【摘要】：“中心法則”中將遺傳信息從mRNA傳遞到蛋白質(zhì)這一過程,主要由大分子機器核糖體負責完成。核糖體是整個蛋白質(zhì)質(zhì)量控制過程的中心,通過校對蛋白合成和下游基因啟動,來調(diào)控新生肽鏈的命運。核糖體翻譯過程中,一系列核糖體蛋白結合因子作用于核糖體,使核糖體以不恒定的速率合成新生肽鏈,并對新生肽鏈進行修飾和輔助折疊,從而參與蛋白質(zhì)質(zhì)量控制。越來越多的研究發(fā)現(xiàn),新生肽鏈在合成過程中,會被暫停于核糖體肽鏈輸出通道,與核糖體組分相互作用參與共轉(zhuǎn)錄基因的表達調(diào)控。大腸桿菌中,SecM是一種170個氨基酸長度的分泌型蛋白,它的C端序列會調(diào)控核糖體翻譯暫停,啟動下游基因的表達。生化與結構研究揭示SecM的C端17個氨基酸序列(150FSTPVWISQAQGIRAGP166)能與23S rRNA和核糖體蛋白uL22、uL4相互作用,以緊湊的構象存在肽鏈輸出通道中,引起肽基轉(zhuǎn)移酶中心(PTC中心)失活從而導致翻譯暫停。然而,SecM誘導翻譯暫停的分子機制仍然不是很清楚。更甚地是,越來越多的研究發(fā)現(xiàn)SecM誘導的翻譯暫停可能是一個動態(tài)變化的過程。因此,我們采用冷凍電子顯微鏡技術來分析SecM暫停核糖體翻譯的機制,并解析出分辨率在3.5~3.7?的兩種翻譯暫停復合物冷凍電鏡結構。通過生化與結構分析,我們發(fā)現(xiàn)SecM暫停翻譯存在兩種不同的機制,并且會暫停在翻譯延伸不同階段。一種是SecM暫停核糖體于非旋轉(zhuǎn)狀態(tài),使肽基轉(zhuǎn)移酶中心失活,不利于氨基-tRNA定位到50S大亞基的A位點并進行自調(diào)整。另一種是SecM暫停核糖體于旋轉(zhuǎn)狀態(tài),延長新生肽鏈-tRNA從雜交A/P位點易位到P/P位點過程。兩種暫停機制中,SecM暫停序列中R163都起到關鍵作用,在暫停序列中其他氨基酸的幫助下,定位于特定位置并與通道組分相互作用,引起50S構象變化而不利于翻譯延伸。綜上所述,本研究首次提出SecM暫停核糖體翻譯存在兩種機制,揭示了SecM與核糖體組分相互作用,對翻譯延伸進行連續(xù)多步調(diào)控的細節(jié)。結合已報道的研究,包括抗生素、引導肽等在內(nèi)的新生肽鏈和/或小分子配體通過不同機制誘導核糖體構象變化,從而微調(diào)核糖體翻譯速率。
[Abstract]:The process of transferring genetic information from mRNA to protein in the Central Law is mainly done by macromolecular machine ribosomes. Ribosome is the center of the whole protein quality control process. Through proofreading protein synthesis and downstream gene initiation, the fate of new peptide chain is regulated. In the process of ribosomal translation, a series of ribosomal protein binding factors act on ribosomes, which make ribosomes synthesize new peptide chains at a constant rate, and modify and assist folding of new peptide chains, thus participating in protein quality control. More and more studies have found that the new peptide chain will be suspended in the ribosomal peptide chain output channel in the process of synthesis, and the interaction with ribosomal components will participate in the regulation of co-transcriptional gene expression. In E. coli, SecM is a secretory protein with the length of 170amino acids. Its C-terminal sequence can regulate ribosomal translation pause and initiate the expression of downstream genes. Biochemical and structural studies have revealed that the C-terminal 17 amino acid sequence (150FSTPVWISQAQGIRAGP166) of SecM can interact with 23s rRNA and ribosomal protein uL22,uL4, and exist in the peptide chain output channel in a compact configuration. It leads to the inactivation of peptidytransferase center (PTC center), which leads to the suspension of translation. However, the molecular mechanism of SecM induced translation suspension is still unclear. More and more studies have found that SecM-induced translation pauses may be a dynamic process. Therefore, we use frozen electron microscope to analyze the mechanism of SecM suspending ribosomal translation, and analyze the resolution of 3.5 鈮，

本文編號：2483478