聚核醣體效應對-1核醣體框架位移效率影響之研究

Abstract

在大腸桿菌中的去氧核醣核酸聚合酶III (DNA polymerase III holoenzyme)中包含著多種次單元 (subunit)，其中的兩種次單元γ及τ單元是藉由-1核醣體框架位移 (-1 ribosomal frameshifting) 在dnaX基因所轉錄之mRNA上轉譯 (translation) 而出。框架位移的效率會被mRNA上的滑動序列 (slippery sequence)、二級結構 (secondary structure)以及Shine-Dalgarno sequence (S-D 序列) 所影響。此外，在高轉譯起始頻率的狀況下，聚核醣體 (polyribosome) 的產生機會增加。而在聚核醣體產生時，下游核醣體將二級結構打開 (unfolding) 後，上游 核醣體於二級結構因序列尚被佔住而無法回復 (refold) 時便已轉譯至框架位移發生處序列，使得其在轉譯時不受二級結構影響，因此大幅降低了框架位移效率。 本實驗主要著重在細胞內 (in vivo) 與細胞外 (in vitro) ，利用修改後的dnaX序列加以研究影響聚核醣體密集程度的各項因子對框架位移效率的影響。在實驗中我們所操控的變因為細胞內異丙基-β-D-硫代半乳糖苷 (Isopropyl β-D-1-thiogalactopyranoside,IPTG) 的誘導時間及濃度；在細胞外則有轉譯時間、加入的核醣核酸或去氧核醣核酸量以及停止密碼子 (stop codon) 的第四個核苷酸 (nucleotide)的種類。在細胞內的實驗中，我們發現若增加IPTG的誘導時間或濃度，則框架位移的效率會有顯著增加。代表隨著IPTG誘導的mRNA量增加時，細胞內的核醣體對mRNA比例降低，因此使得聚核醣體的程度降低，而框架位移的效率增加。在細胞外的實驗也同樣因轉譯時間、加入的核醣核酸或去氧核醣核酸量的增加使得核醣體對mRNA比例降低，增加了框架位移的效率。而停止密碼子的第四個核苷酸則會對於終止效率 (termination efficiency) 產生影響，當其終止速率過慢時即會造成聚核醣體現象，進而降低框架位移效率。

In Esherichia coli, DNA polymerase III holoenzyme includes γ and τ subunits that are synthesized by regulation of the -1 frameshifting translation on mRNA of dnaX. Frameshifting efficiency can be determined by some elements on mRNA, which are the slippery sequence, secondary structure and Shine-Dalgarno sequence (SD sequence). Another element is polyribosomes, which occur at high frequency of initiation and cause the decrease of frameshifting efficiency. The reason that polyribosome decreases the frameshifting is the downstream ribosome blocks the refolding of secondary structures and prevents the upstream ribosome from interacting with the secondary structures. In our study, we use the dnaX frameshifting motif as a model system to study the influence of the density of polyribome to frameshifting efficiency in vivo and in vitro. In these studies, we control the induction time and concentration of Isopropyl β-D-1-thiogalactopyranoside (IPTG) for the in vivo experiments; in in vitro experiments, we change the translation time, the concentration of DNA plasmid or mRNA, and the fourth nucleotide of the stop codon. Our results show that with longer IPTG induction time or higher IPTG concentrations, the frameshifting efficiency was higher in in vivo experiment. For in vitro experiments, we observed that higher frameshifting efficiency comes from longer translation times or higher concentrations of the DNA plasmid or mRNA. Both results can be explained by the polyribosome effect; when mRNA concentration is increased, the ratio of ribosome to mRNA will be decreased, thus the density of polyribosome decreases to make frameshifting efficiency increased. Furthermore, the fourth nucleotide of the stop codon can influence termination efficiency; when the efficiency is too low, then the ribosome will pause longer to cause polyribosome effect and reduce frameshifting efficiency.