Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA.
Nucleic Acids Res. 2018 Nov 16;46(20):10589-10597. doi: 10.1093/nar/gky898.
A previously developed RNA polymerase ribozyme uses nucleoside triphosphates (NTPs) to extend a primer 3'-terminus, templated by an RNA template with good fidelity, forming 3'-5'-phosphordiester bonds. Indirect evidence has suggested that the ribozyme's accessory domain binds the NTP with a highly conserved purine-rich loop. To determine the NTP binding site more precisely we evolved the ribozyme for efficient use of 6-thio guanosine triphosphate (6sGTP). 6sGTP never appeared in the evolutionary history of the ribozyme, therefore it was expected that mutations would appear at the NTP binding site, adapting to more efficient binding of 6sGTP. Indeed, the evolution identified three mutations that mediate 200-fold improved incorporation kinetics for 6sGTP. A >50-fold effect resulted from mutation A156U in the purine-rich loop, identifying the NTP binding site. This mutation acted weakly cooperative with two other beneficial mutations, C113U in the P2 stem near the catalytic site, and C79U on the surface of the catalytic domain. The preference pattern of the ribozyme for different NTPs changed when position 156 was mutated, confirming a direct contact between position 156 and the NTP. The results suggest that A156 stabilizes the NTP in the active site by a hydrogen bond to the Hoogsteen face of the NTP.
先前开发的 RNA 聚合酶核酶使用核苷三磷酸 (NTP) 延伸引物 3' 末端，以 RNA 模板为模板，具有良好的保真度，形成 3' 至 5' 磷酸二酯键。间接证据表明，核酶的辅助结构域与 NTP 结合，形成高度保守的嘌呤丰富环。为了更精确地确定 NTP 结合位点，我们进化了核酶，以有效地利用 6-硫代鸟苷三磷酸 (6sGTP)。6sGTP 从未出现在核酶的进化历史中，因此预计突变会出现在 NTP 结合位点，以适应 6sGTP 更有效的结合。事实上，进化确定了三个突变，这些突变介导了 6sGTP 结合动力学提高了 200 倍。A156U 在嘌呤丰富环中的突变导致了 >50 倍的效果，确定了 NTP 结合位点。该突变与另外两个有益突变（靠近催化位点的 P2 茎中的 C113U 和催化结构域表面的 C79U）弱协同作用。当位置 156 发生突变时，核酶对不同 NTP 的偏好模式发生变化，证实位置 156 与 NTP 之间存在直接接触。结果表明，A156 通过与 NTP 的 Hoogsteen 面形成氢键稳定 NTP 在活性位点中的位置。
