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Genetic dissection of independent and cooperative transcriptional activation by the LysR-type activator ThnR at close divergent promoters.

Rivas-Marín E, Floriano B, Santero E - Sci Rep (2016)

Bottom Line: A mutation at the PB promoter region, which abolishes its independent transcription, does not affect at all PB transcription in the presence of the divergent promoter PC, thus indicating that the complex formed via DNA loop can compensate for the deficiencies in the correct protein-DNA interaction at one of the promoters.Combination of mutations in both promoters identifies a region at PC that is not important for its independent transcription but it is essential for cooperative transcription from both promoters.This work provides new insights into the diversity and complexity of activation mechanisms used by the most abundant type of bacterial transcriptional regulators.

View Article: PubMed Central - PubMed

Affiliation: Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide/Consejo Superior de Investigaciones Científicas/Junta de Andalucía, and Departamento de Biología Molecular e Ingeniería Bioquímica, Universidad Pablo de Olavide, Spain.

ABSTRACT
Regulation of tetralin biodegradation operons is one of the examples of unconventional LysR-type mediated transcriptional regulation. ThnR activates transcription from two divergent and closely located promoters PB and PC. Although ThnR activates each promoter independently, transcription from each one increases when both promoters are together. Mutational analysis of the intergenic region shows that cooperative transcription is achieved through formation of a ThnR complex when bound to its respective sites at each promoter, via formation of a DNA loop. Mutations also defined ThnR contact sites that are important for independent transcriptional activation at each promoter. A mutation at the PB promoter region, which abolishes its independent transcription, does not affect at all PB transcription in the presence of the divergent promoter PC, thus indicating that the complex formed via DNA loop can compensate for the deficiencies in the correct protein-DNA interaction at one of the promoters. Combination of mutations in both promoters identifies a region at PC that is not important for its independent transcription but it is essential for cooperative transcription from both promoters. This work provides new insights into the diversity and complexity of activation mechanisms used by the most abundant type of bacterial transcriptional regulators.

No MeSH data available.


(a) EMSA assays showing complex I and complex II formation at the thnB-thnC divergent promoter region in WT and +4, +6, +11 and +33 bp insertion mutants. (b) Effects of the insertions on thnB expression. (c) Effects of the insertions on thnC expression. The horizontal dotted line represents independent levels of expression. Basal levels of the thnB::lacZ gene fusions ranged between 73 and 137 MU and those for the thnC::lacZ gene fusions ranged between 127 and 364 MU. The increasing concentrations of ThnR tetramers are: 0, 25, 50, 75, 100, 125 and 150 nM.
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f4: (a) EMSA assays showing complex I and complex II formation at the thnB-thnC divergent promoter region in WT and +4, +6, +11 and +33 bp insertion mutants. (b) Effects of the insertions on thnB expression. (c) Effects of the insertions on thnC expression. The horizontal dotted line represents independent levels of expression. Basal levels of the thnB::lacZ gene fusions ranged between 73 and 137 MU and those for the thnC::lacZ gene fusions ranged between 127 and 364 MU. The increasing concentrations of ThnR tetramers are: 0, 25, 50, 75, 100, 125 and 150 nM.

Mentions: The centres of the ThnR primary binding sites for each promoter are separated by 55 bp, corresponding to 5 integral turns of the helix (Fig. 1b), thus both sites are oriented on the same face of the helix. In order to test the effect of further separating these bindings sites or placing them on opposite faces of the helix, +4, +6, +11 and +33 bp were inserted between the binding sites (Fig. 4). These modified promoter regions were used as probes to analyse DNA-protein interactions and to construct thnB and thnC gene fusions to lacZ, which were inserted into the chromosome of the strain T-690 to characterise their effect on transcription. As previously reported22, ThnR initially binds to its highest affinity PBS close to thnC (complex I) but at higher concentrations ThnR binding resulted in the formation of a complex II due to protein-protein interaction between ThnR bound to the sites of each promoter region (Fig. 4a). Insertion of half turns of the helix (+4 and +6 bp) did not affect initial binding but had a dramatic effect on formation of the complex II structure, as evidenced by electrophoretic mobility shift assays (EMSA) (Fig. 4a), whilst insertion of integral turns of the helix (+11 and +33 bp) still allowed it. Footprinting analyses showed that ThnR can bind to both sites B and C in the outphased promoters but they lack the hypersensitive band shown by the wild-type DNA region at position −103 from the PC transcriptional start, between the primary binding sites B and C, which is indicative of a DNA distortion. This band still remains in the +11 and +33 bp mutants (Supplementary Fig. S1). These data strongly suggest that two ThnR molecules bound to their respective sites in the intergenic region form a complex through ThnR protein-protein interactions that results in formation of a DNA loop, which cannot be formed if the ThnR primary binding sites are not aligned in the same face of the helix. These mutant regions were used to construct lacZ gene fusions, and were integrated into the chromosome of T-690 as described above, to analyse expression from each promoter. Insertion of half turns of the helix reduced expression from both promoters down to almost the levels of independent transcription (Fig. 4b,c). On the other hand, insertion of integral turns of the helix allowing formation of the complex did not substantially affect the thnC expression levels (Fig. 4c), thus suggesting that cooperative transcription from PC relies on ThnR interactions when bound to distant sites with the consequent formation of the DNA loop. Interestingly, expression from PB was also reduced to independent levels of expression when the binding sites were separated by 1 or 3 turns of the helix (Fig. 4b), thus indicating that cooperative transcription of thnB is highly sensitive to the distance between the primary binding sites.


