Regulation of gene expression occurs at the level of transcription, processing, transport and mRNA translation. The primary goals of this section are to investigate the transcriptional and translational control mechanisms responsible for regulated gene expression. To identify components required for transcription of mRNA by RNA polymerase II, distinct preinitiation, initiation and elongation transcription complexes have been purified and characterized. The protein factors associated with the specific DNA fragment containing promoter and control sequences are being used to 1) identify transcription factors and 2) to generate monoclonal antibodies to identify their partial functional activities during transcriptional initiation. Factors are being purified by DNA affinity chromatographic techniques, under conditions where we have demonstrated specific interactions between protein and DNA promoter sequences required for transcriptional initiation. A 90 kD polypeptide complex has been purified to apparent homogeneity using these techniques and is currently being evaluated for its function during transcription. The simplicity of globin gene expression in a mouse model of Beta-thalassemia has been useful in examining the regulation of translation and transcription in vivo. Deletion of the Beta-major globin gene is accompanied by a compensatory increase in Beta-minor globin gene expression. The ratio of Beta-minor: Alpha globin, normally 0.2, increases from 0.2 to 0.8. However, compensation is almost totally the result of increased translational initiation on Beta-minor globin mRNA since the ratio of Beta-minor to Alpha transcriptional initiation and mRNA remain at 0.2. To understand the regulation of translation factors, the genes for the Alpha, Beta, and Gamma subunits of eIF-2 are being identified and sequenced. Oligonucleotide probes based on the eIF-2Alpha amino-terminal sequences have tentatively identified genomic clones for eIF-2Alpha. In addition, the mechanism of translational regulation during adenovirus and influenza viral infection has been shown to involve the modulation of eIF-2 dsRNA kinase activity.
