Shimer G H, Woody A Y, Woody R W
Department of Biochemistry, Colorado State University, Fort Collins 80523.
Biochim Biophys Acta. 1988 Sep 7;950(3):354-65. doi: 10.1016/0167-4781(88)90132-7.
The interaction of Escherichia coli RNA polymerase with poly[d(A-T)] and poly[d-(I-C)] was studied by difference absorption spectroscopy at temperatures, from 5 to 45 degrees C in the absence and presence of Mg2+. The effect of KCl concentration, at a fixed temperature, was studied from 12.5 to 400 mM. Difference absorption experiments permitted calculation of the extent of DNA opening induced by RNA polymerase and estimation of the equilibrium constant associated with the isomerization from a closed to an open RNA polymerase-DNA complex. delta H0 and delta S0 for the closed-to-open transition with poly[d(A-T)] or poly[d(I-C)] complexed with RNA polymerase are significantly lower than the values associated with the helix-to-coil transition for the free polynucleotides. For the RNA polymerase complexes with poly[d(A-T)] and poly[d(I-C)] in 50 mM KCl, delta H0 approximately 15-16 kcal/mol (63-67 kJ/mol) and delta S0 approximately 50-57 cal/K per mol (209-239 J/K per mol). The presence of Mg2+ does not change these parameters appreciably for the RNA polymerase-poly[d(A-T)] complex, but for the RNA polymerase-poly[d(I-C)] complex in the presence of Mg2+, the delta H0 and delta S0 values are larger and temperature-dependent, with delta H0 approximately 22 kcal/mol (92 kJ/mol) and delta S0 approximately 72 cal/K per mol (approx. 300 J/K per mol) at 25 degrees C, and delta Cp0 approximately 2 kcal/K per mol (approx. 8.3 kJ/K per mol). The circular dichroism (CD) changes observed for helix opening induced by RNA polymerase are qualitatively consistent with the thermally induced changes observed for the free polynucleotides, supporting the difference absorption method. The salt-dependent studies indicate that two monovalent cations are released upon helix opening. For poly[d(A-T)], the temperature-dependence of enzyme activity correlates well with the helix opening, implying this step to be the rate-determining step. In the case of poly[d(I-C)], the same is not true, and so the rate-determining step must be a process subsequent to helix opening.
通过差示吸收光谱法，研究了大肠杆菌RNA聚合酶与聚[d(A-T)]和聚[d(I-C)]在5至45摄氏度、有无Mg2+存在的条件下的相互作用。在固定温度下，研究了KCl浓度从12.5至400 mM时的影响。差示吸收实验可以计算RNA聚合酶诱导的DNA解链程度，并估算与从闭合型RNA聚合酶-DNA复合物向开放型异构化相关的平衡常数。与RNA聚合酶复合的聚[d(A-T)]或聚[d(I-C)]从闭合向开放转变的ΔH0和ΔS0显著低于游离多核苷酸从螺旋向无规卷曲转变的相关值。对于在50 mM KCl中与聚[d(A-T)]和聚[d(I-C)]形成的RNA聚合酶复合物，ΔH0约为15 - 16 kcal/mol（63 - 67 kJ/mol），ΔS0约为50 - 57 cal/K每摩尔（209 - 239 J/K每摩尔）。Mg2+的存在对RNA聚合酶-聚[d(A-T)]复合物的这些参数没有明显影响，但对于存在Mg2+的RNA聚合酶-聚[d(I-C)]复合物，ΔH0和ΔS0值更大且与温度相关，在25摄氏度时，ΔH0约为22 kcal/mol（92 kJ/mol），ΔS0约为72 cal/K每摩尔（约300 J/K每摩尔），ΔCp0约为2 kcal/K每摩尔（约8.3 kJ/K每摩尔）。观察到的由RNA聚合酶诱导的螺旋解链引起的圆二色性（CD）变化在定性上与游离多核苷酸的热诱导变化一致，支持了差示吸收法。盐依赖性研究表明，螺旋解链时会释放两个单价阳离子。对于聚[d(A-T)]，酶活性的温度依赖性与螺旋解链密切相关，这意味着这一步是速率决定步骤。在聚[d(I-C)]的情况下，情况并非如此，因此速率决定步骤一定是螺旋解链之后的一个过程。
