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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.


Effect of mutations at the −10 regions of PC and PB.(a) Intergenic thnB-C region indicating the −10 and −35 promoter regions of each promoter and the constructed mutations. (b) Expression levels of thnB-lacZ gene fusions. (c) Expression levels of thnC::lacZ gene fusions. The horizontal dotted lines represent the independent levels of expression (short lacZ gene fusion).
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f3: Effect of mutations at the −10 regions of PC and PB.(a) Intergenic thnB-C region indicating the −10 and −35 promoter regions of each promoter and the constructed mutations. (b) Expression levels of thnB-lacZ gene fusions. (c) Expression levels of thnC::lacZ gene fusions. The horizontal dotted lines represent the independent levels of expression (short lacZ gene fusion).

Mentions: Mutations of the putative −10 box of each promoter were constructed (Fig. 3a), and its effect on transcription form its own and divergent promoters, was tested. As shown in Fig. 3b,c, mutations of the −10 boxes severely reduced the expression from their respective promoter, thus showing their importance as part of each promoter. Mutation of the −10 box of PC did not modify induced PB expression levels at all (Fig. 3b), thus indicating that the cooperative effect in PB transcription does not require transcription from PC. On the other hand, PC expression was slightly affected in a negative way by the mutation in the PB −10 box (Fig. 3c). However, importantly, cooperation between the two promoters was detected even in the presence of the PB −10 box mutation, as the expression levels driven by this construct were higher than those of carrying the independent PB or PC promoters (dotted lines in Fig. 3b,c).


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)

Effect of mutations at the −10 regions of PC and PB.(a) Intergenic thnB-C region indicating the −10 and −35 promoter regions of each promoter and the constructed mutations. (b) Expression levels of thnB-lacZ gene fusions. (c) Expression levels of thnC::lacZ gene fusions. The horizontal dotted lines represent the independent levels of expression (short lacZ gene fusion).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Effect of mutations at the −10 regions of PC and PB.(a) Intergenic thnB-C region indicating the −10 and −35 promoter regions of each promoter and the constructed mutations. (b) Expression levels of thnB-lacZ gene fusions. (c) Expression levels of thnC::lacZ gene fusions. The horizontal dotted lines represent the independent levels of expression (short lacZ gene fusion).
Mentions: Mutations of the putative −10 box of each promoter were constructed (Fig. 3a), and its effect on transcription form its own and divergent promoters, was tested. As shown in Fig. 3b,c, mutations of the −10 boxes severely reduced the expression from their respective promoter, thus showing their importance as part of each promoter. Mutation of the −10 box of PC did not modify induced PB expression levels at all (Fig. 3b), thus indicating that the cooperative effect in PB transcription does not require transcription from PC. On the other hand, PC expression was slightly affected in a negative way by the mutation in the PB −10 box (Fig. 3c). However, importantly, cooperation between the two promoters was detected even in the presence of the PB −10 box mutation, as the expression levels driven by this construct were higher than those of carrying the independent PB or PC promoters (dotted lines in Fig. 3b,c).

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.