Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Baddiley-Clark Building, Richardson Road, Newcastle upon Tyne NE2 4AX, UK.
Nucleic Acids Res. 2013 Nov;41(20):9257-65. doi: 10.1093/nar/gkt708. Epub 2013 Aug 9.
Transcription elongation consists of repetition of the nucleotide addition cycle: phosphodiester bond formation, translocation and binding of the next nucleotide. Inhibitor of multi-subunit RNA polymerase tagetitoxin (TGT) enigmatically slows down addition of nucleotides in a sequence-dependent manner, only at certain positions of the template. Here, we show that TGT neither affects chemistry of RNA synthesis nor induces backward translocation, nor competes with the nucleoside triphosphate (NTP) in the active center. Instead, TGT increases the stability of the pre-translocated state of elongation complex, thus slowing down addition of the following nucleotide. We show that the extent of inhibition directly depends on the intrinsic stability of the pre-translocated state. The dependence of translocation equilibrium on the transcribed sequence results in a wide distribution (~1-10(3)-fold) of inhibitory effects of TGT at different positions of the template, thus explaining sequence-specificity of TGT action. We provide biochemical evidence that, in pre-translocated state, TGT stabilizes folded conformation of the Trigger Loop, which inhibits forward and backward translocation of the complex. The results suggest that Trigger Loop folding in the pre-translocated state may serve to reduce back-tracking of the elongation complex. Overall, we propose that translocation may be a limiting and highly regulated step of RNA synthesis.
磷酸二酯键形成、核苷酸的移位和下一个核苷酸的结合。多亚基 RNA 聚合酶标签毒素（TGT）抑制剂以序列依赖性的方式神秘地减缓核苷酸的添加，仅在模板的某些位置。在这里，我们表明 TGT 既不影响 RNA 合成的化学性质，也不诱导向后移位，也不在活性中心与核苷三磷酸（NTP）竞争。相反，TGT 增加延伸复合物的预移位状态的稳定性，从而减缓后续核苷酸的添加。我们表明，抑制程度直接取决于预移位状态的固有稳定性。由于转录序列对移位平衡的影响，TGT 在模板的不同位置产生广泛分布（~1-10（3）倍）的抑制作用，从而解释了 TGT 作用的序列特异性。我们提供了生化证据表明，在预移位状态下，TGT 稳定了触发环的折叠构象，从而抑制了复合物的向前和向后移位。结果表明，延伸复合物的预移位状态中的触发环折叠可能有助于减少延伸复合物的回溯。总体而言，我们提出移位可能是 RNA 合成的限制和高度调节步骤。
