Abstract
Abstract: :
Purpose: NRL (neural retina leucine zipper) is a bZIP transcription factor, which is preferentially expressed in the rod photoreceptors and is essential for rod differentiation. NRL is shown to regulate the expression of rhodopsin and other rod–specific genes working in concert with other transcription factors, such as CRX and SP4. The aim of the present study is to purify the NRL–containing protein complex(es) from the bovine retinal extracts and identify various protein components, in order to understand the mechanism(s) of transcriptional regulation. Methods: Bovine retinal extracts were prepared in phosphate–buffered saline supplemented with protease inhibitors. The extract was subjected to size–exclusion chromatography, and NRL–containing protein fractions were assayed by immunoblotting. These fractions were then immunoprecipitated using anti–NRL antibody and analyzed by immunoblotting. Results: Though NRL monomers are 29–35 kDa, most of the NRL protein was obtained in the high molecular weight fraction (150 – 670 kDa) from the gel–filtration column. Immunoblot analysis of NRL–immunoprecipitate showed that NRL–complex(es) contain a number of proteins, which are components of the basal transcription machinery, such as TBP, TFIIA, TFIIB and PARP (poly–ADP ribose polymerase). Mass–spec analysis of other protein bands is in progress. Conclusions:Our results suggest that NRL may activate the expression of target genes by recruiting basal transcriptional complexes to promoter elements. These studies should identify additional co–activators or repressors that participate in controlling gene expression in mature rod photoreceptors.
Keywords: protein purification and characterization • photoreceptors