PROCESSIVITY IN EARLY STAGES OF TRANSCRIPTION BY T7-RNA POLYMERASE
PROCESSIVITY IN EARLY STAGES OF TRANSCRIPTION BY T7-RNA POLYMERASE
复制标题
DOI：
10.1021/bi00411a012
复制
发表时间：
1988-05-31
期刊：
影响因子：
2.9
通讯作者：
COLEMAN, JE
中科院分区：
文献类型：
--
作者：
MARTIN, CT;MULLER, DK;COLEMAN, JE
转录开始后，T7 RNA聚合酶立即进入一个阶段，在这个阶段，酶- dna -RNA三联体的解离与延伸明显竞争，这一过程在大肠杆菌酶中被称为流产循环[Carpousis， a . J., and Gralla， J. D. (1980) Biochemistry 19, 3245-3253]。在T7 RNA聚合酶系统中，在不同的反应条件和不同的信息序列模板上对这一过程的表征表明，在加入8个碱基后，转化为高度过程化的三元络合物，并且在此之前，解离和延伸之间的相对竞争受到几种不同力的影响。特别是，流产脱落的序列依赖性表明，在UMP掺入后立即发生解离，而在GMP掺入RNA信息后不太可能发生解离。人工合成的寡核苷酸模板在启动子区域是双链的，但在整个信息区域是单链的。这一结果证明，DNA编码区双链的融化和再退火不会导致流产循环。此外，启动子结合减弱一个数量级仅轻微影响流产循环，这表明与启动子的强相互作用并不是流产循环的主要原因。动力学分析表明，在加入8个碱基后转化为高度进展的三元配合物，可能反映了由于新生RNA与DNA模板之间以及RNA与酶之间的接触增加，该配合物的单分子解离率大大降低。最后，证明了在转录初始序列GGG的信息时，，在GTP作为唯一核苷酸存在的情况下，由于RNA信息沿着DNA模板滑动，T7 RNA聚合酶合成了poly [r(G)]转录物的阶梯。这个阶梯大部分截断在14个碱基的长度上，提供了DNA模板上位于+3位置的三元配合物的空间约束信息。
Immediately following initiation of transcription, T7 RNA polymerase enters a phase in which dissociation of the enzyme-DNA-RNA ternary complex significantly competes with elongation, a process referred to in the Escherichia coli enzyme as abortive cycling [Carpousis, A. J., and Gralla, J. D. (1980) Biochemistry 19, 3245-3253]. Characterization of this process in the T7 RNA polymerase system under various reaction conditions and on templates with differing message sequences reveals that conversion to a highly processive ternary complex occurs after incorporation of eight bases and that the relative competition between dissociation and elongation up to this point is influenced by several different forces. In particular, the sequence dependence of abortive falloff suggests that dissociation is favored immediately following incorporation of UMP and less likely following incorporation of GMP into the RNA message. Abortive cycling is unchanged in transcription from a synthetic oligonucleotide template which is double-stranded in the promoter region but single-stranded-throughout the entire message region. This result proves that melting and reannealing of the DNA duplex in the coding region do not contribute to abortive cycling. Furthermore, weakening of promoter binding by an order of magnitude affects abortive cycling only slightly, suggesting that strong interactions with the promoter are not the major cause of abortive cycling. Kinetic analyses show that conversion to a highly processive ternary complex after the incorporation of eight bases may reflect a large decrease in the unimolecular rate of dissociation of the complex due to increased contacts between the nascent RNA and the DNA template and between RNA and enzyme. Finally, it is shown that when transcribing a meassage of initial sequence GGG..., in the presence of GTP as the sole nucleotide, T7 RNA polymerase synthesizes a ladder of poly [r(G)] transcripts due to a slipping of the RNA message along the DNA template. This ladder is largely truncated at a length of 14 bases, providing information on steric constraints in the ternary complex positioned at +3 on the DNA template.