Yoganathan T, Horikoshi M, Hasegawa S, Roeder R G, Sells B H
Department of Molecular Biology and Genetics, College of Biological Science, University of Guelph, Ontario, Canada.
Biochem J. 1992 Aug 1;285 ( Pt 3)(Pt 3):721-3. doi: 10.1042/bj2850721.
We analysed transcription of the gene for the ribosomal protein (rp) L32 of the mouse, which is transcribed in mouse L1210 nuclear extracts in vitro. The rpL32 gene lacks a canonical TATA box. Hence it has been suggested that this gene has an alternative transcription pathway not requiring transcription factor IID (TFIID). Selective inactivation of TFIID in nuclear extract completely abolished the transcription of rpL32 in vitro. Selective inactivation was restored by the addition of cloned and purified yeast TFIID (yTFIID), indicating that this TATA-less rpL32 promoter utilizes TFIID for its transcription initiation. Furthermore, addition of an oligonucleotide-containing TATA sequence interfered with the rpL32 transcription and this was overcome by the addition of yTFIID. To further examine the stage of involvement of TFIID in rpL32 transcription, TATA oligonucleotide was added to nuclear extract before and after the formation of the transcription complex. The results reveal that TFIID associates with the pre-initiation complex and that this complex is largely resistant to added TATA oligonucleotide. Our results show, for the first time, that the TATA-less rpL32 gene utilizes TFIID for transcription initiation.
我们分析了小鼠核糖体蛋白（rp）L32基因的转录情况，该基因在体外可在小鼠L1210核提取物中进行转录。rpL32基因缺乏典型的TATA框。因此，有人提出该基因具有一条不依赖转录因子IID（TFIID）的替代转录途径。核提取物中TFIID的选择性失活完全消除了rpL32在体外的转录。通过添加克隆并纯化的酵母TFIID（yTFIID）可恢复选择性失活，这表明这个无TATA的rpL32启动子利用TFIID进行转录起始。此外，添加含TATA序列的寡核苷酸会干扰rpL32的转录，而添加yTFIID可克服这一干扰。为了进一步研究TFIID参与rpL32转录的阶段，在转录复合物形成之前和之后向核提取物中添加TATA寡核苷酸。结果显示，TFIID与起始前复合物结合，并且该复合物对添加的TATA寡核苷酸具有很大的抗性。我们的结果首次表明，无TATA的rpL32基因利用TFIID进行转录起始。
