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Functional four-base A/T gap core sequence CATTAG of P53 response elements specifically bound tetrameric P53 differently than two-base A/T gap core sequence CATG bound both dimeric and tetrameric P53.

Cai BH, Chen JY, Lu MH, Chang LT, Lin HC, Chang YM, Chao CF - Nucleic Acids Res. (2009)

Bottom Line: The p53-binding affinity and the activity of CATTAG were lower than for the mutant CATATG core sequence.A p53 mutant, A344, forms dimeric p53; it can only bind to CATG, and not to CATATG.Therefore, tetrameric and dimeric p53 can bind to a two-base A/T gap core sequence, but only tetrameric p53 can bind to a four-base A/T gap core sequence.

View Article: PubMed Central - PubMed

Affiliation: National Defense Medical Center, Institute of Life Sciences, Taipei, Taiwan, ROC.

ABSTRACT
The consensus sequence of p53 is repeated half sites of PuPuPuC(A/T)(A/T)GPyPyPy. GtAGCAttAGCCCAGACATGTCC is a 14-3-3sigma promoter p53 regulation site; the first core sequence is CAttAG, and the second is CATG. Both mutants GtAGgAttAGCCCAGACATGTCC and GtAGCAttAGCCCAGACATcTCC can be activated by p53 as a 1.5-fold half site. The original p53 regulated site on the 14-3-3sigma promoter is a whole site, and CATTAG is a functional core sequence. The p53-binding affinity and the activity of CATTAG were lower than for the mutant CATATG core sequence. Wild-type p53 acts as a tetramer to bind to the whole site; however, it also can bind to a half site by one of its dimers. Wild-type p53 can only bind to a half site with core sequence CATG but not to CATATG. The 1.5-fold half site or whole site with core sequence CATATG can be bound by wild-type p53. A p53 mutant, A344, forms dimeric p53; it can only bind to CATG, and not to CATATG. Therefore, tetrameric and dimeric p53 can bind to a two-base A/T gap core sequence, but only tetrameric p53 can bind to a four-base A/T gap core sequence.

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The promoter activity in various A/T gap core sequences of p53 promoter response element. (A) Designed various A/T gap core sequence p53 response elements. m4-1g CAG: fourth quarter-site mutation and first half-site core sequence CAG. m4-2g CATG, m4-3g CATAG, m4-4g CATTAG, m4-4g CATATG, m4-5g CATATAG and d12 as negative control as in Figure 2B. (B) Designed various A/T gap core sequences of p53 promoter response element constructs were cotransfected with p53 expression vector into H1299 cells, and the luciferase promoter activity was compared to pcDNA3.0 as 1. The activity order is: m4-2g CATG > m4-4g CATATG > m4-4g CATTAG. No activity could be detected in m4-1g CAG, m4-3g CATAG, m4-5g CATATAG or d12.
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Figure 3: The promoter activity in various A/T gap core sequences of p53 promoter response element. (A) Designed various A/T gap core sequence p53 response elements. m4-1g CAG: fourth quarter-site mutation and first half-site core sequence CAG. m4-2g CATG, m4-3g CATAG, m4-4g CATTAG, m4-4g CATATG, m4-5g CATATAG and d12 as negative control as in Figure 2B. (B) Designed various A/T gap core sequences of p53 promoter response element constructs were cotransfected with p53 expression vector into H1299 cells, and the luciferase promoter activity was compared to pcDNA3.0 as 1. The activity order is: m4-2g CATG > m4-4g CATATG > m4-4g CATTAG. No activity could be detected in m4-1g CAG, m4-3g CATAG, m4-5g CATATAG or d12.

Mentions: To evaluate if the A/T base gap in the p53 core sequence can be a functional core sequence of the p53 response element. One to five A/T base gaps were synthesized and cloned into 1.5-fold half sites (Figure 3A). The promoter activities were examined by luciferase activity assay. Only two- and four-base gaps can be activated by p53 (Figure 2B). When comparing two-base A/T gap core sequences with four-base A/T gaps, the activities were higher in two-base A/T gaps than in four-base A/T gaps. However, the 4 A/T base gap CATATG activity was higher than CATTAG (Figure 3B).Figure 3.


