Shih M C, Gussin G N
J Mol Biol. 1984 Feb 5;172(4):489-506. doi: 10.1016/s0022-2836(84)80019-4.
Abortive and productive initiation assays were used to study transcription initiation at the PRE promoter of phage lambda in vitro. Two parameters were measured: k2, the rate constant for the transition between closed and open complexes; and KB, the equilibrium constant for the initial binding of RNA polymerase to promoter DNA. In the absence of cII protein (which activates PRE) the PRE promoter was extremely weak as expected, with k2 = 4.0 X 10(-4) S-1 and KB = 1.0 X 10(7) M-1. The addition of cII protein resulted in about a 15-fold increase in KB and a 40-fold increase in k2. Thus, cII activation of PRE results both in enhanced binding of RNA polymerase to DNA to form closed complexes and in an enchanced rate of isomerization of closed to open complexes. In addition, we found that open complexes formed in the presence of cII protein were at least four times as stable as those formed in its absence. This suggests that RNA polymerase and cII protein may remain in close contact even after complexes are formed.
采用流产起始和有效起始试验在体外研究噬菌体λ的PRE启动子处的转录起始。测定了两个参数：k2，即封闭复合物与开放复合物之间转变的速率常数；以及KB，即RNA聚合酶与启动子DNA初始结合的平衡常数。在不存在cII蛋白（激活PRE）的情况下，正如预期的那样，PRE启动子极其微弱，k2 = 4.0×10⁻⁴ s⁻¹，KB = 1.0×10⁷ M⁻¹。添加cII蛋白导致KB增加约15倍，k2增加40倍。因此，cII对PRE的激活既导致RNA聚合酶与DNA结合形成封闭复合物的增强，也导致封闭复合物异构化为开放复合物的速率增强。此外，我们发现，在存在cII蛋白的情况下形成的开放复合物的稳定性至少是在其不存在时形成的开放复合物的四倍。这表明即使在复合物形成后，RNA聚合酶和cII蛋白可能仍保持紧密接触。
