The TATA-less rat GAD65 promoter can be activated by Sp1 through non-consensus elements

Glutamic acid decarboxylase (GAD) 65 is one of two homologous proteins responsible for the synthesis of gamma-aminobutyric acid, the most ubiquitous inhibitory neurotransmitter. In order to characterize the DNA elements responsible for controlling GAD65 expression, we cloned the 5' flanking region of the rat GAD65 gene. A major, proximal and a minor, distal region of transcription initiation were located by RACE experiments. Sequence analysis revealed that the initiation sites are located within a region devoid of TATA boxes. We investigated the functional organization of the promoter by measuring the ability of 5' deletion mutants to drive the expression of a luciferase reporter gene. The major promoter was found to be located in the region encompassing the 100bp immediately upstream of the proximal transcription initiation site. A number of near consensus GC boxes and initiator elements are found in this region, but gel-shift assays suggest that they play only a minor role in transcription initiation. However, gel-shift assays and reporter gene assays suggest that Sp1 can bind to a region devoid of consensus Sp1 binding sites.