Selective and accurate initiation of transcription at the ad2 major late promotor in a soluble system dependent on purified rna polymerase ii and dna
Selective and accurate initiation of transcription at the ad2 major late promotor in a soluble system dependent on purified rna polymerase ii and dna
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DOI：
10.1016/0092-8674(79)90065-5
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发表时间：
1979-10
期刊：
影响因子：
64.5
通讯作者：
P. Weil;D. Luse;J. Segall;R. Roeder
中科院分区：
文献类型：
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作者：
P. Weil;D. Luse;J. Segall;R. Roeder
在补充有外源性（纯化的）RNA聚合酶II的粗细胞提取物存在下，Ad 2 DNA模板的转录选择性地且准确地起始于位于图谱位置16.45处的主要晚期病毒启动子。特异性起始已经通过杂交、核酸酶S1作图、大小和用各种模板产生的转录物的部分序列（指纹）分析的组合来证明。对于完整的Ad 2 DNA，转录在达到26 hb转录单位的末端之前就终止了。使用截短的模板（其含有完整的启动子区和转录区的几百个碱基对片段），观察到预期的流出产物，沿着低水平的提前终止的转录物。显示Sma IF模板的560个核苷酸流出产物（坐标11.6-16.2）含有所有大RNA酶T1寡核苷酸，其是来自该区域的相应体内转录物的特征;此外，5 '末端十一烷核苷酸似乎是加帽的和甲基化的。我们已经研究了影响粗系统中特异性转录水平的各种参数（盐、金属离子和模板浓度），并且已经发现，在最佳条件下，Ad 2 DNA的特异性转录持续数小时。此外，在晚期启动子处的特异性转录起始用来自病毒感染或未感染的KB细胞的提取物和用从人、小牛、鼠或两栖动物细胞分离的II类RNA聚合酶观察到。来自小麦胚芽的RNA聚合酶II在该系统中不起作用。
Transcription of Ad2 DNA templates in the presence of crude cellular extracts supplemented with exogenous (purified) RNA polymerase II is selectively and accurately initiated at the major late viral promoter at map position 16.45. Specific initiation has been demonstrated by a combination of hybridization, nuclease Sl mapping, size and partial sequence (fingerprint) analyses of the transcripts generated with various templates. With intact Ad2 DNA, transcription is terminated well before the end of the 26 hb transcription unit is reached. With truncated templates (which contain intact promoter regions and several hundred base pair segments of the transcribed region) the expected run-off products are observed, along with a low level of prematurely terminated transcripts. The 560 nucleotide run-off product of the Sma IF template (coordinates 11.6-16.2) was shown to contain all the large RNAase Tl oligonucleotides that are characteristic of the corresponding in vivo transcript from this region; in addition, the 5’terminal undecanucleotide appears to be both capped and methylated. We have investigated various parameters(salt, metal ion and template concentrations) that affect the level of specific transcription in the crude system and have found that, under optimal conditions, sps cific transcription of Ad2 DNA continues for several hours. In addition, specific transcription initiation at the late promoter is observed with extracts derived from either virus-infected or uninfected KB cells and with class II RNA polymerases isolated from either human, calf, murine or amphibian cells. RNA polymerase II from wheat germ does not function in this system.