Regulation of gene expression occurs at the level of transcription, processing, transport, and mRNA translation. The primary goal of this section is to investigate the transcriptional and translational control mechanisms responsible for regulated gene expression. To identify components required for transcription of genes by RNA polymerase II, transcriptionally active nuclear extracts were fractionated using FPLC and DNA affinity chromatography. Sequence- specific DNA binding factors required for transcription of the adenovirus 2 major late promoter and the eIF-2 alpha promoter were identified by DNase I footprinting, mobility shift assays, and their function(s) studied using a novel assay system based on binding to synthetic oligonucleotides of their cognate promoter sequences. In addition, a UV protein-DNA crosslinking procedure was developed which is able to directly identify sequence specific DNA-binding factors at very early stages of their purification. Using these methods, trans acting factors binding to new Ad2 MLP promoter elements in addition to those recognizing the TATAA and upstream promoter sequence (UPS) have been identified. Characterization of the eIF-2 alpha promoter also identified new cis-acting promoter elements. During infection by adenoviruses and influenza viruses, activation of host cell ds-RNA dependent eIF-2 alpha kinase is prevented by VA1 RNA and an unknown, but functionally similar, influenza gene product. The mechanisms by which certain cell lines escape viral takeover of their translational factors which participate in virus- host interactions, the genes for subunits of eIF-2 and eIF-2B are being identified and sequenced. In addition, binding of VA1 RNA to interferon-induced P68 eIF-2 alpha kinase has been shown to inhibit activation by double-stranded RNA.
