Department of Molecular Biology, Howard Hughes Medical Institute, Massachusetts General Hospital, Boston, United States.
Department of Genetics, Harvard Medical School, Boston, United States.
Elife. 2018 May 31;7:e36422. doi: 10.7554/eLife.36422.
The importance of genome replication has inspired detailed crystallographic studies of enzymatic DNA/RNA polymerization. In contrast, the mechanism of nonenzymatic polymerization is less well understood, despite its critical role in the origin of life. Here we report the direct observation of nonenzymatic RNA primer extension through time-resolved crystallography. We soaked crystals of an RNA primer-template-dGMP complex with guanosine-5'-phosphoro-2-aminoimidazolide for increasing times. At early times we see the activated ribonucleotides bound to the template, followed by formation of the imidazolium-bridged dinucleotide intermediate. At later times, we see a new phosphodiester bond forming between the primer and the incoming nucleotide. The intermediate is pre-organized because of the constraints of base-pairing with the template and hydrogen bonding between the imidazole amino group and both flanking phosphates. Our results provide atomic-resolution insight into the mechanism of nonenzymatic primer extension, and set the stage for further structural dissection and optimization of the RNA copying process.
基因组复制的重要性激发了对酶促 DNA/RNA 聚合的详细晶体学研究。相比之下，尽管非酶聚合在生命起源中起着关键作用，但人们对其机制的了解却较少。在这里，我们通过时间分辨晶体学直接观察到了非酶 RNA 引物延伸。我们将 RNA 引物-模板-dGMP 复合物的晶体浸泡在鸟苷-5'-磷酸-2-氨基咪唑核苷中，时间逐渐延长。在早期，我们看到了与模板结合的活化核苷酸，然后形成了咪唑鎓桥接的二核苷酸中间物。在稍后的时间里，我们看到引物和新的核苷酸之间形成了新的磷酸二酯键。由于与模板的碱基配对和咪唑氨基与两侧磷酸之间的氢键的限制，中间物预先组织化。我们的结果提供了非酶引物延伸机制的原子分辨率见解，并为进一步的结构剖析和 RNA 复制过程的优化奠定了基础。
