The long term objective of this work is to understand the biochemical reactions responsible for regulated transcription of the allantoin system transport genes. An important component of this catabolite repression. The allantoate permease gene, DAL5, has been chosen as a model system with which to study this type of regulation. We have identified the upstream activation sequence (UAS) responsible for transcriptional activation of DAL5 and shown that it binds one or more proteins. The objective of the present proposals is to significantly refine our understanding of the UAS structure by altering the 12-15 bases that have been shown to be functionally required for transcriptional activation. The effects of these alterations on ability to support transcriptional activation and protein binding to the UAS will be measured along with the effects of purine methylation on protein binding. The gene encoding the protein that binds to the DAL5 UAS will be cloned using one of several proposed strategies. The mechanisms of nitrogen catabolite repression (NCR) will be addressed using multiple strategies involving the use of both the DAL5 and CAR1 genes. The DAL5 UAS has been demonstrated to be the only DNA sequence information required for sensitivity of DAL5 gene expression to NCR. In contrast, an upstream repression element (URS), which behaves like an operator, has been implicated in NCR of the CAR1 gene. We have proposed detailed experiments to ascertain whether the URS is the only cis-acting element of CAR1 required for NCR sensitivity. Experiments are then proposed to determine whether protein phosphorylation is associated with NCR sensitivity of the CAR1 and DAL5 genes.
Showing the most recent 10 out of 113 publications
