探究在轉譯起始階段的核糖體結合位點序列空間

在原核生物的轉譯起始階段，信使核糖核酸(mRNA)會先與核糖體結合後，進行轉譯產生蛋白質，而兩者的親和性會影響到轉譯的效率及蛋白質表現量。在基因起始密碼子的上游位置，有一段核糖體結合位點，其中有段長九個核苷酸的Shine-Dalgarno (SD)序列，和16S 核醣體核糖核酸尾端的序列互補，所以能增加mRNA與核醣體的結合能力，在本實驗中，主要是產生出含有所有可能的SD序列質體，轉型至大腸桿菌中表現，並以質體上接有的綠色螢光蛋白基因來觀測轉譯效率，之後使用流式細胞儀去分選出不同強度螢光的細菌族群，再以次世代定序的技術去定序出不同螢光表現族群其SD序列的組成，以高通量實驗方式獲取大量的實驗數據，並進行分析。而得出的結果發現到，在不同的基因背景下，相同的SD序列對於轉譯效益的幫助可能會有所不同，此外在SD序列中鳥嘌呤(Guanine)的數量多寡對於轉譯起始的速率影響很大，即使整段SD序列的互補程度並不高，由於這種實驗方法能夠大量獲取序列空間的資訊，因此對於研究影響mRNA與核糖體結合的生物物理機制及發展和改善電腦預測模型都將有所助益。

關鍵字

轉譯起始；核糖體結合位點；螢光活化細胞分選；次世代定序；序列空間

並列摘要

In bacterial translation initiation step, the affinity between the mRNA and ribosome is related to the translation efficiency and gene expression. The upstream of the gene start codon, there is a sequence that can improve ribosome binding to mRNA, called ‘’Shine-Dalgatno (SD) sequence.” The SD sequence is up to nine nucleotides long and complementary to the tail of 16S ribosomal RNA. The SD sequence can enhance ribosome binding to mRNA. To realize the contribution of SD sequence to gene expression, I randomized the 9-nt SD sequence and use green fluorescent protein (GFP) as reporter gene. Using a high-throughput approach, including fluorescence-activated cell sorting (FACS) and deep sequencing measures insight into biophysical mechanism about bacterial ribosome and mRNA binding. Then I found the translation efficiency of SD sequence depending on gene background. Because of strong folding structure, in a GC-rich environment, the ribosome and mRNA need higher affinity to binding. Moreover, the G nucleotides ratio of a sequence variant correlate to the expression level. The A nucleotide ratio has no significant different in medium and high levels. It means that G-rich sequences can improve translation initiation even if they are only partly complementary to the anti-SD sequence. I will keep research the sequence space of SD sequence to confirm suppositions and the reliability of high-throughput approach.

並列關鍵字

translation initiation ； ribosome binding site ； fluorescence-activated cell sorting ； next generation sequencing ； sequence space

參考文獻

Kaczanowska, M. and M. Ryden-Aulin (2007). "Ribosome biogenesis and the translation process in Escherichia coli." Microbiol Mol Biol Rev 71(3): 477-494.

Salis, H. M., E. A. Mirsky and C. A. Voigt (2009). "Automated design of synthetic ribosome binding sites to control protein expression." Nat Biotechnol 27(10): 946-950.

Salis, H. M. (2011). "The ribosome binding site calculator." Methods Enzymol 498: 19-42

Espah Borujeni, A. and H. M. Salis (2016). "Translation Initiation is Controlled by RNA Folding Kinetics via a Ribosome Drafting Mechanism." J Am Chem Soc 138(22): 7016-7023.

Omotajo, D., T. Tate, H. Cho and M. Choudhary (2015). "Distribution and diversity of ribosome binding sites in prokaryotic genomes." BMC Genomics 16: 604.

國際替代計量

探究在轉譯起始階段的核糖體結合位點序列空間

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