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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.


Footprints of WT and 2B2 substitution mutant.(a) PC top strand and (b) PB top strand. Black and grey rectangles represent protected regions at the primary and secondary binding sites of each promoter, respectively. Circles represent positions hypersensitive to DNase I treatment upon ThnR binding. Arrows represent the mutations location. The increasing concentrations of ThnR tetramers are: 0, 0.1, 0.5, 1 and 1.5 μM.
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f7: Footprints of WT and 2B2 substitution mutant.(a) PC top strand and (b) PB top strand. Black and grey rectangles represent protected regions at the primary and secondary binding sites of each promoter, respectively. Circles represent positions hypersensitive to DNase I treatment upon ThnR binding. Arrows represent the mutations location. The increasing concentrations of ThnR tetramers are: 0, 0.1, 0.5, 1 and 1.5 μM.

Mentions: On the other hand, the pentanucleotide substitution in mutation 2B2 resulted in barely detectable levels of transcription activation from PB in isolation (Fig. 6a), indicating that independent PB transcriptional activation strictly requires productive interactions of ThnR with these sequences. Strikingly, in the presence of PC, the expression levels from the 2B2 mutant PB were higher than those obtained from the 2B1 mutant and indistinguishable from those obtained with the wild-type region (Fig. 6c). These data indicates that the strong deficiency in the independent transcription activation caused by the 2B2 mutation can be fully compensated by the presence of the divergent PC promoter region, thus making thnB expression in this context strictly dependent on cooperative transcription. Footprinting analysis of the divergent promoters region containing the 2B2 mutation showed that this secondary site B is not protected (Fig. 7), thus suggesting that productive interactions of ThnR with the conserved regions in the secondary site B are not essential for cooperative transcription from PB.


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)

Footprints of WT and 2B2 substitution mutant.(a) PC top strand and (b) PB top strand. Black and grey rectangles represent protected regions at the primary and secondary binding sites of each promoter, respectively. Circles represent positions hypersensitive to DNase I treatment upon ThnR binding. Arrows represent the mutations location. The increasing concentrations of ThnR tetramers are: 0, 0.1, 0.5, 1 and 1.5 μM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Footprints of WT and 2B2 substitution mutant.(a) PC top strand and (b) PB top strand. Black and grey rectangles represent protected regions at the primary and secondary binding sites of each promoter, respectively. Circles represent positions hypersensitive to DNase I treatment upon ThnR binding. Arrows represent the mutations location. The increasing concentrations of ThnR tetramers are: 0, 0.1, 0.5, 1 and 1.5 μM.
Mentions: On the other hand, the pentanucleotide substitution in mutation 2B2 resulted in barely detectable levels of transcription activation from PB in isolation (Fig. 6a), indicating that independent PB transcriptional activation strictly requires productive interactions of ThnR with these sequences. Strikingly, in the presence of PC, the expression levels from the 2B2 mutant PB were higher than those obtained from the 2B1 mutant and indistinguishable from those obtained with the wild-type region (Fig. 6c). These data indicates that the strong deficiency in the independent transcription activation caused by the 2B2 mutation can be fully compensated by the presence of the divergent PC promoter region, thus making thnB expression in this context strictly dependent on cooperative transcription. Footprinting analysis of the divergent promoters region containing the 2B2 mutation showed that this secondary site B is not protected (Fig. 7), thus suggesting that productive interactions of ThnR with the conserved regions in the secondary site B are not essential for cooperative transcription from PB.

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.