Gnatt A, Fu J, Kornberg R D
Department of Structural Biology, Stanford University School of Medicine, Stanford California, 94305, USA.
J Biol Chem. 1997 Dec 5;272(49):30799-805. doi: 10.1074/jbc.272.49.30799.
Minimal templates were devised for the efficient generation of yeast RNA polymerase II transcription elongation complexes. A 33-base pair DNA with a 15-residue dC tail at one 3'-end supported the formation of a complex containing the polymerase paused at nucleotide 11 of the duplex region and an RNA of 14-16 residues. The same template could yield an arrested complex with the enzyme at nucleotide 13-15 and RNA of 15-17 residues. These complexes were stable for at least a week under various conditions and could be resolved by gel electrophoresis or purified by ion exchange chromatography. The purified paused complex formed crystals capable of x-ray diffraction to 3.5 A resolution. The complex remained active in the crystal and, in the presence of nucleoside triphosphates, could efficiently extend the transcript in situ.
设计了最小模板以高效生成酵母RNA聚合酶II转录延伸复合物。一个在3'端之一带有15个残基dC尾的33碱基对DNA支持形成一种复合物，该复合物包含在双链区核苷酸11处暂停的聚合酶以及14 - 16个残基的RNA。相同的模板可以产生一种停滞复合物，其中酶位于核苷酸13 - 15处，RNA为15 - 17个残基。这些复合物在各种条件下至少稳定一周，并且可以通过凝胶电泳解析或通过离子交换色谱纯化。纯化的暂停复合物形成了能够进行X射线衍射至3.5埃分辨率的晶体。该复合物在晶体中保持活性，并且在核苷三磷酸存在的情况下，可以在原位有效地延伸转录本。
