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標題: Spacer長度對於不同刺激子所造成的-1核醣體框架轉移之研究
Effect of spacer-length on -1 programmed ribosomal frameshifting of different stimulator RNAs
作者: 張惠婷
Hui-Ting Chang
關鍵字: 核醣體框架位移;刺激子;-1 programmed ribosomal frameshifting;spacer-length
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核醣體在轉譯過程中根據訊息核糖核酸進行蛋白質的合成,然而存在於訊息核糖核酸上的特殊信號會誘導核醣體進行-1框架位移(-1 programmed ribosomal frameshifting,-1PRF),此信號包括一段由七個核苷酸組成的滑動序列與相隔適當的spacer區域和刺激子RNA (stimulator RNA)。由於核醣體在解旋過程中遇到複雜且穩定的stimulator,造成假結產生抵抗的扭力去抵抗核醣體的解旋,而使
核醣體轉譯速率下降並且在滑動序列上產生延遲。根據目前提出的-1PRF機制,-1PRF的發生可能在當傳遞RNA(tRNA)攜帶胺基酸進入核醣體的accommodation階段或是核醣體進行轉譯位移的時候,因為stimulator RNA位於核醣體mRNA入口處,造成進行轉譯延長的核醣體在mRNA上產生張力(tension),此tension藉由破壞滑動序列上的密碼子與反密碼子間的作用力所釋放,進而讓tRNA在滑動序列上產生滑動並發生-1PRF。由於tension發生在spacer區域,故spacer區域被認為與調控-1PRF的作用有關。且有報告指出,在滑動序列及stimulator RNA間需有正確的spacer距離,證實了spacer距離的改變對-1 PRF效率會造成影響(Brierley et al.,1989)。故本研究探討spacer長度對於不同的stimulator RNA所造成的-1PRF效率之影響。首先構築帶有不同spacer長度的-1 PRF報導基因分析其-1 PRF效率。實驗結果顯示,下游的stimulator RNA替換成SRV假結或是JEV假結時,在不同的spacer長度時測得的-1PRF效率變化不大,而當替換成DU177假結時其-1PRF效率差異較明顯;且DU177假結在不同的真核系統下對於spacer長度的最適距離會因為核醣體的不同而有差異,另外如果在滑動序列上游構築穩定的髮夾結構作為減弱子,也會因為下游spacer長度的不同影響上游減弱子對於-1PRF attenuation的效率。顯示spacer距離的改變對於stimulator RNA刺激-1PRF效率的影響會因為不同的stimulator RNA結構特性而有所差異。

Ribosome decodes the reading frame of messenger RNA (mRNA) to synthesize protein during translation. However, the mRNAs contain the signasl that are sufficient to cause -1 programmed ribosomal frameshifting (-1PRF). The signals include a hepta-nucleotide slippery site, a stimulator RNA structure, and a proper spacer distance. According to the -1PRF model, frameshifting could occur during the accommodation of aminoacyl-tRNA into the A-site or translocation stage of a translation elongation cycle. When the elongating ribosome encounters the stable and complicated stimulator RNA, the attempt to unwind the stimulator provides additional torsional resistance to ribosome movement, and cause the elongating ribosome to pause at the slippery site. Therefore, the stimulator RNA blockage at the entrance tunnel caused the ribosome to build up tension in the spacer region. The tension is released via disrupting the codon-anticodon interaction at the slippery site allowing the movement of tRNA to slip toward -1 direction. Thus, frameshifting efficiency could be correlated to the tension force built up in the spacer region. Previous study reveals that the correct spacing distance must be maintained between the slippery sequence and the stimulator RNA, and changing the spacer distance could affect -1 frameshifting efficiency (Brierley et al., 1989). My research aims to investigate the influence of spacer-length on -1 PRF of different stimulator RNAs. The -1 PRF constructs contain a range of spacer lengths, and then inserted into the reporter gene to measure -1 frameshifting efficiency. Here, we show the -1 frameshifting efficiency stimulated by the DU177 pseudoknot is more sensitive than those of SRV-1 and JEV pseudoknots in response to the variation of spacer lengths. In addition, the optimal spacer-length for efficient -1PRF activity stimulated by DU177 pseudoknot is different between the two different eukaryotic ribosomes tested, and may by caused by difference in the ribosome sizes. Furthermore, we constructed an RNA hairpin upstream of the slippery sequence to function as -1 frameshifting attenuator for the DU177 pseudoknot, and found that -1PRF attenuation efficiency was also affected by the spacer-length. Together, these results indicate that the effect of spacer-length in -1 frameshifting efficiency is also affected by the structural features of the stimulator used.
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