Abstract
摘要:轉譯是核醣體在mRNA的框架上讀取密碼子以合成蛋白質的過程,通常一條mRNA可以同時被多個核醣體作用而形成聚核醣體。核醣體在這聚合物裡是高度組織化的,但是它們彼此之間是否有任何交互作用則是不得而知。單股mRNA的結構在多個核醣體結合時不易形成,但是有些結構對轉譯的調控又是非常重要,像是大腸桿菌dnaX基因轉錄出來的mRNA,上面形成的髮夾結構會造成核醣體的框架位移(FS)。所以在此研究計畫中,我們提出利用已經在實驗室中建立起來的光鉗及單分子螢光技術,以dnaX為範本,來研究聚核醣體的特性以及其如何影響FS。首先,我們將構築含數個dnaX FS序列的質體來測量其在細菌內產生FS的效率,因每個位移點會有部分核醣體被終止反應而釋放出來,其數目會逐漸減少,所以可藉此來觀察聚核醣體對FS效率的影響。其次,我們將利用單分子螢光來計算每一個mRNA上被螢光標定的核醣體數目,並觀察在mRNA加入dnaX FS序列後,是否有更多核醣體因被該結構阻礙而滯留在mRNA上。再來,我們將利用光鉗探討在核醣體作用下dnaX髮夾結構的構形變化,因為核醣體解開結構的效率將會影響其被滯留在mRNA上而產生聚核醣體的程度。最後,我們將繼續用光鉗來測量在mRNA上兩個相鄰核醣體間彼此的作用強度,以模擬在聚核醣體內的狀況。
Abstract: Translation by the ribosome is a process of protein synthesis in the cell. The ribosome decodes consecutive codons on the mRNA; the order of the codons defines a reading frame. Polyribosomes are generally formed during translation, in which one mRNA is translated by several ribosomes at the time. Study of electron microscopy has shown that polyribosomes are highly ordered structures; member ribosomes are usually packed in various geometries. It is not clear if such arrangement in a polyribosome plays any roles in translation and if any interactions happen between neighbor ribosomes in the complex. A single-stranded mRNA is less likely to form secondary structures when bound with several ribosomes. However, some RNA structures are important for translation regulation, such as a special hairpin in the dnaX mRNA from Escherichia coli. The ribosome will undergo frameshifting, with a certain probability, when encountering an RNA motif containing the hairpin. Thus, in this proposal we plan to study the nature of polyribosomes and how they affect frameshifting based on the dnaX system. We will use our established single-molecule detection systems as the main tools, including optical tweezers and single-molecule fluorescence.
First, we will construct a series of plasmids containing tandem repeats of the dnaX frameshift-inducing motif and measure the frameshifting efficiencies in bacteria. Since a fraction of ribosomes will be terminated after each motif, the level of polyribosomes will be decreased gradually, and thus the effects of polyribosomes can be dissected. Our preliminary data suggested that crowded ribosomes would reduce frameshifting efficiencies. Second, we will use single-molecule fluorescence to measure the number of fluorescence-labeled ribosomes on each mRNA template. By introducing the dnaX structure to the mRNA, we can further determine whether ribosomes are piled up around the motif, since it has been shown that the ribosome was paused at the frameshifting site during translation. Third, we will use optical tweezers to study the kinetics and dynamics of the dnaX structure under the helicase action of a stalled ribosome. The helicase activity of the ribosome may determine how likely movement of the ribosome is retarded by a structure and thus to retains more ribosomes on the template. Finally, we will use optical tweezers to investigate the interaction between two neighbor ribosomes on an mRNA by measuring the rupture force. This is to mimic the situation in a polyribosome, and any interactions enhancing or attenuating the physical contact between the ribosomes can be detected. In summary, the molecular mechanism inside polyribosomes is not understood to any details. Here we will use single-molecule approaches to tackle the problems to reveal unforeseen features, which otherwise are difficult to explore by traditional methods. The results will also provide great insight into translation regulation of frameshifting.
Keyword(s)
聚核醣體
核醣體
轉譯
框架位移
單分子
光鉗
螢光
Polyribosome
polysome
ribosome
translation
frameshifting
single-molecule
optical tweezers
fluorescence