細胞內-1核醣體框架位移效率之研究

Abstract

大腸桿菌dnaX基因藉由高效率-1核醣體框架位移(-1 ribosomal frameshifting)表現兩種去氧核醣核酸聚合酶III (DNA polymerase III holoenzyme)所需之次單元：gamma與tau，其框架位移點之效率會受到mRNA上的滑動序列(slippery sequence)、下游的二級結構，以及其相對距離的影響。當帶有框架位移訊號的基因在轉譯時，若提高核醣體的起始頻率，便有可能會縮短mRNA上核醣體彼此之間的距離，聚核醣體(polysome)發生的機會上升，使得核醣體解開二級結構後間隔不夠二級結構回復，而維持解開的狀態，降低上游核醣體位移的效率。 本實驗著重在大腸桿菌細胞內(in vivo)，利用dnaX序列加以修改，研究聚核醣體、造成框架位移的二級結構與終止密碼子位置之間的關係，以及如何對框架位移效率造成影響。我們發現終止密碼子若位於二級結構下游，核醣體在終止的同時，其體積佔據部分二級結構回復的空間，使核醣體等待釋放因子時，上游核醣體已經在框架位移點做選擇，失去二級結構將影響上游核醣體框架位移效率。若降低聚核醣體的形成機會，則框架位移效率就比較不受到終止密碼子位置的影響，而呈現個別核醣體在框架位移點獨立選擇的結果。本實驗的結果支持文獻的推論，同時也發現在特定環境下，不只是造成框架位移的序列本身，核醣體之間的交互作用也可能會影響框架位移效率。

Two subunits (tau and gamma) of the DNA polymerase III holoenzyme in Escherichia coli are produced through efficient -1 programmed ribosomal frameshifting. The efficiency of frameshifting is determined by the elements around the shift site on mRNA, including the slippery sequence, secondary structure and the distance between them. In general, increasing the translation initiation frequency will shorten the space between neighboring ribosomes on the mRNA and then increase the possibility of polysome formation. As a result, once the secondary structure is unfolded by a preceding ribosome, it may remain open until the next ribosome reaches. Thus, the frameshifting efficiency may be decreased. In this study, we use the dnaX frameshifting motif as a model system to explore the relationship between polysome and frameshifting efficiency. Our results suggest that a downstream ribosome unfolds the secondary structure of the mRNA and blocks refolding when it is terminated on the stop codon just after the secondary structure. In the meantime, an upstream ribosome coming to the frameshifting site encounters no secondary structures. Thus, fewer ribosomes are induced to undergo -1 frameshifting. The frameshifting efficiency will be restored while the formation frequency of polysome decreases. Our results support that frameshifting efficiency may be affected not only by the sequence itself, but also by the interaction between ribosomes on the mRNA.