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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.


Cooperative and independent transcription activation from PB (a) and PC (b) promoters, using the large and the short lacZ gene fusions to each promoter. Boxes represent the promoter regions. The arrows downstream of some of the promoters represent the lacZ gene fusions to these promoters. Basal and tetralin-induced (THN) expression levels are shown.
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f2: Cooperative and independent transcription activation from PB (a) and PC (b) promoters, using the large and the short lacZ gene fusions to each promoter. Boxes represent the promoter regions. The arrows downstream of some of the promoters represent the lacZ gene fusions to these promoters. Basal and tetralin-induced (THN) expression levels are shown.

Mentions: These thn::lacZ gene fusions and others that bore substitutions in each primary binding site B and C were previously constructed and their effects on transcription from their cognate or the divergent promoters when ThnR and ThnY were highly expressed from the heterologous Ptac promoter, were reported22. The results of similar experiments but producing the activators from their own promoter in a low copy number plasmid are shown in Fig. 2. Low expression of both ThnR and ThnY in T-690 is enough to induce thn genes expression in the presence of tetralin. Independent expression from each promoter, i. e. in the absence of its divergent partner, was obvious after induction with tetralin. Expression from PB was lower than that from PC, as previously described19. However, expression levels from both promoters increased when the divergent promoter was also present (146% and 165% for PB and PC, respectively).


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)

Cooperative and independent transcription activation from PB (a) and PC (b) promoters, using the large and the short lacZ gene fusions to each promoter. Boxes represent the promoter regions. The arrows downstream of some of the promoters represent the lacZ gene fusions to these promoters. Basal and tetralin-induced (THN) expression levels are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Cooperative and independent transcription activation from PB (a) and PC (b) promoters, using the large and the short lacZ gene fusions to each promoter. Boxes represent the promoter regions. The arrows downstream of some of the promoters represent the lacZ gene fusions to these promoters. Basal and tetralin-induced (THN) expression levels are shown.
Mentions: These thn::lacZ gene fusions and others that bore substitutions in each primary binding site B and C were previously constructed and their effects on transcription from their cognate or the divergent promoters when ThnR and ThnY were highly expressed from the heterologous Ptac promoter, were reported22. The results of similar experiments but producing the activators from their own promoter in a low copy number plasmid are shown in Fig. 2. Low expression of both ThnR and ThnY in T-690 is enough to induce thn genes expression in the presence of tetralin. Independent expression from each promoter, i. e. in the absence of its divergent partner, was obvious after induction with tetralin. Expression from PB was lower than that from PC, as previously described19. However, expression levels from both promoters increased when the divergent promoter was also present (146% and 165% for PB and PC, respectively).

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.