Study of Polyribosome-induced Frameshifting in vivo

• Main Authors: Jian-Zhou Wang, 汪健州
• Other Authors: Jin-Der Wen
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/02145111575295975440

碩士 === 國立臺灣大學 === 分子與細胞生物學研究所 === 104 === 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.