Functional organization of the Aspergillus nidulans trpC promoter

Abstract

We investigated the functional organization of the Aspergillus nidulans trpC promoter by the sequential removal of sequences upstream of the major trpC mRNA cap site (+1). DNA fragments containing promoter mutations were fused to the Escherichia coli lacZ gene, and a novel method was used to select for integration of the fusion gene at the Aspergillus argB locus. beta-Galactosidase assays and S1 nuclease protection experiments demonstrated that the promoter mutations affected gene expression in three ways: (i) 5' deletions up to -82 resulted in variable increases in beta-galactosidase activity, depending on the growth conditions; (ii) a deletion from -67 to -11 did not alter the level of beta-galactosidase activity, but did give rise to mRNAs with aberrant 5' ends; and (iii) a 5' deletion with an endpoint at -11 and an internal deletion from -142 to -11 abolished gene expression. These results indicate that sequences upstream of -82 reduce transcription of the trpC gene and that distinct DNA sequence elements are required for expression versus correct initiation of transcription of the trpC gene. The sequences essential for trpC expression do not include the common eucaryotic promoter elements CCAAT and TATAAA. To our knowledge, this is the first functional analysis of a promoter from a fungus other than Saccharomyces cerevisiae.