Signal transduction pathways converge ultimately at the level of transcriptional activation to produce specific patterns of gene expression in response to environmental stimuli. The initiation of transcription mediated by these signalling pathways is regulated by the coordinate expression and/ or activation of specific transcription factors that bind to the control regions of genes. Specific insights into the mechanisms underlying transcriptional activation have recently arisen from the studies of the structure and functions of these transcription factors. The CREB/ATF family of transcriptional transactivating proteins has only recently been discovered and appears to provide a link between the regulation of gene expression in response to activators of cellular signalling pathways and the regulation of gene expression by viral transactivating proteins. Understanding the nature and importance of protein/protein interactions in the mechanisms of eukaryotic gene expression is essential to understanding the normal and aberrant regulation of gene transcription. We have recently developed a rapid and sensitive technique which allows the detection of these interactions between mammalian transcription factors. Utilizing 125I-labelled recombinant CREB and ATF-2 peptides to probe western blots of HeLa nuclear extracts, we have identified multiple separate nuclear factors that form specific protein/protein interactions with these leucine zipper-containing transcriptional regulatory proteins. In this application, we propose to utilize this technique to isolate cDNA clones expressing proteins that form specific protein/protein interactions with CREB and/or ATF-2. In addition, we will perform detailed analyses of the DNA-binding and transcriptional characteristics of several CREB/ATF proteins. The assay of protein/protein interactions we have developed will allow a systematic approach to identifying and cloning proteins involved in the control of eukaryotic transcriptional regulation. The identification and characterization of the components of eukaryotic transcription complexes will allow studies that address the molecular mechanisms of normal and aberrant control of cellular gene expression.
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