Design of a genetic screen to identify novel components of the transcriptional machinery.

摘要

We designed and engineered a yeast genetic screen to isolate novel transcriptional machinery components such as elongation factors and chromatin remodelling components. When artificially recruited to a promoter, these components can stimulate gene expression. For example, fusion of known machinery components to the bacterial DNA binding domain (DBD) of LexA results in activation of test genes bearing a binding site for LexA. In a previous study, the coding region of the LexA DBD was fused to random bacterial DNA sequences and, surprisingly, 1% of all random sequences tested were able to activate transcription simply because they were negatively charged. By inserting the LexA site downstream of the transcriptional start site of our reporter genes, we predict that we will exclude these same negatively charged sequences, which do not have a specific role in transcription. The rationale is that at this location classical activators, which are negatively charged, cannot activate transcription. The ability of transcriptional machinery components to activate transcription from a downstream location, however, was demonstrated. In order to isolate transcriptional machinery components, we prepared a yeast genomic DNA library fused to DNA encoding the LexA DBD for transformation into a yeast strain containing reporters bearing a downstream LexA operator site. Using this system, we may isolate novel components of the transcriptional machinery as well as already identified components. This genetic screen should also be able to isolate transcriptional machinery components from higher eukaryotic organisms.