A Modified Reverse One-Hybrid Screen Identifies Transcriptional Activation Domains in PHYTOCHROME-INTERACTING FACTOR 3.

ABSTRACT: Transcriptional activation domains (TADs) are difficult to predict and identify, since they are not conserved and have little consensus. Here, we describe a yeast-based screening method that is able to identify individual amino acid residues involved in transcriptional activation in a high throughput manner. A plant transcriptional activator, PIF3 (phytochrome interacting factor 3), was fused to the yeast GAL4-DNA-binding Domain (BD), driving expression of the URA3 (Orotidine 5'-phosphate decarboxylase) reporter, and used for negative selection on 5-fluroorotic acid (5FOA). Randomly mutagenized variants of PIF3 were then selected for a loss or reduction in transcriptional activation activity by survival on FOA. In the process, we developed a strategy to eliminate false positives from negat

SUBMITTER: Dalton JC 

PROVIDER: S-EPMC4911399 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature