影響轉譯框架位移的核醣核酸結構和此結構與聚核醣體相互作用之研究

Abstract

核醣體負責將信使核醣核酸(mRNA)轉譯成蛋白質。一條mRNA的轉譯起始位點可能會有核醣體接續結合上來一起轉譯，形成聚核醣體。核醣體按照mRNA上每三個核苷酸作為一個密碼子的方式轉譯成一個胺基酸。這種對應方式稱為閱讀框架，也是0轉譯框架。當核醣體轉譯到一個由滑動序列和類夏因達爾加諾(Shine-Dalgarno, SD)序列或mRNA二級結構組成的框架位移單元時，有些核醣體會發生框架位移滑動到其他閱讀框架上(−1或+1)繼續做出不同蛋白質。由此，相同的mRNA序列可以被轉譯成不同的胺基酸序列。 在本論文的研究中，我們探討大腸桿菌聚核醣體現象對框架位移的影響。我們使用核醣體逐漸脫離的設計測試不同的聚核醣體密集程度和框架位移效率的關聯。在−1框架位移單元上的核醣體數量越少框架位移效率越高。兩個核醣體會互相靠近形成一組通過框架位移單元，限制了框架位移單元的mRNA二級結構的折疊，導致第二個核醣體的框架位移效率下降。成組的核醣體還能通過相互碰撞引起對方的框架位移。我們發現插入序列IS2的框架位移可能受到聚核醣體現象的調控。 聚核醣體除了能佔據mRNA序列影響其折疊，還能沿著5’往3’方向梳理mRNA，特別是假結結構。核醣體會沿著5’往3’方向打開假結結構並以相同的順序釋放mRNA序列讓其重新沿著5’往3’方向折疊，導致假結結構3’部分的序列可以沿著不同的方向繞上已形成的5’部分的髮夾結構。我們使用光鉗技術觀察單個核醣核酸(RNA)分子從5’方向開始折疊以及重新從5’髮夾結構開始打開的過程。假結結構的3’序列沿著右手螺旋方向繞上5’髮夾結構形成的第一種折疊方式變得更穩定，這是由於3’序列和髮夾結構的序列平行而容易形成更多氫鍵。假結結構3’序列沿著左手螺旋跨過5’髮夾結構的RNA鏈和凹槽形成的第二種折疊方式會把已形成的5’髮夾結構夾住，導致再次解開5’髮夾結構時會發生更多的結構變化。通過核醣體梳理形成的兩種不同的假結結構折疊方式可能在各自的框架位移位點發揮不同的作用。

The ribosome translates mRNA to synthesize protein. Ribosomes continuously initiate on the ribosome binding site to start translation on one mRNA, forming structures known as polyribosomes. The ribosome decodes three nucleotides of the mRNA as a codon corresponding to an amino acid. This pattern is called a reading frame (the 0 frame). When translating to a programmed ribosomal frameshifting motif, usually containing a slippery sequence and a stimulator, such as an internal Shine-Dalgarno (iSD) sequence and a secondary structure, some ribosomes may slide to another frame (−1 or +1) to continue translation to produce protein that is different from the original product. Therefore, the same mRNA sequence can be translated into two amino acid sequences. In this study, we investigate how polyribosomes interfere with the mRNA frameshifting motif. We used the ribosome abscission design to test the effect of polyribosome enrichment on the frameshifting motif. Frameshifting efficiency showed anti-correlation with the ribosome load on the −1 frameshifting motif. Two ribosomes may translate one after another through the frameshifting motif as a group to restrain refolding of the mRNA secondary structure to reduce the frameshifting efficiency. The group effect may also stimulate frameshifting via collision between two ribosomes; we found that this is a promising frameshifting mechanism of the insertion element IS2. Besides occupation of structure sequence, polyribosomes also organize the structural folding of mRNA from the 5’ end to the 3’ end, especially for pseudoknots. The ribosome unfolds a pseudoknot from the 5’ to 3’ orientation and then releases the sequence to allow pseudoknot refolding in the same orientation. Through this way, the 3’ side sequence may wrap the preformed hairpin at the 5’ side in different directions. We used optical tweezers to study the orientational refolding and unfolding of pseudoknots at the single molecule point of view. The folding pattern that the 3’ sequence wraps in a right-handed twist appeared more stable, since the 3’ sequence was aligned along the major groove of the double helix of the 5’ hairpin and form more hydrogen bonds. The alternative folding pattern that the 3’ sequence wraps left-handed to cross the helix of the 5’ hairpin results in reduced stability of the 5’ hairpin and varied pseudoknot conformations. These two folding patterns organized by ribosomes may have different frameshift-stimulating capabilities.