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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 activities of the pentapeptide and its tandem repeats. (A) Transcriptional assays. The pentapeptide, WYDWW, and its tandem repeats were expressed as fusions to the specific DNA-binding protein, LexA, and the abilities of the resulting fusion proteins to turn on the reporter genes (LEU2 and lacZ), which are controlled by distinct LexA-responsive promoters, were tested. (B) Quantitative assays of β-gal activity. (C) Western blot analysis of the expressions of LexA fusion proteins. Upper panel, LexA fusion protein; lower panel, PGK (as a loading control). The numbers (circled) in A, B, and C represent the constructs shown in A.
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Figure 2: Assays of the transcriptional activities of the pentapeptide and its tandem repeats. (A) Transcriptional assays. The pentapeptide, WYDWW, and its tandem repeats were expressed as fusions to the specific DNA-binding protein, LexA, and the abilities of the resulting fusion proteins to turn on the reporter genes (LEU2 and lacZ), which are controlled by distinct LexA-responsive promoters, were tested. (B) Quantitative assays of β-gal activity. (C) Western blot analysis of the expressions of LexA fusion proteins. Upper panel, LexA fusion protein; lower panel, PGK (as a loading control). The numbers (circled) in A, B, and C represent the constructs shown in A.

Mentions: While the pentapeptide, WYDWW, can function as an AD, its activity was relatively low, only ~50% of that of the positive control and ~30% of that of the segment containing residues 98~115 of the N domain (Figure 1B, numbers 1, 4, and 8). To enhance its activity, two or three tandem repeats of the pentapeptide sequence were cloned, and the activities of the resulting constructs were tested. As shown in Figure 2A and 2B, duplication of the pentapeptide, resulting in (WYDWW)2, strongly enhanced the activity (~3-fold increase relative to that of a single pentapeptide) (compare numbers 3 and 4). However, an additional replication of the sequence, resulting in (WYDWW)3, did not further enhance the activity; (WYDWW)3 exhibited activity comparable to that of (WYDWW)2 (compare numbers 4 and 5). Western blotting assays showed that these constructs expressed similar levels of LexA fusion proteins. Thus, changes in the transcriptional activity of these fusion constructs did not result from different protein expression levels (Figure 2C, numbers 3~5).


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 activities of the pentapeptide and its tandem repeats. (A) Transcriptional assays. The pentapeptide, WYDWW, and its tandem repeats were expressed as fusions to the specific DNA-binding protein, LexA, and the abilities of the resulting fusion proteins to turn on the reporter genes (LEU2 and lacZ), which are controlled by distinct LexA-responsive promoters, were tested. (B) Quantitative assays of β-gal activity. (C) Western blot analysis of the expressions of LexA fusion proteins. Upper panel, LexA fusion protein; lower panel, PGK (as a loading control). The numbers (circled) in A, B, and C represent the constructs shown in A.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Assays of the transcriptional activities of the pentapeptide and its tandem repeats. (A) Transcriptional assays. The pentapeptide, WYDWW, and its tandem repeats were expressed as fusions to the specific DNA-binding protein, LexA, and the abilities of the resulting fusion proteins to turn on the reporter genes (LEU2 and lacZ), which are controlled by distinct LexA-responsive promoters, were tested. (B) Quantitative assays of β-gal activity. (C) Western blot analysis of the expressions of LexA fusion proteins. Upper panel, LexA fusion protein; lower panel, PGK (as a loading control). The numbers (circled) in A, B, and C represent the constructs shown in A.
Mentions: While the pentapeptide, WYDWW, can function as an AD, its activity was relatively low, only ~50% of that of the positive control and ~30% of that of the segment containing residues 98~115 of the N domain (Figure 1B, numbers 1, 4, and 8). To enhance its activity, two or three tandem repeats of the pentapeptide sequence were cloned, and the activities of the resulting constructs were tested. As shown in Figure 2A and 2B, duplication of the pentapeptide, resulting in (WYDWW)2, strongly enhanced the activity (~3-fold increase relative to that of a single pentapeptide) (compare numbers 3 and 4). However, an additional replication of the sequence, resulting in (WYDWW)3, did not further enhance the activity; (WYDWW)3 exhibited activity comparable to that of (WYDWW)2 (compare numbers 4 and 5). Western blotting assays showed that these constructs expressed similar levels of LexA fusion proteins. Thus, changes in the transcriptional activity of these fusion constructs did not result from different protein expression levels (Figure 2C, numbers 3~5).

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