SYNTHETIC SITES FOR TRANSCRIPTION TERMINATION AND A FUNCTIONAL COMPARISON WITH TRYPTOPHAN OPERON TERMINATION SITES INVITRO
SYNTHETIC SITES FOR TRANSCRIPTION TERMINATION AND A FUNCTIONAL COMPARISON WITH TRYPTOPHAN OPERON TERMINATION SITES INVITRO
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DOI：
10.1073/pnas.78.7.4180
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发表时间：
1981-01-01
期刊：
影响因子：
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通讯作者：
PLATT, T
中科院分区：
文献类型：
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作者：
CHRISTIE, GE;FARNHAM, PJ;PLATT, T
大肠杆菌RNA聚合酶在体外终止转录似乎主要取决于终止位点的两个结构特征；二体对称的富含G + C（鸟嘌呤、胞嘧啶）的区域和一系列末端尿嘧啶残基。确定这两个特征是否足以指定。rho。通过在一个色氨酸（trp）操纵子结构基因中引入新的序列，建立了两个具有这些特征的位点。野生型RNA聚合酶体外转录表明，在这些新位点之一发生离散终止，尽管水平较低。使用突变RNA聚合酶rpo203，它比野生型酶对某些弱终止子更敏感，增加了两个位点的终止。我们合成的终止子的活性与大肠杆菌色氨酸操纵子中几个终止位点的活性进行了比较。在体外正常转录条件下，随着RNA发夹茎长度的增加或连续尿苷残基数量的增加，终止变得更有效。用rpo203聚合酶和核糖核苷酸类似物转录得到的变化与这些总体趋势一致。一个涉及RNA发夹和尿苷延伸在转录本中独立但重要作用的终止模型得到了支持。
Termination of transcription by Escherichia coli RNA polymerase in vitro appears to depend primarily on 2 structural features of the termination site; a G + C(guanine, cytosine)-rich region of dyad symmetry and a series of terminal uridine residues in the transcript. To determine whether these 2 features are sufficient to specify .rho.-independent termination in vitro, new sequences were introduced within a tryptophan (trp) operon structural gene to create 2 sites with these characteristics. Transcription with wild-type RNA polymerase in vitro demonstrates that discrete termination occurs at one of these new sites, although at a low level. Use of the mutant RNA polymerase rpo203, which is more sensitive to certain weak terminators than is the wild-type enzyme, increases termination at both sites. The activity of our synthetic terminators was compared with those of several termination sites in the E. coli trp operon. Under normal conditions of transcription in vitro, termination becomes more efficient with an increase in the length of the stem in the RNA hairpin or an increase in the number of consecutive uridine residues. Transcription with the rpo203 polymerase and with ribonucleotide analogs gives changes consistent with these general trends. A model for termination involving separate but essential roles for the RNA hairpin and the stretch of uridines in the transcript is supported.