Landick R, Yanofsky C
Department of Biological Sciences, Stanford University, CA 94305.
J Mol Biol. 1987 Jul 20;196(2):363-77. doi: 10.1016/0022-2836(87)90697-8.
Transcription pausing is a key step in many prokaryotic transcription attenuation mechanisms. Pausing is thought to occur when an RNA hairpin forms near the 3' end of a growing transcript. We report here the isolation of the trp leader paused transcription complex containing a defined 92-nucleotide nascent transcript. Digestion of isolated paused complexes with RNase T1 suggests that the trp leader RNA hairpin designated 1:2 forms in the paused transcription complex. The transcription factor NusA alters the RNase T1 digestion pattern of the 92-nucleotide pause transcript in the complex but not the cleavage patterns of purified pause RNA, suggesting that NusA specifically affects the 1:2 hairpin in the paused transcription complex. The isolated paused transcription complex retains the ability to resume transcription. Kinetic studies on the resumption of elongation suggest that NusA is a non-competitive inhibitor of paused complex release and that the Ks for GTP is around 300 microM. RNA polymerase in the paused transcription complex protects approximately 30 base-pairs on both DNA strands from exonuclease digestion.
转录暂停是许多原核生物转录衰减机制中的关键步骤。当RNA发夹在正在生长的转录本3'端附近形成时，就会发生暂停。我们在此报告了含有特定92个核苷酸新生转录本的色氨酸操纵子前导序列暂停转录复合物的分离。用核糖核酸酶T1消化分离的暂停复合物表明，在暂停转录复合物中形成了指定为1:2的色氨酸操纵子前导RNA发夹。转录因子NusA改变了复合物中92个核苷酸暂停转录本的核糖核酸酶T1消化模式，但不改变纯化的暂停RNA的切割模式，这表明NusA特异性地影响了暂停转录复合物中的1:2发夹。分离的暂停转录复合物保留了恢复转录的能力。对延伸恢复的动力学研究表明，NusA是暂停复合物释放的非竞争性抑制剂，并且GTP的Ks约为300微摩尔。暂停转录复合物中的RNA聚合酶保护两条DNA链上约30个碱基对不被核酸外切酶消化。
