RHO-INDEPENDENT TERMINATION - DYAD SYMMETRY IN DNA CAUSES RNA-POLYMERASE TO PAUSE DURING TRANSCRIPTION INVITRO
RHO-INDEPENDENT TERMINATION - DYAD SYMMETRY IN DNA CAUSES RNA-POLYMERASE TO PAUSE DURING TRANSCRIPTION INVITRO
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DOI：
10.1093/nar/9.3.563
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发表时间：
1981-01-01
影响因子：
14.9
通讯作者：
PLATT, T
中科院分区：
文献类型：
--
作者：
FARNHAM, PJ;PLATT, T
RNA聚合酶在rho非依赖性位点的转录终止似乎主要取决于两个结构特征，即终止点之前的DNA中富含GC的二联体对称区域和转录物3′端的一段尿苷。前者可能是减缓延伸的原因，这一可能性促使我们对E.色氨酸操纵子。延伸速率显著减慢或停止的区域是可识别的，因为它们在凝胶上产生离散的转录物条带。与终止位点不同，来自暂停位点的物种是短暂的，仅在转录的前两分钟内可检测到，因为聚合酶最终会恢复伸长。在两个多模式终止位点（trp a135和trp a1419），在体外终止不完全或缺失，但观察到实质性的停顿。同样，在trp t（操纵子末端的终止位点）处也发生了显着的暂停。我们的实验还揭示了一个主要的暂停网站在tripleader序列的位置90左右，刚刚过去的二分体对称区域。对应于该位点的RNA发夹是富含U的，并且通过掺入BrUTP而强烈增强暂停。相比之下，这种类似物不影响富含GC的发夹在衰减子或终止子位点处的暂停。这些结果有力地支持了这一假设，即暂停的聚合酶是一个强制性的前奏rho独立终止。此外，终止事件显然是连续的，但离散的反应，这些网站的单独的结构特征的结果。
Termination of transcription by RNA polymerase at rho-independent sites appears to depend primarily upon two structural features, a region of GC-rich dyad symmetry in the DNA preceding the stop point and a stretch of uridines at the 3′ end of the transcript. The possibility that the former might be responsible for slowing elongation prompted us to perform a kinetic analysis of transcription across the leader and terminator regions of theE. colitryptophan(trp)operon. Regions where the elongation rate is dramatically slowed or stopped are identifiable because they generate discrete transcript bands on a gel. Species derived from pause sites, unlike those resulting from termination sites, are transient and detectable only within the first two minutes of transcription, since polymerase eventually resumes elongation. At two mutanttrpattenuator sites (trp a135andtrp a1419), where termination is incomplete or absentin vitro, a substantial pause is nevertheless observed. Likewise, a significant pause occurs attrp t, the termination site at the end of the operon. Our experiments also reveal a major pause site at about position 90 in thetrpleader sequence, just past a region of dyad symmetry. The RNA hairpin corresponding to this site is U-rich, and pausing is strongly enhanced by incorporation of BrUTP. In contrast, this analog does not affect pausing at the attenuator or terminator sites with hairpins that are GC-rich. These results strongly support the hypothesis that pausing of the polymerase is an obligatory prelude to rho-independent termination. Moreover, the termination event evidently results from consecutive but discrete responses to separate structural features of these sites.