It has been found that the non-B form DNA structures, like G-quadruplex (G4) and i-motif, are involved in many important biological processes. Our previous study showed that the silkworm transcription factor BmLARK binds to the G4 structure in the promoter of the transcription factor BmPOUM2 and regulates its promoter activity. However, the binding mechanism between BmLARK and BmPOUM2 G4 structure remains unclear. In this study, binding domains and key amino acid residues involved in the interaction between BmLARK and BmPOUM2 G4 were studied. The electrophoretic mobility shift assay results indicated that the two RNA-recognition motifs (RRM) of BmLARK are simultaneously required for the binding with the G4 structure. Either RRM1 or RRM2 alone could not bind with the G4 structure. The zinc-finger motif was not involved in the binding. A series of mutant proteins with specific amino acid mutations were expressed and used to identify the key amino acid residues involving the interaction. The results indicated that β sheets, especially the β1 and β3 sheets, in the RRM domains of BmLARK played critical roles in the binding with the G4 structure. Several amino acid mutations of RRM1/2 in ribonucleoprotein domain 1 (RNP1) (motif in β3 strand) and RNP2 (motif in β1 strand) caused loss of binding ability, indicating that these amino acids are the key sites for the binding. All the results suggest that RRM domains, particularly their the RNP1 and RNP2 motifs, play important roles not only in RNA recognition, but also in the G4 structure binding.

中文翻译：

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家蚕 BmPOUM2 启动子中 BmLARK 与 G4 结构相互作用的结合域和关键氨基酸的鉴定

已经发现非 B 型 DNA 结构，如 G-四链体 (G4) 和 i-motif，参与许多重要的生物过程。我们以前的研究表明，家蚕转录因子BmLARK结合到G4结构的转录因子的启动子BmPOUM2并调节其启动子活性。然而，BmLARK 与BmPOUM2 G4 结构之间的结合机制尚不清楚。在本研究中，参与 BmLARK 和BmPOUM2相互作用的结合域和关键氨基酸残基G4进行了研究。电泳迁移率变化分析结果表明 BmLARK 的两个 RNA 识别基序 (RRM) 同时需要与 G4 结构的结合。RRM1 或 RRM2 单独不能与 G4 结构结合。锌指基序不参与结合。表达了一系列具有特定氨基酸突变的突变蛋白，并用于鉴定涉及相互作用的关键氨基酸残基。结果表明，BmLARK 的 RRM 结构域中的 β 折叠，尤其是 β1 和 β3 折叠在与 G4 结构的结合中起关键作用。核糖核蛋白结构域1（RNP1）（β3链基序）和RNP2（β1链基序）中RRM1/2的几个氨基酸突变导致结合能力丧失，表明这些氨基酸是结合的关键位点。