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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.


Transcriptional arrangement of the thn operons with all detected operons (a) and a schematic zoom of the intergenic thnBC region showing the ThnR primary and secondary binding sites at each promoter region, and the BglII restriction site used for isolation of each promoter region for independent transcription analysis (b).
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f1: Transcriptional arrangement of the thn operons with all detected operons (a) and a schematic zoom of the intergenic thnBC region showing the ThnR primary and secondary binding sites at each promoter region, and the BglII restriction site used for isolation of each promoter region for independent transcription analysis (b).

Mentions: One of such unconventional systems is that regulating the biodegradation of the organic solvent tetralin in Sphingopyxis granuli (formerly Sphingopyxis macrogoltabida) strain TFA. The genes coding for the degradation pathway are grouped in four operons (Fig. 1a) induced in response to tetralin and subjected to catabolite repression2021. ThnR is an LTTR essential for transcription activation from the four promoters PC, PB, PH and PM2221. Expression of thn operons also requires ThnY23, a ferredoxin reductase-like protein that has evolved to function as a co-activator of the thn operons24, which mediates a unique modulation system that prevents gratuitous induction of the thn operons by molecules that, being similar to tetralin, are not substrates of the pathway14. ThnY slightly increases binding affinity of ThnR to the promoter regions but it does not bind DNA by itself and does not alter the footprint pattern generated by ThnR at these regions. Rather, it co-activates transcription by activating ThnR via protein-protein interactions24. The regulatory proteins are expressed from both a constitutive promoter (PR) and the tetralin inducible PC.


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)

Transcriptional arrangement of the thn operons with all detected operons (a) and a schematic zoom of the intergenic thnBC region showing the ThnR primary and secondary binding sites at each promoter region, and the BglII restriction site used for isolation of each promoter region for independent transcription analysis (b).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Transcriptional arrangement of the thn operons with all detected operons (a) and a schematic zoom of the intergenic thnBC region showing the ThnR primary and secondary binding sites at each promoter region, and the BglII restriction site used for isolation of each promoter region for independent transcription analysis (b).
Mentions: One of such unconventional systems is that regulating the biodegradation of the organic solvent tetralin in Sphingopyxis granuli (formerly Sphingopyxis macrogoltabida) strain TFA. The genes coding for the degradation pathway are grouped in four operons (Fig. 1a) induced in response to tetralin and subjected to catabolite repression2021. ThnR is an LTTR essential for transcription activation from the four promoters PC, PB, PH and PM2221. Expression of thn operons also requires ThnY23, a ferredoxin reductase-like protein that has evolved to function as a co-activator of the thn operons24, which mediates a unique modulation system that prevents gratuitous induction of the thn operons by molecules that, being similar to tetralin, are not substrates of the pathway14. ThnY slightly increases binding affinity of ThnR to the promoter regions but it does not bind DNA by itself and does not alter the footprint pattern generated by ThnR at these regions. Rather, it co-activates transcription by activating ThnR via protein-protein interactions24. The regulatory proteins are expressed from both a constitutive promoter (PR) and the tetralin inducible PC.

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.