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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.


(a) Alignment of the thn promoters showing the difference between PC and the other promoters, and mutations constructed in the PC promoter region. Effect of each spacer mutation on independent transcription from PC (b) or on coordinated transcription from PC (c) and PB (d). Effect of the SBS substitutions on independent transcription from PC (e) or on coordinated transcription from PC (f) and PB (g). The horizontal dotted lines represent the independent levels of expression.
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f5: (a) Alignment of the thn promoters showing the difference between PC and the other promoters, and mutations constructed in the PC promoter region. Effect of each spacer mutation on independent transcription from PC (b) or on coordinated transcription from PC (c) and PB (d). Effect of the SBS substitutions on independent transcription from PC (e) or on coordinated transcription from PC (f) and PB (g). The horizontal dotted lines represent the independent levels of expression.

Mentions: The arrangement of ThnR binding sites at the PB, PH and PM promoter regions is conventional whilst at the PC promoter region is unusual (Figs 1b and 5a). First, its PBS, which is essential for ThnR binding and ThnR-mediated activation of PC, is centred at −76, one helix turn further upstream of the transcription start site compared to the conventional arrangement. Secondly, a palindromic motif resembling the PBS is found 29 bp downstream of the primary site, which is also protected from DNase I digestion by ThnR binding22 (Supplementary Fig. S1). This SBS, though located in the same position relative to the transcription start site as the conserved regions in the PB, PH and PM promoters, does not show similarity to the conserved sequence found in them (Fig. 5a).


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)

(a) Alignment of the thn promoters showing the difference between PC and the other promoters, and mutations constructed in the PC promoter region. Effect of each spacer mutation on independent transcription from PC (b) or on coordinated transcription from PC (c) and PB (d). Effect of the SBS substitutions on independent transcription from PC (e) or on coordinated transcription from PC (f) and PB (g). The horizontal dotted lines represent the independent levels of expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: (a) Alignment of the thn promoters showing the difference between PC and the other promoters, and mutations constructed in the PC promoter region. Effect of each spacer mutation on independent transcription from PC (b) or on coordinated transcription from PC (c) and PB (d). Effect of the SBS substitutions on independent transcription from PC (e) or on coordinated transcription from PC (f) and PB (g). The horizontal dotted lines represent the independent levels of expression.
Mentions: The arrangement of ThnR binding sites at the PB, PH and PM promoter regions is conventional whilst at the PC promoter region is unusual (Figs 1b and 5a). First, its PBS, which is essential for ThnR binding and ThnR-mediated activation of PC, is centred at −76, one helix turn further upstream of the transcription start site compared to the conventional arrangement. Secondly, a palindromic motif resembling the PBS is found 29 bp downstream of the primary site, which is also protected from DNase I digestion by ThnR binding22 (Supplementary Fig. S1). This SBS, though located in the same position relative to the transcription start site as the conserved regions in the PB, PH and PM promoters, does not show similarity to the conserved sequence found in them (Fig. 5a).

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.