Reisbig R R, Woody A Y, Woody R W
J Biol Chem. 1979 Nov 25;254(22):11208-17.
We have studied the circular dichroism and ultraviolet difference spectra of T7 bacteriophage DNA and various synthetic polynucleotides upon addition of Escherichia coli RNA polymerase. When RNA polymerase binds nonspecifically to T7 DNA, the CD spectrum shows a decrease in the maximum at 272 but no detectable changes in other regions of the spectrum. This CD change can be compared with those associated with known conformational changes in DNA. Nonspecific binding to RNA polymerase leads to an increase in the winding angle, theta, in T7 DNA. The CD and UV difference spectra for poly[d(A-T)] at 4 degrees C show similar effects. At 25 degrees C, binding of RNA polymerase to poly[d(A-T)] leads to hyperchromicity at 263 nm and to significant changes in CD. These effects are consistent with an opening of the double helix, i.e. melting of a short region of the DNA. The hyperchromicity observed at 263 nm for poly[d(A-T)] is used to determine the number of base pairs disrupted in the binding of RNA polymerase holoenzyme. The melting effect involves about 10 base pairs/RNA polymerase molecule. Changes in the CD of poly(dT) and poly(dA) on binding to RNA polymerase suggest an unstacking of the bases with a change in the backbone conformation. This is further confirmed by the UV difference spectra. We also show direct evidence for differences in the template binding site between holo- and core enzyme, presumably induced by the sigma subunit. By titration of the enzyme with poly(dT) the physical site size of RNA polymerase on single-stranded DNA is approximately equal to 30 bases for both holo- and core enzyme. Titration of poly[d(A-T)] with polymerase places the figure at approximately equal to 28 base pairs for double-stranded DNA.
我们研究了加入大肠杆菌RNA聚合酶后T7噬菌体DNA及各种合成多核苷酸的圆二色性和紫外差光谱。当RNA聚合酶非特异性结合到T7 DNA上时，圆二色光谱显示272处的最大值降低，但光谱其他区域未检测到变化。这种圆二色性变化可与DNA中已知构象变化相关的变化进行比较。与RNA聚合酶的非特异性结合导致T7 DNA的缠绕角θ增加。4℃时聚[d（A-T）]的圆二色光谱和紫外差光谱显示出类似的效应。在25℃时，RNA聚合酶与聚[d（A-T）]的结合导致263nm处的增色效应以及圆二色性的显著变化。这些效应与双螺旋的打开一致，即DNA短区域的解链。聚[d（A-T）]在263nm处观察到的增色效应用于确定RNA聚合酶全酶结合时被破坏的碱基对数。解链效应涉及约10个碱基对/RNA聚合酶分子。聚（dT）和聚（dA）与RNA聚合酶结合时圆二色性的变化表明碱基堆积的破坏以及主链构象的改变。紫外差光谱进一步证实了这一点。我们还直接证明了全酶和核心酶之间模板结合位点的差异，推测是由σ亚基诱导的。通过用聚（dT）滴定酶，全酶和核心酶在单链DNA上的物理位点大小约等于30个碱基。用聚合酶滴定聚[d（A-T）]时，双链DNA的数值约等于28个碱基对。
