Initial transcribed sequence mutations specifically affect promoter escape properties
Initial transcribed sequence mutations specifically affect promoter escape properties
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DOI：
10.1021/bi060247u
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发表时间：
2006-07-25
期刊：
影响因子：
2.9
通讯作者：
Ahn, Colette
中科院分区：
文献类型：
--
作者：
Hsu, Lilian M.;Cobb, Ingrid M.;Ahn, Colette
大肠杆菌RNA聚合酶的启动子逃逸效率由核心启动子和初始转录序列(ITS)共同决定。在这里，我们定量检测了43个噬菌体T5 N25启动子的随机初始序列变体的逃逸特性。与野生型N25的+11/+12接合点不同，所有N25-其变异体上的启动子逃逸位置都发生在+15/+16接合点。这些变种在逃逸效率上还表现出25倍的差异。其有利于启动子逃逸的变化显示出与初始位置的核苷酸底物结合亲和力无关的组成偏向。比较所有的变异体，自然的N25启动子进化出了一个最适合启动子逃逸的启动子，在经历了最短的流产程序后，提供了高水平的生产性合成。我们将Greb添加到转录反应中，以更好地了解体内流产的起始和启动子逃逸。GREB通过改变败育RNA的模式，在不改变短的(2-5个核苷酸)和超长的(16-20个核苷酸)败育RNA的水平的情况下，降低中等(6-10个核苷酸)到长(11-15个核苷酸)的败育RNA的丰度，从而将产量提高2-5倍。短流产RNA产生的格雷布折射性质可能反映了维持初始或回溯复合体的稳定性所需的RNA-DNA杂交的最小长度为4-5个碱基对。很长的流产RNA没有受到Greb的影响，这表明它们不太可能是聚合酶回溯的产物。在初始转录复合体的结构背景下，讨论了ITS如何影响早期转录的过程。
Promoter escape efficiency of E. coli RNA polymerase is guided by both the core promoter and the initial transcribed sequence (ITS). Here, we quantitatively examined the escape properties of 43 random initial sequence variants of the phage T5 N25 promoter. The position for promoter escape on all N25-ITS variants occurred at the +15/+16 juncture, unlike the +11/+12 juncture for the wild type N25. These variants further exhibited a 25-fold difference in escape efficiency. ITS changes favoring promoter escape showed a compositional bias that is unrelated to nucleotide substrate binding affinity for the initial positions. Comparing all variants, the natural N25 promoter emerges as having evolved an ITS optimal for promoter escape, giving a high level of productive synthesis after undergoing the shortest abortive program. We supplemented GreB to transcription reactions to better understand abortive initiation and promoter escape in vivo. GreB supplementation elevated productive RNA synthesis 2-5-fold by altering the abortive RNA pattern, decreasing the abundance of the medium (6-10 nt) to long (11-15 nt) abortive RNAs without changing the levels of short (2-5 nt) and very long abortive RNAs (16-20 nt). The GreB-refractive nature of short abortive RNA production may reflect a minimum length requirement of 4-5 bp of the RNA-DNA hybrid for maintaining the stability of initial or backtracked complexes. That the very long abortive RNAs are unaffected by GreB suggests that they are unlikely to be products of polymerase backtracking. How the ITS might influence the course of early transcription is discussed within the structural context of an initial transcribing complex.