GlnR has been characterized as a central regulator governing most nitrogen metabolisms in many important actinomycetes. So far, the GlnR binding consensus sequences have been extensively studied, but with different motifs proposed, which has therefore brought confusion and impeded the understanding of the in-depth molecular mechanisms of GlnR-mediated transcriptional regulation. Here, a 30-nt GlnR-protected DNA sequence in the promoter of ginA in Amycolatopsis mediterranei was employed for precise characterization of GlnR binding consensus sequences. Site-by-site mutagenesis strategy combining with the Electrophoretic Mobility Shift Assay were employed, and a 5-nt GlnR Box was precisely defined as the basic unit for GInR binding. (C) 2015 Elsevier Inc. All rights reserved.