細胞內聚核醣體引起轉譯框架位移之研究

Study of Polyribosome-induced Frameshifting in vivo

汪健州(Jian-Zhou Wang)

指導教授 ： 温進德

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201600584

摘要

核醣體是生物製造蛋白質的工廠。一條mRNA上通常有多個核醣體同時進行轉譯，稱作聚核醣體。核醣體在轉譯過程中遇到特殊的mRNA序列時會發生框架位移，從一段mRNA序列轉譯出不同的蛋白質產物。大腸桿菌的dnaX基因框架位移位點由滑動序列、上游的內部SD序列和下游的髮夾結構三個部分組成。當核醣體轉譯到滑動序列時，受到下游髮夾結構的阻礙和上游SD序列的拉扯，有機會往上游方向滑動，從0轉譯框架進入-1轉譯框架。兩個轉譯框架分別做出DNA聚合酶III的γ和τ次單元。
本文在大腸桿菌內研究聚核醣體現象對轉譯框架位移的影響。我們用dnaX轉譯框架位移位點作為材料，通過西方墨點法定量兩個轉譯框架的蛋白質，進而計算出框架位移比例。我們發現核醣體在遇到SD序列和二級結構時轉譯速度減慢，可能會被上游的核醣體追上。此時，下游核醣體代替原本框架位移位點的髮夾結構，促使上游核醣體發生-1框架位移。如果去掉下游的SD序列或二級結構，使核醣體不再聚集，框架位移比例都會下降。另外，內部SD序列還可以抓住游離的30S次單元，同樣能夠阻礙上游核醣體引起轉移框架位移。通過這些研究，我們成功觀測到聚核醣體中相鄰核醣體會互相影響，並且對轉譯結果產生顯著的影響。

關鍵字

核醣體 ； 框架位移 ； 轉譯 ； 聚核醣體 ； dnaX

並列摘要

The ribosome is a ubiquitous protein manufactory for all living creatures. There are usually copious ribosomes, also called polyribosome, co-translating on one mRNA. The ribosome has a chance to shift its reading frame while encountering a special mRNA sequence, like the frameshifting site of dnaX gene in Escherichia coli, and thus translates the downstream mRNA sequence to another different protein product. The dnaX frameshifting site consists of a slippery sequence, an upstream internal Shine-Dalgarno sequence and a downstream hairpin. Because of being obstructed by hairpin and drawn by the SD sequence, the ribosome may shift one base towards upstream on the slippery sequence from the 0 frame to -1 frame. As a consequence, different codons on two frames are respectively translated into γ and τ subunits of DNA polymerase III.
In this thesis, we investigate how polyribosome affects translation frameshifting in E. coli in vivo. We use the dnaX frameshifting site as a model, quantify protein products from two frames by Western blot, and calculate frameshifting efficiency. We find that a ribosome can be temporarily stalled by a strong SD sequence or secondary structure and then caught up by following ribosomes. At this time, the stalled ribosome may mimic the role of the hairpin and promote the following ribosome to shift to the -1 frame on the slippery sequence. When we mutate the downstream SD sequence or secondary structure, ribosomes are unable to accumulate, resulting in a reduction of frameshifting efficiency. Moreover, not only an elongating ribosome but also a free 30S ribosomal subunit can trigger upstream ribosomes frameshifting by binding to the internal SD sequence. Through our research, we have successfully observed the interaction between ribosomes, which may remarkably affect translation outcome.

並列關鍵字

ribosome ； frameshifting ； translation ； polyribosome ； dnaX

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