Guajardo R, Lopez P, Dreyfus M, Sousa R
Dept. of Biochemistry, University of Texas Health Science Ctr., 7703 Floyd Curl Drive, San Antonio, TX 78284-7760, USA.
J Mol Biol. 1998 Sep 4;281(5):777-92. doi: 10.1006/jmbi.1998.1988.
The hypothesis that active site translocation during initial transcription occurs by a passive sliding mechanism which allows the pre- and post-translocated states to equilibrate on the time scale of bond formation was tested by evaluating the effects of NTP concentration on individual transcript extension steps in the presence of translocation roadblocks created by proteins bound immediately downstream of a T7 promoter, as well as by evaluating the effects of NTP concentration on competing transcript extension pathways (iterative synthesis and "normal" extension). Results are consistent with a passive sliding mechanism for translocation which is driven by NTP binding, and are inconsistent with mechanisms in which the pre- and post-translocated states fail to equilibrate with each other on the time scale of bond formation or in which translocation is driven by NTP hydrolysis. We also find, in agreement with many previous studies, that divergence from consensus in the ITS (initially transcribed sequence) of the T7 promoter decreases productive initiation. However, this appears to be largely due to increases in the NTP concentration requirements for efficient transcription on the divergent ITSs.
通过评估NTP浓度对T7启动子下游紧邻区域结合的蛋白质所产生的转位障碍存在时单个转录本延伸步骤的影响，以及评估NTP浓度对竞争性转录本延伸途径（迭代合成和“正常”延伸）的影响，来检验以下假设：初始转录过程中活性位点转位是通过被动滑动机制发生的，该机制允许转位前和转位后的状态在键形成的时间尺度上达到平衡。结果与由NTP结合驱动的被动滑动转位机制一致，而与转位前和转位后的状态在键形成的时间尺度上无法相互平衡的机制，或与由NTP水解驱动转位的机制不一致。我们还发现，与许多先前的研究一致，T7启动子的ITS（初始转录序列）与共有序列的差异会降低有效起始。然而，这似乎主要是由于在不同的ITS上进行有效转录所需的NTP浓度增加所致。
