts the downstream part of site R, with residue 273 interacting with the base pair at position 13. To confirm this, we used a preparation of purified MelR that had been labelled with an inorganic DNA cleavage reagent at residue 269, adjacent to helix–turn–helix 2 (16). The reagent, p-bromoacetamidobenzyl-EDTA-Fe (Fe-BABE), generates a pulse of hydroxyl radicals that can be used to find the location of DNA binding of a protein and its orientation (26–28), and previously we exploited this to determine the orientation of MelR binding at sites 1′, 1, 2 and 2′ (16). Figure 4A shows the pattern of DNA cleavage due to Fe-BABE covalently attached at residue 269 of purified MelR, bound at site R. As expected, two sets of bands are observed. These result from cleavage at minor grooves on either side of the site where helix–turn–helix 2 is bound, which occurs as a pulse of hydroxyl radicals encounters the neighbouring DNA. Figure 4B shows the location of these sites of cleavage in the context of MelR-binding site R. The results confirm that helix—turn–helix 2 of MelR binds to the downstream half of site R and hence MelR must bind in opposite orientations at site R and site 2 (Figure 1A).Figure 4.

Bottom Line: Optimal repression requires MelR binding to a site that overlaps the melR transcription start point and to upstream sites.In this work, we have investigated the different determinants needed for optimal repression and their spatial requirements.We show that repression requires a complex involving four DNA-bound MelR molecules, and that the global CRP regulator plays little or no role.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.

ABSTRACT
The Escherichia coli MelR protein is a transcription activator that autoregulates its own promoter by repressing transcription initiation. Optimal repression requires MelR binding to a site that overlaps the melR transcription start point and to upstream sites. In this work, we have investigated the different determinants needed for optimal repression and their spatial requirements. We show that repression requires a complex involving four DNA-bound MelR molecules, and that the global CRP regulator plays little or no role.

Show MeSH
Related in: MedlinePlus