Genetic dissection of independent and cooperative transcriptional activation by the LysR-type activator ThnR at close divergent promoters.

Rivas-Marín E, Floriano B, Santero E - Sci Rep (2016)

(a) EMSA assays showing complex I and complex II formation at the thnB-thnC divergent promoter region in WT and +4, +6, +11 and +33 bp insertion mutants. (b) Effects of the insertions on thnB expression. (c) Effects of the insertions on thnC expression. The horizontal dotted line represents independent levels of expression. Basal levels of the thnB::lacZ gene fusions ranged between 73 and 137 MU and those for the thnC::lacZ gene fusions ranged between 127 and 364 MU. The increasing concentrations of ThnR tetramers are: 0, 25, 50, 75, 100, 125 and 150 nM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834489&req=5

f4: (a) EMSA assays showing complex I and complex II formation at the thnB-thnC divergent promoter region in WT and +4, +6, +11 and +33 bp insertion mutants. (b) Effects of the insertions on thnB expression. (c) Effects of the insertions on thnC expression. The horizontal dotted line represents independent levels of expression. Basal levels of the thnB::lacZ gene fusions ranged between 73 and 137 MU and those for the thnC::lacZ gene fusions ranged between 127 and 364 MU. The increasing concentrations of ThnR tetramers are: 0, 25, 50, 75, 100, 125 and 150 nM.
Mentions: The centres of the ThnR primary binding sites for each promoter are separated by 55 bp, corresponding to 5 integral turns of the helix (Fig. 1b), thus both sites are oriented on the same face of the helix. In order to test the effect of further separating these bindings sites or placing them on opposite faces of the helix, +4, +6, +11 and +33 bp were inserted between the binding sites (Fig. 4). These modified promoter regions were used as probes to analyse DNA-protein interactions and to construct thnB and thnC gene fusions to lacZ, which were inserted into the chromosome of the strain T-690 to characterise their effect on transcription. As previously reported22, ThnR initially binds to its highest affinity PBS close to thnC (complex I) but at higher concentrations ThnR binding resulted in the formation of a complex II due to protein-protein interaction between ThnR bound to the sites of each promoter region (Fig. 4a). Insertion of half turns of the helix (+4 and +6 bp) did not affect initial binding but had a dramatic effect on formation of the complex II structure, as evidenced by electrophoretic mobility shift assays (EMSA) (Fig. 4a), whilst insertion of integral turns of the helix (+11 and +33 bp) still allowed it. Footprinting analyses showed that ThnR can bind to both sites B and C in the outphased promoters but they lack the hypersensitive band shown by the wild-type DNA region at position −103 from the PC transcriptional start, between the primary binding sites B and C, which is indicative of a DNA distortion. This band still remains in the +11 and +33 bp mutants (Supplementary Fig. S1). These data strongly suggest that two ThnR molecules bound to their respective sites in the intergenic region form a complex through ThnR protein-protein interactions that results in formation of a DNA loop, which cannot be formed if the ThnR primary binding sites are not aligned in the same face of the helix. These mutant regions were used to construct lacZ gene fusions, and were integrated into the chromosome of T-690 as described above, to analyse expression from each promoter. Insertion of half turns of the helix reduced expression from both promoters down to almost the levels of independent transcription (Fig. 4b,c). On the other hand, insertion of integral turns of the helix allowing formation of the complex did not substantially affect the thnC expression levels (Fig. 4c), thus suggesting that cooperative transcription from PC relies on ThnR interactions when bound to distant sites with the consequent formation of the DNA loop. Interestingly, expression from PB was also reduced to independent levels of expression when the binding sites were separated by 1 or 3 turns of the helix (Fig. 4b), thus indicating that cooperative transcription of thnB is highly sensitive to the distance between the primary binding sites.

Bottom Line: A mutation at the PB promoter region, which abolishes its independent transcription, does not affect at all PB transcription in the presence of the divergent promoter PC, thus indicating that the complex formed via DNA loop can compensate for the deficiencies in the correct protein-DNA interaction at one of the promoters.Combination of mutations in both promoters identifies a region at PC that is not important for its independent transcription but it is essential for cooperative transcription from both promoters.This work provides new insights into the diversity and complexity of activation mechanisms used by the most abundant type of bacterial transcriptional regulators.

View Article: PubMed Central - PubMed

Affiliation: Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide/Consejo Superior de Investigaciones Científicas/Junta de Andalucía, and Departamento de Biología Molecular e Ingeniería Bioquímica, Universidad Pablo de Olavide, Spain.

ABSTRACT
Regulation of tetralin biodegradation operons is one of the examples of unconventional LysR-type mediated transcriptional regulation. ThnR activates transcription from two divergent and closely located promoters PB and PC. Although ThnR activates each promoter independently, transcription from each one increases when both promoters are together. Mutational analysis of the intergenic region shows that cooperative transcription is achieved through formation of a ThnR complex when bound to its respective sites at each promoter, via formation of a DNA loop. Mutations also defined ThnR contact sites that are important for independent transcriptional activation at each promoter. A mutation at the PB promoter region, which abolishes its independent transcription, does not affect at all PB transcription in the presence of the divergent promoter PC, thus indicating that the complex formed via DNA loop can compensate for the deficiencies in the correct protein-DNA interaction at one of the promoters. Combination of mutations in both promoters identifies a region at PC that is not important for its independent transcription but it is essential for cooperative transcription from both promoters. This work provides new insights into the diversity and complexity of activation mechanisms used by the most abundant type of bacterial transcriptional regulators.

No MeSH data available.