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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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Promoter activity assay for four-base A/T gap core sequence with p53 2X and 1.5X-fold p53 half site. (A) Designed four-base A/T gap core sequence p53 response element constructs and its mutants: 2× CATATG: whole site with CATATG core sequence; 2× CATTAG: whole site with CATTAG core sequence; m1-4g CATATG: mutation of first-quarter site with CATATG core sequence; m1-4g CATTAG: mutation of first-quarter site with CATTAG core sequence; and d12 is a negative control. (B) Designed four-base A/T gap core sequence p53 response element and mutant promoter constructs were co-transfected with p53 expression vectors into H1299 cells. The folds induction of luciferase promoter activity compared to the pcDNA3.0 as 1. Both 2- and 1.5-fold half-site core sequences of CATATG promoter activity were higher than CATTAG core sequence.
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Figure 4: Promoter activity assay for four-base A/T gap core sequence with p53 2X and 1.5X-fold p53 half site. (A) Designed four-base A/T gap core sequence p53 response element constructs and its mutants: 2× CATATG: whole site with CATATG core sequence; 2× CATTAG: whole site with CATTAG core sequence; m1-4g CATATG: mutation of first-quarter site with CATATG core sequence; m1-4g CATTAG: mutation of first-quarter site with CATTAG core sequence; and d12 is a negative control. (B) Designed four-base A/T gap core sequence p53 response element and mutant promoter constructs were co-transfected with p53 expression vectors into H1299 cells. The folds induction of luciferase promoter activity compared to the pcDNA3.0 as 1. Both 2- and 1.5-fold half-site core sequences of CATATG promoter activity were higher than CATTAG core sequence.

Mentions: The whole p53 response element sequence activation by p53 was higher than for the 1.5-fold half site (5). Is the four-base A/T gap core sequence as functional in the whole site as in the 1.5-fold half site as previously demonstrated? The four-base A/T gap mutants in the whole p53 response element sequence were cloned, and the promoter activity was examined by a luciferase activity assay (Figure 4A). The results indicated that the core CATATG activity activated by p53 is higher than for CATTAG in both the whole site and the 1.5-fold half site (Figure 4B). Two super p53 mutants, p53F46 and p53Δ364–393 (10,11), also can activate the p53 whole site with the CATATG core sequence, even more so than wild-type p53 (Figure 5).Figure 4.


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)

Promoter activity assay for four-base A/T gap core sequence with p53 2X and 1.5X-fold p53 half site. (A) Designed four-base A/T gap core sequence p53 response element constructs and its mutants: 2× CATATG: whole site with CATATG core sequence; 2× CATTAG: whole site with CATTAG core sequence; m1-4g CATATG: mutation of first-quarter site with CATATG core sequence; m1-4g CATTAG: mutation of first-quarter site with CATTAG core sequence; and d12 is a negative control. (B) Designed four-base A/T gap core sequence p53 response element and mutant promoter constructs were co-transfected with p53 expression vectors into H1299 cells. The folds induction of luciferase promoter activity compared to the pcDNA3.0 as 1. Both 2- and 1.5-fold half-site core sequences of CATATG promoter activity were higher than CATTAG core sequence.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2665222&req=5

Figure 4: Promoter activity assay for four-base A/T gap core sequence with p53 2X and 1.5X-fold p53 half site. (A) Designed four-base A/T gap core sequence p53 response element constructs and its mutants: 2× CATATG: whole site with CATATG core sequence; 2× CATTAG: whole site with CATTAG core sequence; m1-4g CATATG: mutation of first-quarter site with CATATG core sequence; m1-4g CATTAG: mutation of first-quarter site with CATTAG core sequence; and d12 is a negative control. (B) Designed four-base A/T gap core sequence p53 response element and mutant promoter constructs were co-transfected with p53 expression vectors into H1299 cells. The folds induction of luciferase promoter activity compared to the pcDNA3.0 as 1. Both 2- and 1.5-fold half-site core sequences of CATATG promoter activity were higher than CATTAG core sequence.
Mentions: The whole p53 response element sequence activation by p53 was higher than for the 1.5-fold half site (5). Is the four-base A/T gap core sequence as functional in the whole site as in the 1.5-fold half site as previously demonstrated? The four-base A/T gap mutants in the whole p53 response element sequence were cloned, and the promoter activity was examined by a luciferase activity assay (Figure 4A). The results indicated that the core CATATG activity activated by p53 is higher than for CATTAG in both the whole site and the 1.5-fold half site (Figure 4B). Two super p53 mutants, p53F46 and p53Δ364–393 (10,11), also can activate the p53 whole site with the CATATG core sequence, even more so than wild-type p53 (Figure 5).Figure 4.

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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