The objectives of this grant are to locate functionally the transcription termination signal(s) of the galactose operon of E.coli; to determine the nucleotide sequence of the signal(s); to assay the efficiency of termination of signal(s); if more than one signal is found, to study the relationship of the signals; and to compare the signal(s) to ones previously characterized. Since preliminary studies suggest the possibility of an as yet uncharacterized gene on the galactose operon, a further objective is to determine if such a gene exists. Sections of DNA generated from restriction enzyme digests will be inserted into a plasmid (pKG) developed for assaying transcription termination efficiency. Once the transcription termination signal(s) is/are functionally located, the nucleotide sequence of the termination region will be determined. In the event multiple transcription termination signals are discovered, their relationships in termination efficiency will be studied using the pKG plasmid. The possibility of an uncharacterized gene on this operon will be explored by inserting sections of DNA containing open reading frames into plasmids developed to generate proteins. The proteins will be injected into rabbits, which will produce antibodies against the proteins. The antibodies will then be used to probe E.coli proteins to determine if such a protein exists. The project is significant in contributing to an understanding of the role of transcription termination in the regulation of gene expression.
