Expression of qa-1F activator protein: identification of upstream binding sites in the qa gene cluster and localization of the DNA-binding domain.

Abstract

The qa-1F regulatory gene of Neurospora crassa encodes an activator protein required for quinic acid induction of transcription in the qa gene cluster. This activator protein was expressed in insect cell culture with a baculovirus expression vector. The activator binds to 13 sites in the gene cluster that are characterized by a conserved 16-base-pair sequence of partial dyad symmetry. One site is located between the divergently transcribed qa-1F and qa-1S regulatory genes, corroborating prior evidence that qa-1F is autoregulated and controls expression of the qa-1S repressor. Multiple upstream sites located at variable positions 5' to the qa structural genes appear to allow for greater transcriptional control by qa-1F. Full-length and truncated activator peptides were synthesized in vitro, and the DNA-binding domain was localized to the first 183 amino acids. A 28-amino acid sequence within this region shows striking homology to N-terminal sequences from other lower-eucaryotic activator proteins. A qa-1F(Ts) mutation is located within this putative DNA-binding domain.