The minimal transactivation domain of the basic motif–leucine zipper protein NRL interacts with TATA–binding protein

Abstract

Abstract: : Purpose: To determine the minimum functional region of the transactivation domain for the rod photoreceptor specific bZIP protein NRL, in order to delineate biochemical mechanisms of transcriptional activation. Methods: A series of progressive N–terminal NRL deletions were generated and cloned into the yeast two hybrid vector pHybLexZeo (Invitrogen). Six large and six small internal fragments of NRL, and one internal deletion of NRL were also generated. These constructs were used to transform yeast strain L40, which were subsequently grown under selective conditions (minus–His, +zeo, +50mM 3–AT, +X–gal). Several of the NRL fragments were transferred into the pGBKT7 vector to verify their ability to activate a reporter gene in a different strain of yeast. Interaction of NRL regions with TBP was examined using in vivo and in vitro co–immunoprecipitation assays. Results: Experiments using NRL deletion constructs in yeast demonstrated that NRL transactivation was detected even when amino acids 1 to 30 were removed. Subsequent deletions after amino acid 75 showed little or no transactivation ability when grown under selection on X–gal containing media. The smallest internal NRL fragment containing transactivation function comprised of amino acids 40–74. NRL amino acids 1 to 57 were unable to transactivate in the yeast assay. Full length NRL and TBP were present in the same complex in bovine retinal nuclear extracts. The N–terminal region of NRL was observed to interact with the C–terminal region of TBP. Conclusions: The minimal transactivation domain of NRL is from amino acids 40 to 74 and a TBP binding domain overlaps with this region. Our data suggests that the function of NRLâ|*128*|^TMs transactivation domain is to recruit the general transcription machinery to the promoter. These studies should provide a better understanding of NRLâ|*128*|^TMs function on the regulation of photoreceptor specific genes.

Keywords: gene/expression • retinal development • photoreceptors