鼠伤寒沙门菌核糖体乙酰化修饰与翻译功能间的关系研究

Acetylation is a reversible protein post-translational modification and conserved in both prokaryotes and eukaryotes. In our preliminary study, we isolated ribosomal proteins from Salmonella Typhimurium and determined their acetylation levels using quantitative mass spectrometry. The results showed that the ribosomal proteins were acetylated extensively, and acetyltransferase Pat, deacetylase CobB and metabolic intermediate acetyl phosphate were involved in regulating of acetylation of ribosomal proteins. The acetylation of ribosomal proteins varied with bacterial growth phases. Interestingly, increase of ribosomal protein L2 (RplB) acetylation level inhibited its rRNA binding ability. Furthermore, a total of 11 acetylated lysine residues on ribosomal proteins involved in interaction between ribosomal protein and rRNA were identified based on the quantitative mass spectrometry results and ribosomal three-dimensional structure information. Therefore, we speculate that acetylation of ribosomal proteins might exert key effects on the interaction between ribosomal protein and rRNA by changing the structure and charge of lysine-side chain. Acetylation could consequently influence ribosome assembly and translational efficiency. To test this speculation, we will use both in vitro and in vivo strategies to determine the role of acetylation in interaction between ribosome proteins and rRNA. Then an in vitro translation assay will be employed to explore the mechanism behind ribosome translational activities regulated by acetylation. The information derived from this study will provide a new perspective for understanding the regulation of ribosome assembly and translational efficiency.

乙酰化修饰作为一种普遍的蛋白质翻译后修饰形式，在原核和真核生物中都具有非常重要的调控作用。我们分离了鼠伤寒沙门菌核糖体蛋白并进行乙酰化修饰定量质谱检测。结果显示核糖体蛋白普遍存在乙酰化修饰，且受到乙酰基转移酶、去乙酰化酶、小分子代谢物乙酰磷酸等因素多重调控。进一步研究发现核糖体蛋白L2(RplB)的乙酰化水平升高后，其与rRNA的结合能力下降。此外质谱结果及核糖体蛋白结构信息分析结果显示，11个关键赖氨酸位点可能参与乙酰化修饰调控核糖体蛋白与rRNA的结合。因此我们推测核糖体蛋白赖氨酸残基上的乙酰化修饰能改变蛋白的电荷状态并影响其与rRNA的结合，进而影响核糖体的组装和核糖体的翻译功能。为此本课题将采用定点乙酰化修饰及染色体基因敲入法等手段获得关键赖氨酸位点突变蛋白，检测核糖体蛋白的赖氨酸位点乙酰化修饰对于核糖体组装及翻译活性的影响。本申请将为理解核糖体功能调控机制提供新的思路。

乙酰化修饰作为一种普遍的蛋白质翻译后修饰形式，在原核和真核生物中都具有非常重要的调控作用。我们分离了鼠伤寒沙门菌核糖体蛋白并进行乙酰化修饰定量质谱检测。结果显示核糖体蛋白普遍存在乙酰化修饰，且受到乙酰基转移酶、去乙酰化酶、小分子代谢物乙酰磷酸等因素多重调控。通过蔗糖梯度离心，我们发现AcP调控的核糖体蛋白乙酰化能够影响核糖体的组装。进一步的研究中通过凝胶电泳迁移实验，发现AcP调控的核糖体蛋白乙酰化能够影响核糖体蛋白与rRNA的结合，进而影响核糖体的组装。此外我们分离了野生型沙门菌及乙酰化相关基因的敲除株的多聚核糖体，随后通过Western blotting实验表明，结合在多聚核糖体上的翻译延长因子EF-Tu、EF-Ts及EF-G等均减少。由于翻译延长因子结合的减少，进一步的体外翻译系统发现这几株敲除株相较野生型菌株而言，翻译效率均降低。说明Pat和AcP调控的乙酰化修饰能够影响核糖体的翻译功能。该研究将为理解核糖体功能调控机制提供新的思路，为核糖体靶向抗生素研发提供理论基础。