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A tryptophan-rich peptide acts as a transcription activation domain.

Lin CH, Lin G, Chang CP, Wang CC - BMC Mol. Biol. (2010)

Bottom Line: While many sequence patterns and motifs have been defined for DBDs, ADs do not share easily recognizable motifs or structures.Mutations which substituted tryptophan residues for both of the non-tryptophan residues in the pentapeptide (resulting in W5) significantly enhanced its activity (~1.8-fold), while mutations which substituted aromatic residues with alanine residues severely impaired its activity.Since W7 shares no sequence homology or features with any known transcription activators, it may represent a novel class of AD.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Life Science, National Central University, Jung-li 32001, Taiwan.

ABSTRACT

Background: Eukaryotic transcription activators normally consist of a sequence-specific DNA-binding domain (DBD) and a transcription activation domain (AD). While many sequence patterns and motifs have been defined for DBDs, ADs do not share easily recognizable motifs or structures.

Results: We report herein that the N-terminal domain of yeast valyl-tRNA synthetase can function as an AD when fused to a DNA-binding protein, LexA, and turn on reporter genes with distinct LexA-responsive promoters. The transcriptional activity was mainly attributed to a five-residue peptide, WYDWW, near the C-terminus of the N domain. Remarkably, the pentapeptide per se retained much of the transcriptional activity. Mutations which substituted tryptophan residues for both of the non-tryptophan residues in the pentapeptide (resulting in W5) significantly enhanced its activity (~1.8-fold), while mutations which substituted aromatic residues with alanine residues severely impaired its activity. Accordingly, a much more active peptide, pentatryptophan (W7), was produced, which elicited ~3-fold higher activity than that of the native pentapeptide and the N domain. Further study indicated that W7 mediates transcription activation through interacting with the general transcription factor, TFIIB.

Conclusions: Since W7 shares no sequence homology or features with any known transcription activators, it may represent a novel class of AD.

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Assays of the transcriptional activity of W7 using the Gal4-based system. (A) Transcriptional assays. W7 and (WYDWW)2 were expressed as fusions to the Gal4-DBD, and the abilities of the resulting fusion proteins to turn on the reporter genes (HIS3 and MEL1), which are controlled by distinct Gal4-responsive promoters, were tested. (B) Quantitative assays of β-gal activity. (C) Western blot analysis of the expressions of Gal4-DBD fusion proteins. Upper panel, LexA fusion protein; lower panel, PGK (as a loading control). The numbers (circled) in A, B, and C denote the constructs shown in A.
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Figure 5: Assays of the transcriptional activity of W7 using the Gal4-based system. (A) Transcriptional assays. W7 and (WYDWW)2 were expressed as fusions to the Gal4-DBD, and the abilities of the resulting fusion proteins to turn on the reporter genes (HIS3 and MEL1), which are controlled by distinct Gal4-responsive promoters, were tested. (B) Quantitative assays of β-gal activity. (C) Western blot analysis of the expressions of Gal4-DBD fusion proteins. Upper panel, LexA fusion protein; lower panel, PGK (as a loading control). The numbers (circled) in A, B, and C denote the constructs shown in A.

Mentions: We next tested whether the transcriptional activities of W7 and (WYDWW)2 were promoter-specific, and whether they were affected by the DBD used. To this end, W7 and (WYDWW)2 were assayed in a Gal4-based system, where the AD was fused in-frame to the Gal4-DBD cloned in pGBKT7 (which carries a TRP1 marker), and the reporter genes used were HIS3 and MEL1 (which encodes α-galactosidase) under the control of two completely heterologous Gal4-responsive upstream activating sequences and promoter elements, GAL1 and MEL1, respectively. As shown in Figure 5, both Gal4-DBD fusion proteins turned on the reporter genes; transformants carrying either of these two fusion constructs (Gal4-DBD-W7 or Gal4-DBD-(WYDWW)2) grew robustly and turned blue on selection medium containing X-α-gal but lacking tryptophan and histidine (Figure 5A, numbers 3 and 4), suggesting that both of these peptides acted as ADs in Gal4-DBD fusion proteins. Thus, the transcriptional activities of these two peptides were non-promoter-specific and were operational in both LexA- and Gal4-DBD-responsive reporter genes.


