Reisbig R R, Woody A Y, Woody R W
Biochim Biophys Acta. 1981 Feb 26;652(2):294-302. doi: 10.1016/0005-2787(81)90119-2.
To characterize the interactions of RNA polymerase with DNA, we have investigated the thermal transition of poly[d(A-T] bound to RNA polymerase from Escherichia coli and the aggregation properties of the enzyme with DNA. The melting curve of the DNA-enzyme complex demonstrates a sharply lowered melting temperature for part of the DNA, whereas for another fraction the double helix is stabilized. This indicates that the DNA-binding site of RNA polymerase serves two functions: (1) to disrupt the double helix at one point, and (2) to maintain the duplex form at other points. The aggregation of DNA and RNA polymerase has been monitored by turbidity measurements, and conditions have been delineated under which aggregation is minimized. Holoenzyme added to double-stranded DNA or single-stranded DNA has little or no tendency to aggregate under most conditions. Core enzyme, on the other hand, aggregate extensively with double-stranded DNA, the only under conditions of low salt (10 mM KCl), without Mg2+, or at high salt (300 mM KCl), with or without Mg2+, can this aggregation be eliminated. Core enzyme also does not aggregate in the presence of single-stranded DNA. These aggregation properties are interpreted as evidence for more than one DNA-binding site on RNA polymerase.
为了描述RNA聚合酶与DNA的相互作用，我们研究了与来自大肠杆菌的RNA聚合酶结合的聚[d(A - T)]的热转变以及该酶与DNA的聚集特性。DNA - 酶复合物的解链曲线表明，部分DNA的解链温度急剧降低，而另一部分双螺旋则得到稳定。这表明RNA聚合酶的DNA结合位点具有两种功能：（1）在某一点破坏双螺旋，（2）在其他点维持双链形式。通过浊度测量监测了DNA和RNA聚合酶的聚集情况，并确定了聚集最小化的条件。在大多数情况下，添加到双链DNA或单链DNA中的全酶几乎没有或没有聚集倾向。另一方面，核心酶与双链DNA广泛聚集，只有在低盐（10 mM KCl）、无Mg2 +的条件下，或者在高盐（300 mM KCl）、有或无Mg2 +的条件下，这种聚集才能消除。核心酶在单链DNA存在下也不会聚集。这些聚集特性被解释为RNA聚合酶上存在多个DNA结合位点的证据。
