Structural and functional analysis of the tRNA-modifying enzymes DNMT2 and tRNA-guanine-transglycosylase

至少93个转录后修饰的核苷已经在tRNAs中被确定。TRNA修饰酶必须识别它们的同源tRNA(S)，并在tRNA内识别修饰的位置。对于这些酶中的许多，导致其高度特异性的结构要求尚不清楚。真核细胞甲基转移酶DNMT2引入了C38的一个重要修饰，其特异性的结构基础将通过对DNMT2-tRNA复合体的晶体结构分析来揭示。这也将为研究反密码子34位的高度修饰的tRNA核苷队列糖对甲基转移酶活性的刺激机制提供依据。在这方面，我们还将研究真核tRNA-鸟氨酸-转糖基酶(TGT)，它通过取代鸟氨酸在tRNA中引入队列。人类TGT的晶体结构是我们刚刚确定的，它揭示了细菌TGT在四级结构上出人意料的不同。将分析由此产生的关于tRNA结合的后果。特别是关于拟议的通过磷酸化调节TGT的公开问题将被讨论。

At least 93 post-transcriptionally modified nucleosides have been identified in tRNAs. tRNA-modifying enzymes have to identify their cognate tRNA(s) and within the tRNA the site of modification. For many of these enzymes the structural requirements leading to their high specificity are yet unknown. The structural basis for the specificity of the eukaryotic methyltransferase DNMT2, which introduces a functionally important modification of C38, will be unraveled by the crystal structure analysis of a DNMT2-tRNA complex. This will also provide insights into the mechanism of stimulation of the methyltransferase activity by the hypermodified tRNA nucleoside queuosine at position 34 in the anticodon. With that respect, we will also study the eukaryotic tRNA-guanine-transglycosylase (TGT), which introduces queuine in tRNAs by replacing the guanine. The crystal structure of human TGT was just determined by us, and revealed unexpected differences in the quaternary structure with regard to the bacterial TGT. The resulting consequences concerning tRNA binding will be analyzed. Particularly open questions regarding the proposed regulation of TGT by phosphorylation will be addressed.