A tryptophan-rich peptide acts as a transcription activation domain.

Lin CH, Lin G, Chang CP, Wang CC - BMC Mol. Biol. (2010)

Assays of the transcriptional activity of W7 using the Gal4-based system. (A) Transcriptional assays. W7 and (WYDWW)2 were expressed as fusions to the Gal4-DBD, and the abilities of the resulting fusion proteins to turn on the reporter genes (HIS3 and MEL1), which are controlled by distinct Gal4-responsive promoters, were tested. (B) Quantitative assays of β-gal activity. (C) Western blot analysis of the expressions of Gal4-DBD fusion proteins. Upper panel, LexA fusion protein; lower panel, PGK (as a loading control). The numbers (circled) in A, B, and C denote the constructs shown in A.
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Related In: Results  -  Collection

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Figure 5: Assays of the transcriptional activity of W7 using the Gal4-based system. (A) Transcriptional assays. W7 and (WYDWW)2 were expressed as fusions to the Gal4-DBD, and the abilities of the resulting fusion proteins to turn on the reporter genes (HIS3 and MEL1), which are controlled by distinct Gal4-responsive promoters, were tested. (B) Quantitative assays of β-gal activity. (C) Western blot analysis of the expressions of Gal4-DBD fusion proteins. Upper panel, LexA fusion protein; lower panel, PGK (as a loading control). The numbers (circled) in A, B, and C denote the constructs shown in A.
Mentions: We next tested whether the transcriptional activities of W7 and (WYDWW)2 were promoter-specific, and whether they were affected by the DBD used. To this end, W7 and (WYDWW)2 were assayed in a Gal4-based system, where the AD was fused in-frame to the Gal4-DBD cloned in pGBKT7 (which carries a TRP1 marker), and the reporter genes used were HIS3 and MEL1 (which encodes α-galactosidase) under the control of two completely heterologous Gal4-responsive upstream activating sequences and promoter elements, GAL1 and MEL1, respectively. As shown in Figure 5, both Gal4-DBD fusion proteins turned on the reporter genes; transformants carrying either of these two fusion constructs (Gal4-DBD-W7 or Gal4-DBD-(WYDWW)2) grew robustly and turned blue on selection medium containing X-α-gal but lacking tryptophan and histidine (Figure 5A, numbers 3 and 4), suggesting that both of these peptides acted as ADs in Gal4-DBD fusion proteins. Thus, the transcriptional activities of these two peptides were non-promoter-specific and were operational in both LexA- and Gal4-DBD-responsive reporter genes.

Bottom Line: While many sequence patterns and motifs have been defined for DBDs, ADs do not share easily recognizable motifs or structures.Mutations which substituted tryptophan residues for both of the non-tryptophan residues in the pentapeptide (resulting in W5) significantly enhanced its activity (~1.8-fold), while mutations which substituted aromatic residues with alanine residues severely impaired its activity.Since W7 shares no sequence homology or features with any known transcription activators, it may represent a novel class of AD.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Life Science, National Central University, Jung-li 32001, Taiwan.

ABSTRACT

Background: Eukaryotic transcription activators normally consist of a sequence-specific DNA-binding domain (DBD) and a transcription activation domain (AD). While many sequence patterns and motifs have been defined for DBDs, ADs do not share easily recognizable motifs or structures.

Results: We report herein that the N-terminal domain of yeast valyl-tRNA synthetase can function as an AD when fused to a DNA-binding protein, LexA, and turn on reporter genes with distinct LexA-responsive promoters. The transcriptional activity was mainly attributed to a five-residue peptide, WYDWW, near the C-terminus of the N domain. Remarkably, the pentapeptide per se retained much of the transcriptional activity. Mutations which substituted tryptophan residues for both of the non-tryptophan residues in the pentapeptide (resulting in W5) significantly enhanced its activity (~1.8-fold), while mutations which substituted aromatic residues with alanine residues severely impaired its activity. Accordingly, a much more active peptide, pentatryptophan (W7), was produced, which elicited ~3-fold higher activity than that of the native pentapeptide and the N domain. Further study indicated that W7 mediates transcription activation through interacting with the general transcription factor, TFIIB.

Conclusions: Since W7 shares no sequence homology or features with any known transcription activators, it may represent a novel class of AD.

Show MeSH