A single point mutation in TFIIA suppresses NC2 requirement in vivo

Negative cofactor 2 (NC2) is a dimeric histone-fold complex that represses RNA polymerase II transcription through binding to TATA-box-binding protein (TBP) and inhibition of the general transcription factors TFIIA and TFIIB. Here we study molecular mechanisms of repression by human NC2 in vivo in yeast. Yeast NC2 genes are essential and can be exchanged with human NC2. The physiologically rele– vant regions of NC2 have been determined and shown to match the histone-fold dimerization motif. A suppressor screen based upon limiting concentrations of NC2β yielded a cold-sensitive mutant in the yeast TFIIA subunit Toa1. The single point mutation in Toa1 alleviates the requirement for both subunits of NC2. Biochemical characterization indicated that mutant (mt)-Toa1 dimerizes well with Toa2; it supports specific recognition of the TATA box by TBP but forms less stable TBP–TFIIA–DNA complexes. Wild-type but not the mt-Toa1 can relieve NC2 effects in purified trans– cription systems. These data provide evidence for a dimeric NC2 complex that is in an equilibrium with TFIIA after the initial binding of TBP to promoter TATA boxes

The evolution of diverse biological responses to DNA damage: insights from yeast and p53

The cellular response to ionizing radiation provides a conceptual framework for understanding how a yeast checkpoint system, designed to make binary decisions between arrest and cycling, evolved in a way as to allow reversible arrest, senescence or apoptosis in mammals. We propose that the diversity of responses to ionizing radiation in mammalian cells is possible because of the addition of a new regulatory control module involving the tumour-suppressor gene p53. We review the complex mechanisms controlling p53 activity and discuss how the p53 regulatory module enables cells to grow, arrest or die by integrating DNA damage checkpoint signals with the response to normal mitogenic signalling and the aberrant signalling engendered by oncogene activation