Somesh Baggavalli P, Reid James, Liu Wei-Feng, Søgaard T Max M, Erdjument-Bromage Hediye, Tempst Paul, Svejstrup Jesper Q
Cancer Research UK London Research Institute, Clare Hall Laboratories, Blanche Lane, South Mimms, Herts EN6 3LD, United Kingdom.
Cell. 2005 Jun 17;121(6):913-23. doi: 10.1016/j.cell.2005.04.010.
In order to study mechanisms and regulation of RNA polymerase II (RNAPII) ubiquitylation and degradation, highly purified factors were used to reconstitute RNAPII ubiquitylation in vitro. We show that arrested RNAPII elongation complexes are the preferred substrates for ubiquitylation. Accordingly, not only DNA-damage-dependent but also DNA-damage-independent transcriptional arrest results in RNAPII ubiquitylation in vivo. Def1, known to be required for damage-induced degradation of RNAPII, stimulates ubiquitylation of RNAPII only in an elongation complex. Ubiquitylation of RNAPII is dependent on its C-terminal repeat domain (CTD). Moreover, CTD phosphorylation at serine 5, a hallmark of the initiating polymerase, but not at serine 2, a hallmark of the elongating polymerase, completely inhibits ubiquitylation. In agreement with this, ubiquitylated RNAPII is hypophosphorylated at serine 5 in vivo, and mutation of the serine 5 phosphatase SSU72 inhibits RNAPII degradation. These results identify several mechanisms that confine ubiquitylation of RNAPII to the forms of the enzyme that arrest during elongation.
为了研究RNA聚合酶II（RNAPII）泛素化和降解的机制及调控，我们使用高度纯化的因子在体外重建RNAPII泛素化过程。我们发现停滞的RNAPII延伸复合物是泛素化的优先底物。因此，不仅DNA损伤依赖性转录停滞，而且DNA损伤非依赖性转录停滞都会在体内导致RNAPII泛素化。已知Def1是损伤诱导的RNAPII降解所必需的，它仅在延伸复合物中刺激RNAPII的泛素化。RNAPII的泛素化依赖于其C末端重复结构域（CTD）。此外，起始聚合酶的标志——丝氨酸5处的CTD磷酸化，但延伸聚合酶的标志——丝氨酸2处的磷酸化，完全抑制泛素化。与此一致的是，体内泛素化的RNAPII在丝氨酸5处低磷酸化，并且丝氨酸5磷酸酶SSU72的突变会抑制RNAPII降解。这些结果确定了几种机制，这些机制将RNAPII的泛素化限制在延伸过程中停滞的酶形式上。
