The Role of TFIIB in Transcription Activation

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DESCRIPTION (provided by applicant): Gene expression is regulated mainly at the level of transcription and transcription in turn, is regulated primarily at the level of initiation. Proper gene regulation is a critical event to maintain the homeostatic state of an organism. This is exemplified by diseases such as cancer, where improper gene regulation leads to uncontrolled cellular growth. Throughout the funding period of this proposal relevant information towards understanding the involvement of the general transcription factor TFIIB in the process of gene activation will be obtained. To comprehend how activator proteins work, a genetic strategy to identify physiological targets of the Pho4 and Adr1 transcription activators has been devised. Both proteins interact with TFIIB as part of their activation mechanism. The approach relies in the isolation of extragenic suppressors of the activation defect conferred by the deletion of these genes. To analyze the general role of TFIIB in transcription, a whole genome microarray analysis will be performed. The experiment will utilize TFIIB mutants at positions E62 and R78 that produce downstream shifts in transcription start site selection. To shed some light on the molecular basis of how do mutations at positions E62 and R78 of TFIIB affect gene expression, the conformational integrity of these mutants will be studied by means of circular dichroism (CD) analysis and by analyzing their susceptibility to proteases.