Reynolds R, Bermúdez-Cruz R M, Chamberlin M J
Division of Biochemistry and Molecular Biology, University of California, Berkeley 94720.
J Mol Biol. 1992 Mar 5;224(1):31-51. doi: 10.1016/0022-2836(92)90574-4.
Escherichia coli RNA polymerase can terminate transcription efficiently at rho-independent terminators in a purified transcription system in the absence of accessory factors. This process of "intrinsic termination" involves direct recognition of the terminator by the core RNA polymerase, and provides an important model system for the study of the molecular interactions involved in the switch between elongation and termination. We have analyzed the intrinsic termination efficiency (%T) of 13 rho-independent terminators, under a variety of in vitro reaction conditions. Although all of these sites share the general sequence features of typical rho-independent terminators, we find a wide range of %T (2% to 90%) for the different sites under our standard transcription conditions. While %T for a particular site is characteristic of that site, the efficiency can be altered considerably by the nature and concentration of salts in the reaction, by alteration of the concentrations of the nucleoside triphosphate substrates, or by transcription from supercoiled rather than linear templates. Surprisingly, different conditions can alter %T to a different extent for different terminators. For neutral salts such as potassium chloride or potassium glutamate, changes in the range from 0.1 to 1 M affect %T for different terminators in a distinct manner, depending on the terminator and the anion involved. At some sites, %T is greatly increased by Cl- concentrations up to 1 M, while at other sites %T is reduced or unaffected by these conditions. At some sites K+ concentrations up to 1 M give a modest increase in %T, while at other sites %T is slightly reduced under the same conditions. Thus the actual values of %T, as well as the order of terminator sites ranked according to %T, can be altered greatly according to the choice of reaction conditions. Reduction of the Mg2+ concentration below 1 mM has a dramatic and quite different effect, enhancing termination to approximately 100% for all terminators tested. Transcription of supercoiled DNA templates gives somewhat reduced %T as compared with linear DNA templates. However, the effect is no greater than twofold. Our results are not consistent with those expected for models in which %T is determined by the differential stability of DNA, RNA and hybrid duplex structures at the melted region in the transcription complex. Thus, the Cl anion does not affect the stability of nucleic acid duplexes even at 1 M concentrations, but can enhance termination tenfold. Also, the alterations of monovalent cation concentration that affect %T are not expected to have a differential effect on Tm for DNA, RNA and hybrid duplexes.(ABSTRACT TRUNCATED AT 400 WORDS)
在没有辅助因子的情况下，大肠杆菌RNA聚合酶能够在纯化的转录系统中于不依赖ρ因子的终止子处高效终止转录。这种“内在终止”过程涉及核心RNA聚合酶对终止子的直接识别，为研究延伸与终止转换过程中涉及的分子相互作用提供了一个重要的模型系统。我们在多种体外反应条件下分析了13个不依赖ρ因子的终止子的内在终止效率（%T）。尽管所有这些位点都具有典型不依赖ρ因子终止子的一般序列特征，但在我们的标准转录条件下，不同位点的%T范围很广（2%至90%）。虽然特定位点的%T是该位点的特征，但反应中盐的性质和浓度、核苷三磷酸底物浓度的改变，或从超螺旋而非线性模板进行转录，都能显著改变终止效率。令人惊讶的是，不同条件对不同终止子的%T改变程度不同。对于中性盐如氯化钾或谷氨酸钾，0.1至1 M范围内的变化以不同方式影响不同终止子的%T，这取决于终止子和所涉及的阴离子。在某些位点，高达1 M的Cl⁻浓度可使%T大幅增加，而在其他位点，这些条件会降低或不影响%T。在某些位点，高达1 M的K⁺浓度会使%T适度增加，而在其他位点，相同条件下%T会略有降低。因此，根据反应条件的选择，%T的实际值以及根据%T排列的终止子位点顺序会有很大改变。将Mg²⁺浓度降至1 mM以下会产生显著且截然不同的效果，使所有测试的终止子的终止率提高到约100%。与线性DNA模板相比，超螺旋DNA模板的转录使%T有所降低。然而，这种影响不超过两倍。我们的结果与那些认为%T由转录复合物中解链区域的DNA、RNA和杂交双链结构的差异稳定性决定的模型预期不一致。因此，即使在1 M浓度下，Cl⁻阴离子也不会影响核酸双链的稳定性，但可使终止效率提高十倍。此外，影响%T的单价阳离子浓度的改变预计不会对DNA、RNA和杂交双链的解链温度产生不同影响。（摘要截选至400字）
