The objective of the work described in this proposal is to gain an understanding of the control mechanisms operative in the regulation of eukaryotic gene expression, using adenovirus type 5 (Ad5) early region 2A as the model system. The gene product of early region 2A is a 529 amino acid single-stranded DNA binding protein (DBP) that is involved in viral DNA replication, the host range of the virus, virus assembly, the transcription of early region 4 (E4) and the stability of mRNAs from early regions 1A and 1B. The immediate goal of the proposed work is to precisely define within the DBP domains that are involved in DNA binding and DNA replication. Three regions have been identified to date, and it is possible that other regions of the protein are also involved. Each region that is identified will be subjected to saturation mutagenesis to define the contribution of individual amino acids to the function of the DBP in DNA binding and DNA replication. The mutant DBPs will be assayed for their ability to participate in the initiation and elongation reactions of adenovirus DNA replication in vitro. DBP mutants will be used to examine the role of the protein in controlling transcription from the E4 promoter and the stability of mRNAs from E1A and E1B. Steady-state levels of RNAs will be measured by Northern blot analysis, and rates of synthesis and decay of mRNAs measured by nuclear run-on and continuous labeling techniques, respectively. A possible role for the DBP in autoregulation will be explored by examining the affinity of the protein for homologous and heterologous mRNAs, and its effect on the in vitro translation of these RNAs. RNAs will be synthesized in vitro, binding to various regions of E2A and other mRNAs measured by filter binding assays, and the effect of the DBP on the in vitro translation of these mRNAs examined. An association between the DBP and the adenovirus DNA polymerase has been postulated to be important in the precessive nature of replication, but such an association has never been demonstrated. We will use synthetic peptides corresponding to regions of the DBP to determine if this potential association can be disrupted, and if so, which regions of the DBP are involved. The successful outcome of these experiments will provide insight into the molecular mechanisms by which eukaryotic gene expression is regulated.
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