Nonsense suppressortRNAs in the study of class II and class III gene expression and regulation.

摘要

The initial aim of this work involved the development and use of mammalian systems of nonsense mutation suppression to examine the mechanisms of eukaryotic class II gene expression and regulation. Herpes simplex virus type-1 (HSV-1) was chosen as a model system. A natural progression of this work involved the study of the molecular mechanisms of mammalian polIII transcription in vivo and in vitro.;Initial work involved the identification and propagation of conditional-lethal nonsense mutants of HSV-1 using a mammalian cell line which can be induced to express high levels of a human amber su;A novel means of efficiently regulating the expression of a su;The second aspect of this thesis involved an examination of the molecular mechanisms that govern transcription by polIII. A novel approach was adopted and involved use of the lac repressor protein as a reagent to probe various stages of polIII transcription to define the promoter disposition and functional properties of mammalian polIII transcription complexes. The effect on tRNA gene transcription, of varying the position of lac repressor protein upstream and downstream of the tRNA gene, was examined in vitro in HeLa cell nuclear extracts. Lac repressor differentially and reversibly interfered with different stages of transcription complex assembly and productive initiation. The mammalian polIII transcription complex extends at least 35 nucleotides (nts) upstream, and to within 10 nts downstream of the tRNA gene. Moreover, sequences directly upstream of the coding region remain accessible to DNA binding proteins throughout multiple rounds of transcription.;The lac operator/repressor system has also afforded a novel means of examining transcription elongation and termination by mammalian polIII. Lac repressor protein, appropriately positioned downstream of the tRNA gene coding region, reversibly blocked elongation by polIII in vitro in HeLa cell nuclear extracts. The results demonstrate that DNA binding proteins can modulate elongation and termination by polIII in vitro, and suggest that conditional factor-mediated blocks to elongation by polIII may function in vivo to attenuate transcription of class III genes. The ability to selectively arrest elongation by polIII at defined positions within the tRNA gene transcription unit has permitted the identification of discrete functional properties of paused mammalian polIII ternary complexes. (Abstract shortened by UMI.).