Abstract:
:VP16, a protein encoded by herpes simplex virus, has a well-characterized 78 amino acid acidic activation domain. When tethered to DNA, tandem repeats of an eight amino acid motif taken from this region stimulate the transcription of a nearby gene. This work addresses how these minimal activation motifs interact with a putative target, the general transcription factor TATA box binding protein (TBP), and the biological relevance of this mechanism of action. I developed novel biophysical techniques to discriminate among three possible mechanistic models that describe how reiterated peptide motifs could synergistically effect transcription: 1) the peptide motifs simultaneously bind to quasi-identical sites on TBP, producing a high-affinity bivalent interaction that holds the general transcription factor near the start site of transcription; 2) the binding of one recognition motif causes an allosteric effect that enhances the subsequent binding of additional peptide motifs; or 3) a high-affinity interaction between the peptide repeats and TBP does occur, but rather than being the result of a "bivalent" interaction, it results from the summation of multiple interactions between the target protein and the entire length of the peptide. I generated self-assembled monolayers (SAMs) that presented different densities of the activation motif peptide in a two-dimensional array to test for avidity effects. Surface plasmon resonance (SPR) was used to measure the amount of target (TBP) binding as a function of the peptide density; a marked increase in avidity above a characteristic, critical peptide surface density was found. Competitive inhibition experiments were performed to compare the avidity of peptide motifs, tandemly repeated two or four times, and single motifs separated by a flexible linker. Four iterations of the motif, preincubated with TBP, inhibited its binding to high-density peptide surfaces approximately 250-fold better than two iterations. Single peptide motifs joined by a flexible amino acid linker inhibited TBP binding to surface peptide nearly as well as four tandem repeats. The results favor mechanistic model 1: reiterated activation motifs interact with TBP through a high-affinity interaction that is the result of the cooperative effect of single motifs simultaneously binding to separate sites on TBP. This finding is consistent with the idea that DNA-bound activation domains trigger the transcription of a nearby gene by tethering the general transcription factor, TBP, near the start site of transcription.
journal_name
Biophys Jjournal_title
Biophysical journalauthors
Bamdad Cdoi
10.1016/S0006-3495(98)77640-4subject
Has Abstractpub_date
1998-10-01 00:00:00pages
1989-96issue
4eissn
0006-3495issn
1542-0086pii
S0006-3495(98)77640-4journal_volume
75pub_type
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