Transcriptional regulation of the nitrate reductase gene in Chlorella vulgaris: identification of regulatory elements controlling expression

Nitrate reductase (NR), the rate-limiting and primary control point of the nitrate assimilation pathway, is regulated at transcriptional and post-tran scriptional levels. To better understand how NR is regulated at the transcr iptional level in Chlorella vulgaris, studies were performed to identify th e factors regulating NR expression. Sequence analysis of the NR promoter id entified a number of potential sites that were investigated by mobility shi ft assays. Of the protein-binding sites found, several, such as USF and E2F are likely involved in the basal NR gene transcription. An indirect repeat sequence with similarity to the sequence recognized by the common plant re gulatory factor was identified and shown to bind a Chlorella protein in vit ro. Mobility shift assays of a consensus GATA element indicated that protei ns able to specifically bind this element are constitutive, regardless of t he nitrogen source. Mutational analysis revealed that the GATA core is requ ired for protein binding in vitro. Additionally, a NIT2 zinc-finger domain/ glutathione S-transferase fusion protein was found to bind in a sequence-sp ecific manner to this site. In Neurospora crassa and Aspergillus nidulans, consensus GATA elements are bound by the NIT2 protein, which has a major ro le in the expression of nitrogen-metabolizing genes. The ability of the GAT A element to function as a nitrogen response element (NRE) was further exam ined by in vivo footprinting. The protection of guanines in the GATA core a nd surrounding region was observed only in cells grown in the presence of n itrate. These data confirm that a single GATA element has a role in regulat ing the expression of NR in C. vulgaris.