The first step in the initiation of protein synthesis is catalyzed by the eukaryotic translation initiation factor 2 (eIF-2). Regulation of the activity of eIF-2 is a common final step in a number of pathways that coordinate the overall rate of translation with the metabolic needs of the cell. Increased phosphoryla-tion of eIF-2 alpha and translational repression have been described in viral infection, growth and differentiation, and metabolic alterations. To examine the regulation of eIF-2 expression at the transcriptional level, we have cloned and characterized the promoter for the gene for the alpha subunit of eIF-2. Analysis of the promoter using a variety of techniques has revealed evidence for a number of specific protein-DNA interactions. We have identified a cluster of five DNase I hypersensitive sites within a 25 kb region surrounding the promoter. None of these elements shares sequence homology with the binding sites of known regulatory factors, suggesting that eIF-2 alpha transcription may be regulated through a novel series of regulatory elements and factors. The most prominent element consists of two adjacent protein binding sites composed of palindromic sequences. Mutation of this sites reduces expression of eIF-2 alpha. The protein binding to this element, termed the alpha palindrome binding protein or alpha-PAL, has been purified to homogeneity from K562 and TIL nuclear extracts using conventional and DNA affinity chromatography. Several cDNA clones are now being sequenced and characterized. Using homogenous alpha-PAL, we have identified a consensus binding sequence by selection from a random oligonucleotide pool. Search of the Genebank database has identified over 40 TATA-less housekeeping genes that utilize the alpha- PAL binding sequence. The work described in this study is directly aimed at understanding transcriptional regulation. eIF-2 alpha is an essential housekeeping gene; as a group, housekeeping genes have been relatively little studied. A detailed understanding of transcriptional regulation may also facilitate the identification of therapeutic agents which act through alterations in specific gene expression.
