Ramprakash Jayanthi, Schwarz Frederick P
Center for Advanced Research in Biotechnology/National Institute of Standards and Technology, 9600 Gudelsky Drive, Rockville, Maryland 20850, USA.
Biophys Chem. 2008 Dec;138(3):91-8. doi: 10.1016/j.bpc.2008.09.007. Epub 2008 Sep 18.
The thermodynamics of RNA polymerase (RNAP) binding to a 108 base pair (bp) synthetic promoter with consensus sequences at the -35 and -10 bp binding regions upstream from the transcription start point were determined using isothermal titration calorimetry (ITC). The binding constant at 25 degrees C is 2.37+/-0.18x10(7) M(-1), which is reduced to 0.17+/-0.06x10(7) M(-1) with mutations in the -10 bp region but remained the same with mutations in the -35 binding region. The binding reactions were enthalpically-driven with exothermic binding enthalpies ranging from -57+/-6 kJ mol(-1) at 15 degrees C to -271+/-20 kJ mol(-1) at 35 degrees C yielding a large binding heat capacity change of -10.7+/-1.9 kJ mol(-1) K(-1), indicating a conformational change upon binding to the RNAP. Differential scanning calorimetry (DSC) scans of the thermal unfolding of RNAP and the promoter-RNAP complex exhibited an unfolding transition at 55.5+/-0.6 degrees C and at 58.9+/-0.5 degrees C for the RNAP but only one transition at 60.5+/-1.1 degrees C for the complex with van't Hoff enthalpy to transition enthalpy ratios of, resp., 3.2+/-0.3 and 4.3+/-0.5. The single transition of the complex results from a shift to 60.5 degrees C of the low temperature transition upon promoter binding to the structural unit unfolding at the lower temperature in RNAP. The large transition enthalpy ratios indicate that the sigma, alpha, alpha, beta, and beta' subunits unfold as almost independent entities. The dissociation thermodynamics of short transcription "bubble" duplexes of 7 promoters sequenced from -1 to -12 bp were determined from ITC and DSC measurements. The free energy change of the promoter binding to the RNAP and the free energy requirement for formation of the transcription bubble at the low promoter concentrations in the cell are sufficient to drive the initiation of transcription through the isomerization of the closed to the open form step of the RNAP-promoter complex.
使用等温滴定量热法（ITC）测定了RNA聚合酶（RNAP）与一个108碱基对（bp）的合成启动子的结合热力学，该启动子在转录起始点上游的-35和-10 bp结合区域具有共有序列。25℃时的结合常数为2.37±0.18×10⁷ M⁻¹，在-10 bp区域发生突变时降至0.17±0.06×10⁷ M⁻¹，但在-35结合区域发生突变时保持不变。结合反应是由焓驱动的，结合焓为放热，范围从15℃时的-57±6 kJ mol⁻¹到35℃时的-271±20 kJ mol⁻¹，产生了-10.7±1.9 kJ mol⁻¹ K⁻¹的大结合热容量变化，表明与RNAP结合时发生了构象变化。对RNAP以及启动子-RNAP复合物的热解折叠进行差示扫描量热法（DSC）扫描，结果显示RNAP在55.5±0.6℃有一个解折叠转变，在58.9±0.5℃有一个解折叠转变，而复合物在60.5±1.1℃只有一个转变，范特霍夫焓与转变焓的比值分别为3.2±0.3和4.3±0.5。复合物的单一转变是由于启动子与RNAP中在较低温度下展开的结构单元结合后，低温转变向60.5℃移动所致。大的转变焓比值表明σ、α、α、β和β'亚基几乎作为独立的实体展开。通过ITC和DSC测量确定了7个从-1到-12 bp测序的启动子的短转录“泡”双链体的解离热力学。在细胞中低启动子浓度下，启动子与RNAP结合的自由能变化以及形成转录泡的自由能需求足以通过RNAP-启动子复合物从封闭形式到开放形式步骤的异构化来驱动转录起始。
