Borukhov S, Sagitov V, Josaitis C A, Gourse R L, Goldfarb A
Public Health Research Institute, New York, New York 10016.
J Biol Chem. 1993 Nov 5;268(31):23477-82.
The rrnB P1 promoter of Escherichia coli (starting sequence C-4-A-3-C-2-C-1-A+1-C+2-U+3-G+4) forms a binary complex with RNA polymerase that is highly unstable and requires the presence of transcription substrates ATP and CTP for stabilizing the enzyme-DNA association (Gourse, R. L. (1988) Nucleic Acids Res. 16, 9789-9809). We show that in the absence of UTP and GTP the stabilization is accomplished by short RNA oligomers synthesized in an unusual "-3-->" mode whereby the primer initiated at the +1 site presumably slips back by three nucleotides into the -3 site and is then extended yielding stable ternary complexes. By contrast, short oligomers initiated in the conventional "+1-->" mode without slippage do not exert the stabilization effect and are readily aborted from the promoter complex. The stable -3-->ternary complexes carry sigma factor but otherwise resemble elongation complexes in their high salt stability and in the fact that they are formed with a mutant RNA polymerase deficient in promoter binding. A model is proposed explaining the stability of the -3-->ternary complexes by RNA slipping into a putative "tight RNA binding site" in RNA polymerase which is normally occupied by RNA during elongation.
大肠杆菌的rrnB P1启动子（起始序列为C-4-A-3-C-2-C-1-A+1-C+2-U+3-G+4）与RNA聚合酶形成一种高度不稳定的二元复合物，并且需要转录底物ATP和CTP的存在来稳定酶与DNA的结合（古尔塞，R.L.（1988年）《核酸研究》16卷，9789 - 9809页）。我们发现，在没有UTP和GTP的情况下，稳定作用是由以一种不寻常的“-3--->”模式合成的短RNA寡聚物实现的，即引物从+1位点起始，大概向后滑移三个核苷酸进入-3位点，然后延伸形成稳定的三元复合物。相比之下，以传统的“+1--->”模式起始且不发生滑移的短寡聚物不会产生稳定作用，并且很容易从启动子复合物中终止。稳定的-3--->三元复合物携带σ因子，但在高盐稳定性方面以及它们由缺乏启动子结合能力的突变RNA聚合酶形成这一事实上，类似于延伸复合物。我们提出了一个模型，通过RNA滑移到RNA聚合酶中一个假定的“紧密RNA结合位点”来解释-3--->三元复合物的稳定性，该位点在延伸过程中通常被RNA占据。
