Krummel B, Chamberlin M J
Department of Biochemistry, University of California, Berkeley 94720.
Biochemistry. 1989 Sep 19;28(19):7829-42. doi: 10.1021/bi00445a045.
We have studied the properties and structures of a series of Escherichia coli RNA polymerase ternary complexes formed during the initial steps of RNA chain initiation and elongation. Five different templates were used that contained the bacteriophage T7 A1 promoter or the E. coli Tac or the lac UV5 promoter, as well as variant templates with alterations in the initial transcribed regions. The majority of ternary complexes bearing short transcripts (from two to nine nucleotides) are highly unstable and cannot be easily studied. This includes transcripts from the phage T7 A1 promoter, for which the stability of complexes bearing transcripts as short as four nucleotides has previously been postulated. However, with one Tac promoter template, RNA polymerase forms ternary complexes with transcripts as short as five nucleotides that are stable enough for biochemical study. We describe several approaches to identifying and isolating such stable complexes and show that stringent criteria are needed in carrying out such experiments if the results are to be meaningful. Deoxyribonuclease I (DNase I) footprinting has been used to probe the general structure of the stable ternary complexes formed as the polymerase begins transcription and moves away from the start site. The enzyme undergoes a sequence of structural changes during initiation and transition to an elongating complex. Complexes with five to eight nucleotide transcripts, designated initial transcribing complexes (ITC), have identical footprints; they all retain the sigma factor and have a slightly extended DNase I footprint (-57 to +24) as compared to the open promoter complex (-57 to +20). ITC complexes all show a region of marked DNase I hypersensitivity in the -25 region that may reflect bending or distortion of the DNA template. Complexes with 10 or 11 nucleotide transcripts, designated initial elongating complexes (IEC), have lost the sigma factor and have a slightly reduced and shifted DNase I footprint (-32 to +30). However, these IEC have not yet achieved the much smaller footprint (approximately 30 bp) reported as characteristic of elongating ternary complexes bearing longer RNA chains. During the initial phase of transcription, the RNA polymerase does not move monotonically along the DNA template as RNA chains are extended, but instead, the upstream and downstream contacts remain more or less fixed as the nascent transcript is elongated up to about eight nucleotides in length. Only after incorporation of 10 nucleotides is there significant movement of the enzyme away from the promoter region and a commitment to elongation.
我们研究了一系列在RNA链起始和延伸的初始步骤中形成的大肠杆菌RNA聚合酶三元复合物的性质和结构。使用了五种不同的模板，其中包含噬菌体T7 A1启动子、大肠杆菌Tac或lac UV5启动子，以及在初始转录区域有改变的变体模板。大多数携带短转录本（两到九个核苷酸）的三元复合物非常不稳定，难以进行研究。这包括来自噬菌体T7 A1启动子的转录本，此前曾推测携带短至四个核苷酸转录本的复合物的稳定性。然而，对于一个Tac启动子模板，RNA聚合酶可形成携带短至五个核苷酸转录本的三元复合物，其稳定性足以用于生化研究。我们描述了几种鉴定和分离此类稳定复合物的方法，并表明如果要使结果有意义，在进行此类实验时需要严格的标准。脱氧核糖核酸酶I（DNase I）足迹法已被用于探测聚合酶开始转录并离开起始位点时形成的稳定三元复合物的总体结构。在起始和转变为延伸复合物的过程中，该酶经历一系列结构变化。携带五到八个核苷酸转录本的复合物，称为初始转录复合物（ITC），具有相同的足迹；与开放启动子复合物（-57至+20）相比，它们都保留了σ因子，并且DNase I足迹略有延伸（-57至+24）。ITC复合物在-25区域均显示出显著的DNase I超敏区域，这可能反映了DNA模板的弯曲或扭曲。携带10或11个核苷酸转录本的复合物，称为初始延伸复合物（IEC），已失去σ因子，并且DNase I足迹略有减小和移位（-32至+30）。然而，这些IEC尚未达到报道的携带更长RNA链的延伸三元复合物特有的小得多的足迹（约30 bp）。在转录的初始阶段，随着RNA链的延伸，RNA聚合酶不会沿着DNA模板单调移动，相反，随着新生转录本延长至约八个核苷酸的长度，上游和下游接触点或多或少保持固定。只有在掺入10个核苷酸后，酶才会从启动子区域发生显著移动并进入延伸阶段。
