tRNA修飾ランドスケープが新規タンパク質機能の進化に与える影響

在拟议研究的三个目标中，我成功地完成了目标1并发表了结果。目标2和3的工作仍在进行中。在目标1中，我们研究了细菌中tRNA修饰m1G37的功能。由tRNA甲基转移酶TrmD引入的这种修饰被认为是细菌生长所必需的，因为它可以防止对蛋白质功能产生有害影响的移码错误。然而，许多其他tRNA修饰在防止移码错误方面具有类似的作用，因此尚不清楚为什么m1G37特别重要。为了解决这个问题，我们进行了一个进化修复实验，研究大肠杆菌的trmD基因突变的进化适应。令人惊讶的是，即使m1G37被完全消除，突变细菌也显示出缓慢的生长，并且在几周的进化后几乎完全恢复生长。全基因组测序表明，这主要是通过复制和突变的基因proS，这是负责氨基酰化的脯氨酸tRNA。这表明m1G37的主要功能与氨酰化有关，而不是错误翻译，我们使用生物化学和生长测定证实了这一点。这些结果进一步证明了细菌对蛋白质错误翻译具有高耐受性，表明进化修复可以揭示必需基因的隐藏功能，并对开发靶向trmD的抗生素具有意义

Out of the three aims of the proposed research, I completed Aim 1 successfully and published the results. Work on Aims 2 and 3 is still ongoing.In Aim 1, we studied the function of the tRNA modification m1G37 in bacteria. This modification, introduced by the tRNA methyltransferase TrmD, is thought to be essential for growth in bacteria because it prevents frameshift errors that have a deleterious effect on protein function. However, many other tRNA modifications have a similar role in preventing frameshift errors, so it was unclear why m1G37 is particularly essential. To address this question, we performed an evolutionary repair experiment, studying the evolutionary adaptation of Escherichia coli to mutations in the trmD gene. Surprisingly, the mutant bacteria showed slow growth even if m1G37 was completely abolished, and showed almost full recovery of growth after several weeks of evolution. Whole-genome sequencing showed that this was achieved mainly through duplication and mutation of the gene proS, which is responsible for aminoacylation of proline tRNA. This suggested that the main function of m1G37 is related to aminoacylation rather than mistranslation, which we confirmed using biochemical and growth assays. These results provide further evidence that bacteria have a high tolerance for protein mistranslation, show that evolutionary repair can reveal hidden functions of essential genes, and have implications for development of antibiotics targeting trmD