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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 additional mutations in PC on the strictly cooperative transcription from the 2B2 mutant PB promoter.(a) Schematic representation of the mutations in the divergent promoters region. 1C is a mutation in the PC PBS22 that virtually abolishes transcription from PC in this system (not shown). (b) Expression levels from PB when the 2B2 mutation is combined with others in the PC promoter region.
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f8: Effect of additional mutations in PC on the strictly cooperative transcription from the 2B2 mutant PB promoter.(a) Schematic representation of the mutations in the divergent promoters region. 1C is a mutation in the PC PBS22 that virtually abolishes transcription from PC in this system (not shown). (b) Expression levels from PB when the 2B2 mutation is combined with others in the PC promoter region.

Mentions: As shown in Fig. 8, either insertion of half turn of the helix or the presence of the 1C or 2C1 mutations fully prevented transcription from the PB promoter bearing the 2B2 mutation. This result clearly indicates that compensation of the deficiency provoked by the 2B2 mutation by the PC promoter region strictly requires ThnR binding at its site in PC, complex formation and proper interactions of ThnR with its palindromic SBS at the divergent PC promoter, thus establishing important elements of PC 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)

Effect of additional mutations in PC on the strictly cooperative transcription from the 2B2 mutant PB promoter.(a) Schematic representation of the mutations in the divergent promoters region. 1C is a mutation in the PC PBS22 that virtually abolishes transcription from PC in this system (not shown). (b) Expression levels from PB when the 2B2 mutation is combined with others in the PC promoter region.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f8: Effect of additional mutations in PC on the strictly cooperative transcription from the 2B2 mutant PB promoter.(a) Schematic representation of the mutations in the divergent promoters region. 1C is a mutation in the PC PBS22 that virtually abolishes transcription from PC in this system (not shown). (b) Expression levels from PB when the 2B2 mutation is combined with others in the PC promoter region.
Mentions: As shown in Fig. 8, either insertion of half turn of the helix or the presence of the 1C or 2C1 mutations fully prevented transcription from the PB promoter bearing the 2B2 mutation. This result clearly indicates that compensation of the deficiency provoked by the 2B2 mutation by the PC promoter region strictly requires ThnR binding at its site in PC, complex formation and proper interactions of ThnR with its palindromic SBS at the divergent PC promoter, thus establishing important elements of PC 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.