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-transcriptional levels. To better understand how NR is regulated at the transcriptional level in Chlorella vulgaris, studies were performed to identify the factors regulating NR expression. Sequence analysis of the NR promoter identified a number of potential sites that were investigated by mobility shift 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 regulatory factor was identified and shown to bind a Chlorella protein in vitro. Mobility shift assays of a consensus GATA element indicated that proteins able to specifically bind this element are constitutive, regardless of the nitrogen source. Mutational analysis revealed that the GATA core is required for protein binding in vitro. Additionally, a NIT2 zinc-finger domain/glutathione S-transferase fusion protein was found to bind in a sequence-specific manner to this site. In Neurospora crassa and Aspergillus nidulans, consensus GATA elements are bound by the NIT2 protein, which has a major role in the expression of nitrogen-metabolizing genes. The ability of the GATA element to function as a nitrogen response element (NRE) was further examined by in vivo footprinting. The protection of guanines in the GATA core and surrounding region was observed only in cells grown in the presence of nitrate. These data confirm that a single GATA element has a role in regulating the expression of NR in C. vulgaris.