Lyakhov D L, He B, Zhang X, Studier F W, Dunn J J, McAllister W T
Morse Institute for Molecular Genetics, Health Science Center at Brooklyn, State University of New York, 450 Clarkson Avenue, Brooklyn, Box 44, NY 11203-2098, USA.
J Mol Biol. 1998 Jul 10;280(2):201-13. doi: 10.1006/jmbi.1998.1854.
Two types of sites are known to cause pausing and/or termination by bacteriophage T7 RNA polymerase (RNAP). Termination at class I sites (typified by the signal found in the late region of T7 DNA, TPhi) involves the formation of a stable stem-loop structure in the nascent RNA ahead of the point of termination, and results in termination near runs of U. Class II sites, typified by a signal first identified in the cloned human preproparathyroid hormone (PTH) gene, generate no evident structure in the RNA but contain a conserved sequence ahead of the point of termination, and also contain runs of U. Termination at class I and class II sites may involve non-equivalent mechanisms, as mutants of T7 RNA polymerase have been identified that fail to recognize class II sites yet continue to recognize class I sites. In this work, we have analyzed pausing and termination at several class II sites, and variants of them. We conclude that the 7 bp sequence ATCTGTT (5' to 3' in the non-template strand) causes transcribing T7 or T3 RNA polymerase to pause. Termination 6 to 8 bp past this sequence is favored by the presence of runs of U, perhaps because they destabilize an RNA:DNA hybrid. The effects of T7 lysozyme on pausing and termination are consistent with the idea that termination involves a reversion of the polymerase from the elongation to the initiation conformation, and that lysozyme inhibits the return to the elongation conformation. A kinetic model of pausing and termination is presented that provides a consistent interpretation of our results.
已知有两类位点会导致噬菌体T7 RNA聚合酶（RNAP）暂停和/或终止。I类位点的终止（以T7 DNA晚期区域发现的信号TPhi为代表）涉及在终止点之前的新生RNA中形成稳定的茎环结构，并导致在连续的U处附近终止。II类位点以首次在克隆的人甲状旁腺激素原（PTH）基因中鉴定出的信号为代表，在RNA中不产生明显结构，但在终止点之前包含一个保守序列，并且也包含连续的U。I类和II类位点的终止可能涉及不同的机制，因为已鉴定出T7 RNA聚合酶的突变体，它们无法识别II类位点，但仍能识别I类位点。在这项工作中，我们分析了几个II类位点及其变体处的暂停和终止。我们得出结论，7个碱基对的序列ATCTGTT（非模板链上从5'到3'）会导致转录中的T7或T3 RNA聚合酶暂停。在该序列之后6至8个碱基对处的终止受到连续U的存在的促进，这可能是因为它们使RNA:DNA杂交体不稳定。T7溶菌酶对暂停和终止的影响与终止涉及聚合酶从延伸构象转变为起始构象，并且溶菌酶抑制恢复到延伸构象的观点一致。提出了一个暂停和终止的动力学模型，该模型对我们的结果提供了一致的解释。
