Farnham P J, Greenblatt J, Platt T
Cell. 1982 Jul;29(3):945-51. doi: 10.1016/0092-8674(82)90457-3.
Termination of transcription at the end of the tryptophan (trp) operon of E. coli at the trp t site is very efficient in vivo, but is only 25% efficient in vitro. To try to resolve this discrepancy, we have altered numerous parameters and report here on the modifications that bring the in vitro results into closer agreement with the in vivo ones. Lowering the concentration of UTP (but not ATP, CTP or GTP) in the transcription mix can greatly improve termination at trp t. With three other terminators structurally similar to trp t, there is no detectable effect of reducing the concentration of any of the four triphosphates. This response at trp t to low UTP is therefore both nucleotide-specific and terminator-specific, suggesting that apparently minor structural differences may still have profound effects upon termination. Increased specificity and sensitivity may also be provided by the NusA protein, which causes RNA polymerase to pause at trp t and at the 1:2 stem of the trp attenuator. NusA protein also enhances termination at trp t, an effect similar to the low UTP response. Further, termination can be slightly improved by including rho factor, resulting in an overall efficiency of almost 100% at trp t.
在大肠杆菌色氨酸（trp）操纵子末端的trp t位点处，转录终止在体内非常高效，但在体外只有25%的效率。为了试图解决这一差异，我们改变了许多参数，并在此报告那些使体外结果与体内结果更趋一致的修饰。降低转录混合物中UTP（而不是ATP、CTP或GTP）的浓度，可以极大地提高trp t处的终止效率。对于其他三个在结构上与trp t相似的终止子，降低四种三磷酸核苷酸中任何一种的浓度都没有可检测到的影响。因此，trp t对低UTP的这种反应既是核苷酸特异性的，也是终止子特异性的，这表明明显微小的结构差异可能仍然对终止有深远影响。NusA蛋白也可能提供更高的特异性和敏感性，它会使RNA聚合酶在trp t和trp弱化子的1:2茎处暂停。NusA蛋白还增强了trp t处的终止作用，这一效应与低UTP反应类似。此外，加入rho因子可以稍微提高终止效率，从而使trp t处的总体效率几乎达到100%。
