Hepatitis B virus (HBV) X protein activates many viral and cellular genes in trans and functional disruption of the p53 tumor suppressor gene product occurs when X protein is transiently expressed in the cytoplasm of cultured cells. We have carried out investigations to determine the exact location of X protein in X gene transfected cells by using a¯uorescent staining technique as well as by biochemical analyses. Aggregation of mitochondrial structures became evident at the periphery of nucleus in the cytoplasm of X transfected cells. X protein was found associated with the aggregated mitochondrial structures. Furthermore, transiently expressed p53 protein co-localized with X protein in X transfected cells. However, the appearance of aggregated mitochondrial structures at the nuclear periphery was independent of the presence of p53 protein in X transfected cells. X protein expression also caused an appearance of TUNEL positive nucleus, cytochrome c release from mitochondrial, the decrease of mitochondrial membrane potential and the membrane blebbing of X transfected cells, which are characteristic of cell death. Our data suggest that X protein causes an abnormal aggregation of mitochondrial structures in the cell, which may be eventually connected with cell death.

Eukaryotic cells are thought to contain a single TATA-binding protein (TBP) that directs transcription by cellular RNA polymerases. Here we report a cell type-specific TBP-related factor (TRF) that can form a stable TRF/IIA/IIB TATA DNA complex and substitute for TBP in directing RNA polymerase II transcription in vitro. Transfection studies reveal that TRF can differentially mediate activation by some enhancer proteins but not others. Like TBP, TRF forms a stable complex containing multiple novel subunits, nTAFs. Antibody staining of embryos and polytene chromosomes reveals cell type-specific expression and gene-selective properties consistent with the shaker/male sterile phenotype of trf mutants. These findings suggest TRF is a homolog of TBP that functions to direct tissue- and gene-specific transcription.

It has been generally accepted that the TATA binding protein (TBP) is a universal mediator of transcription by RNA polymerase I, II, and III. Here we report that the TBP-related factor TRF1 rather than TBP is responsible for RNA polymerase III transcription in Drosophila. Immunoprecipitation and in vitro transcription assays using immunodepleted extracts supplemented with recombinant proteins reveals that a TRF1:BRF complex is required to reconstitute transcription of tRNA, 5S and U6 RNA genes. In vivo, the majority of TRF1 is complexed with BRF and these two proteins colocalize at many polytene chromosome sites containing RNA pol III genes. These data suggest that in Drosophila, TRF1 rather than TBP forms a complex with BRF that plays a major role in RNA pol III transcription.