氨基糖苷类抗生素核糖开关翻译调控机制的深入研究

Riboswitch was structured RNA that regulate the expressing of downstream gene by sensing small metabolites without intermediate proteins. As first co-author, the applicant published a paper in CELL journal in January, 2013 reporting the first antibiotic sensing riboswitch--aminoglycoside antibiotic sensing riboswitch. The riboswitch located in the 5’UTR of aac and aad gene mRNA, it can specifically recognize amimoglycoside antibiotics to change the RNA structure which gave a result of an ‘exposure’ of SD sequence for the ribosome binding to regulate aac and aad gene expressing. The applicant had proposed a translational regulation modle for the aac riboswitch which was inferred from the in vivo reporter data and Real Time PCR data. But there was evidence lacking to validate the translational regulation mechanism. Based on published data, we will continue to find direct proof by in vivo luciferase reporter system and in vitro translation technique to confirm the aac riboswitch mechanism works on translational level. This study will reveal the whole spectrum of molecular regulation mechanism and explore a full potential of application for medical and clinical research.

核糖开关是位于mRNA非编码区域的具有特殊结构的RNA序列，这类RNA可直接特异性识别生物代谢小分子来调控下游基因表达，不依赖于任何蛋白质因子的参与。项目申请人于2013年1月在CELL杂志上报道了世界首例抗生素类核糖开关--氨基糖苷类抗生素核糖开关。该核糖开关与抗生素的特异性结合能够导致核糖体结合位点从‘隐蔽’状态转换为‘暴露’状态，从而调控基因的表达。该文章研究虽已推断出该核糖开关的调控作用发生在翻译水平，但是，目前这一调控机制尚缺少直接有效的实验数据。本项目将在以往工作的基础之上，综合利用报告基因诱导表达系统以及体外翻译体系技术，对氨基糖苷类抗生素核糖开关对下游基因的调控机制进行研究，在进一步明确该核糖开关的调控作用发生在翻译水平之后，深入探讨、细化已建立的分子调控机制，为完善该核糖开关的生物调控模型提供更为直接有效的科学证据。

核糖开关是位于mRNA非编码区域的具有特殊结构的RNA序列，这类RNA可直接特异性识别生物代谢小分子来调控下游基因表达，不依赖于任何蛋白质因子的参与。本项目在以往工作的基础之上，首先构建了氨基糖苷类抗生素抗性基因mRNA 5’UTR区域的进化树，在此基础之上，结合二级结构模型预测软件CM finder等生物信息学手段，预测并提出了氨基糖苷类抗生素抗药性基因的分子调控模型。然后综合利用报告基因诱导表达系统，化学探测技术，微量热泳动技术，进一步确认氨基糖苷类抗生素耐药性基因的表达受到核糖开关分子机制的诱导调控。该研究结果进一步丰富了氨基糖苷类核糖开关的调控模型，为深入研究氨基糖苷类抗生素的耐药性机制提供了更多理论依据。