用單分子技術研究核醣體對訊息核醣核酸二級結構的影響

Abstract

生物體中，細胞藉由核醣體轉譯訊息核醣核酸的方式合成蛋白質。轉譯的過程中，訊息核醣核酸上的二級結構會降低轉譯速率，甚至中斷轉譯作用的進行。而這些作用在蛋白質生成的調控上扮演重要角色，像是蛋白質的折疊或是轉譯框架位移等等。在大腸桿菌Escherichia coli中，dnaX基因負責表現去氧核醣核酸聚合酶(DNA polymerase III)的兩種子蛋白((tau-subunit and gamma-subunit)。dnaX基因會藉由框架位移(-1 frameshifting)的方式調整兩種不同子蛋白的生成。在核醣核酸序列上，產生框架位移的結構主要由三個部分所組成：Shine-Dalgarno序列、滑動序列(slippery sequence)和下游的髮夾二級結構(hairpin)。在細胞中，一條訊息核醣核酸上會有很多核醣體同時進行轉譯作用，稱作多聚核醣體(polyribosome)。先前研究顯示，在多聚核醣體的狀況下框架位移的調控效率會受到影響，而這跟二級結構的穩定性有很大的關係。然而對於核醣體如何影響序列二級結構穩定，目前還不是很清楚。這篇研究中，我們利用單分子光鉗技術測量二級結構的動力學參數，再探討核醣體存在時對這些參數的影響。目前為止，我們發現核醣體不只增加二級結構解開的速度，更會降低結構閉合的速度。藉由這兩種方式，減少解開二級結構所需施加的力。

The ribosome translates nucleotide triplets in single-stranded mRNA into polypeptide sequence for protein synthesis. During translation, mRNA secondary structures can slow down or even halt protein synthesis, which may play a role in cotranslational protein folding and programmed frameshifting. The Escherichia coli dnaX gene, which encodes DNA polymerase III subunits (tau-subunit and gamma-subunit) and is regulated by -1 frameshifting, contains an internal Shine-Dalgarno sequence, a slippery sequence and a 30 nt hairpin downstream in the mRNA. Previous research showed the frameshifting efficiency, which was correlated to the structural stability, would be changed in polyribosome, but how the ribosome influences the stability of mRNA secondary structure is still unknown. In this study, we characterize the structural dynamics of the dnaX mRNA hairpin by using optical tweezers, and then compare the results to the same hairpin with a stalled ribosome. We found the ribosome not only increases hairpin unfolding rates but also decreases refolding rates at the same time. Overall, the force required to unwind the hairpin is significantly decreased in the present of the ribosome.