Emili A, Kobayashi R, Ingles C J
Banting and Best Department of Medical Research and Department of Molecular and Medical Genetics, University of Toronto, Toronto, Canada M5G 1L6.
Proc Natl Acad Sci U S A. 1998 Sep 15;95(19):11122-7. doi: 10.1073/pnas.95.19.11122.
A sensitive in vitro crosslinking technique using a photoactive derivative of the chimeric activator LexA-E2F-1 was used to identify yeast proteins that might influence the response of RNA polymerase II to transcriptional activators. We found that a novel yeast protein, Xtc1p, could be covalently crosslinked to the activation domain of LexA-E2F-1 when this derivatized activator was bound to DNA upstream of an activator-responsive RNA polymerase II promoter. Because affinity chromatography experiments showed that Xtc1p also bound directly and specifically to the activation domains of E2F-1, the viral activator VP16, and the yeast activator Gal4p and copurified with the RNA polymerase II holoenzyme complex, Xtc1p may modulate the response of RNA polymerase II to multiple activators. Consistent with this notion, yeast strains deleted for the XTC1 gene exhibited pleiotropic growth defects, including temperature sensitivity, galactose auxotrophy, and a heightened sensitivity to activator overexpression, as well as an altered response to transcriptional activators in vivo.
利用一种使用嵌合激活因子LexA-E2F-1的光活性衍生物的灵敏体外交联技术，来鉴定可能影响RNA聚合酶II对转录激活因子反应的酵母蛋白。我们发现，当这种衍生化的激活因子与激活因子响应性RNA聚合酶II启动子上游的DNA结合时，一种新的酵母蛋白Xtc1p能够与LexA-E2F-1的激活结构域共价交联。由于亲和层析实验表明Xtc1p也直接且特异性地结合E2F-1、病毒激活因子VP16和酵母激活因子Gal4p的激活结构域，并与RNA聚合酶II全酶复合物共纯化，所以Xtc1p可能调节RNA聚合酶II对多种激活因子的反应。与此观点一致的是，缺失XTC1基因的酵母菌株表现出多效性生长缺陷，包括温度敏感性、半乳糖营养缺陷型以及对激活因子过表达的敏感性增加，以及在体内对转录激活因子的反应改变。
