| Control of gene expression has proven to be a complex process with many facets of regulation. Transcription elongation is emerging as an important aspect of gene regulation though a thorough understanding of its role is not clear. The transcription elongation factor TFIIS, encoded by the PPR2 gene, has been well-characterized in vitro regarding its ability to facilitate cleavage and read-through by RNA polymerase II arrested at a block to elongation. However the in vivo role of TFIIS remains to be elucidated. Using two-hybrid analysis, a library of genomic fragments was screened for interaction with TFIIS. The screen identified three candidates, MAC1, RAD51, and MDJ1 for in vivo interaction with TFIIS. Each of the candidates also interacted with TFIIS in vitro when expressed as fusions to GST. The MAC1 gene encodes a metal-dependent transcription factor involved in regulation of copper and iron homeostasis within the cell. RAD51 is involved in repair of double strand breaks and homologous recombination. MDJ1 is a mitochondrial DnaJ homolog required for proper mitochondrial function.; Damage to DNA in the form of chemical lesions is capable of causing RNA polymerase to arrest, which may require THIS as part of the repair process. An investigation of the response of cells to DNA damaging agents revealed a novel phenotype for THIS null mutants (ppr2Δ). Cells missing TFIIS exhibit decreased sensitivity to high levels of hydrogen peroxide and tert-butyl peroxide. The oxidative stress phenotype is evident during both exponential and stationary phase growth. Catalase activity appears to be identical in wt and ppr2Δ cultures.; While THIS is not an essential gene, ppr2Δ mutants exhibit sensitivity to the nucleotide analog 6-azauracil (6AU), which depletes cellular nucleotide pools by inhibiting IMP dehydrogenase. DNA microarray analysis was performed to look globally at transcriptional differences between wt and ppr2Δ cells in the presence or absence of 6AU. Both wt and ppr2Δ cells exhibited similar induction of IMP dehydrogenase, as well as other genes involved in nucleotide metabolism, in response to 6AU exposure. In addition, genes involved in sterol and sulfur metabolism were identified as differentially expressed between wt and ppr2Δ cells. |