Functional four-base A/T gap core sequence CATTAG of P53 response elements specifically bound tetrameric P53 differently than two-base A/T gap core sequence CATG bound both dimeric and tetrameric P53.

Cai BH, Chen JY, Lu MH, Chang LT, Lin HC, Chang YM, Chao CF - Nucleic Acids Res. (2009)

The promoter activity in various A/T gap core sequences of p53 promoter response element. (A) Designed various A/T gap core sequence p53 response elements. m4-1g CAG: fourth quarter-site mutation and first half-site core sequence CAG. m4-2g CATG, m4-3g CATAG, m4-4g CATTAG, m4-4g CATATG, m4-5g CATATAG and d12 as negative control as in Figure 2B. (B) Designed various A/T gap core sequences of p53 promoter response element constructs were cotransfected with p53 expression vector into H1299 cells, and the luciferase promoter activity was compared to pcDNA3.0 as 1. The activity order is: m4-2g CATG > m4-4g CATATG > m4-4g CATTAG. No activity could be detected in m4-1g CAG, m4-3g CATAG, m4-5g CATATAG or d12.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 3: The promoter activity in various A/T gap core sequences of p53 promoter response element. (A) Designed various A/T gap core sequence p53 response elements. m4-1g CAG: fourth quarter-site mutation and first half-site core sequence CAG. m4-2g CATG, m4-3g CATAG, m4-4g CATTAG, m4-4g CATATG, m4-5g CATATAG and d12 as negative control as in Figure 2B. (B) Designed various A/T gap core sequences of p53 promoter response element constructs were cotransfected with p53 expression vector into H1299 cells, and the luciferase promoter activity was compared to pcDNA3.0 as 1. The activity order is: m4-2g CATG > m4-4g CATATG > m4-4g CATTAG. No activity could be detected in m4-1g CAG, m4-3g CATAG, m4-5g CATATAG or d12.
Mentions: To evaluate if the A/T base gap in the p53 core sequence can be a functional core sequence of the p53 response element. One to five A/T base gaps were synthesized and cloned into 1.5-fold half sites (Figure 3A). The promoter activities were examined by luciferase activity assay. Only two- and four-base gaps can be activated by p53 (Figure 2B). When comparing two-base A/T gap core sequences with four-base A/T gaps, the activities were higher in two-base A/T gaps than in four-base A/T gaps. However, the 4 A/T base gap CATATG activity was higher than CATTAG (Figure 3B).Figure 3.

Bottom Line: The p53-binding affinity and the activity of CATTAG were lower than for the mutant CATATG core sequence.A p53 mutant, A344, forms dimeric p53; it can only bind to CATG, and not to CATATG.Therefore, tetrameric and dimeric p53 can bind to a two-base A/T gap core sequence, but only tetrameric p53 can bind to a four-base A/T gap core sequence.

View Article: PubMed Central - PubMed

Affiliation: National Defense Medical Center, Institute of Life Sciences, Taipei, Taiwan, ROC.

ABSTRACT
The consensus sequence of p53 is repeated half sites of PuPuPuC(A/T)(A/T)GPyPyPy. GtAGCAttAGCCCAGACATGTCC is a 14-3-3sigma promoter p53 regulation site; the first core sequence is CAttAG, and the second is CATG. Both mutants GtAGgAttAGCCCAGACATGTCC and GtAGCAttAGCCCAGACATcTCC can be activated by p53 as a 1.5-fold half site. The original p53 regulated site on the 14-3-3sigma promoter is a whole site, and CATTAG is a functional core sequence. The p53-binding affinity and the activity of CATTAG were lower than for the mutant CATATG core sequence. Wild-type p53 acts as a tetramer to bind to the whole site; however, it also can bind to a half site by one of its dimers. Wild-type p53 can only bind to a half site with core sequence CATG but not to CATATG. The 1.5-fold half site or whole site with core sequence CATATG can be bound by wild-type p53. A p53 mutant, A344, forms dimeric p53; it can only bind to CATG, and not to CATATG. Therefore, tetrameric and dimeric p53 can bind to a two-base A/T gap core sequence, but only tetrameric p53 can bind to a four-base A/T gap core sequence.

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