Introduction

Translation is an important process in cells that takes place on a ribosome, a huge complex comprised of RNAs and proteins. Troubles in the translation process would cause serious problems: Not only cells cannot synthesize proteins but also premature translation products are harmful and disturb other normal processes in cells. Therefore,regulatory systems are necessary to monitor and maintain the translation process in cells.

Generally, the untranslated region encoded in mRNA regulates the translation process.Hence, the lack of such regulatory information in mRNA makes troubles for the translation system. In a eukaryotic system, a regulatory process called nonsensemediated mRNA decay degrades mRNA that has a stop codon at an incorrect position inside the open reading frame. Another process called no-go decay also degrades mRNA that is stalled on a ribosome by the strong secondary conformation. By these systems, stalled ribosomes are released from process error and returned to be in a normal process.

Defective mRNA also causes problems in a prokaryotic system. Transcriptional error or non-specific mRNA cleavage produces mRNA without a stop codon. If such mRNA is translated, the ribosome is arrested at a 3'-terminous of mRNA. In Escherichia coli, a trans-translation system is known as a regulatory process for rescuing such error. SsrA RNA is a central factor in the trans-translation system. The outline of the reaction mechanism of the trans-translation has been elucidated by several reports, but some questions about its detailed mechanisms still remain. In E. coli, SsrA RNA mediated trans-translation cannot deal with all kinds of the translation arrest. Previous researches have shown that the stalled ribosome complex arrested in the middle of mRNA cannot proceed to the trans-translation and the mRNA cleavage is necessary prior to the transtranslation.In addition, it had been shown that the SsrA RNA is not essential to the cell viability. Therefore, it is likely that there are some other processes that can assist with trans-translation or are alternative to the trans-translation system.

Here, I investigated the rescue mechanism of stalled ribosome in E. coli by using the reconstituted cell-free protein synthesis system, the PURE system. The system is constructed with specific factors and enzymes necessary for the translation. Since the components are all identified in the PURE system, it is suitable for the analysis of the molecular mechanism of translation machinery. By using this system, I analyzed the detailed mechanism of trans-translation system and explored a novel pathway for rescuing the stalled ribosome.

1. Ribosome protein S1 function in the trans-translation system

In Escherichia coli, the trans-translation system is known as a unique process to release the translation stalling. SsrA RNA and SmpB protein are the two necessary components of the trans-translation system. SsrA RNA, also called as tmRNA, has a unique feature that it can act as both tRNA and mRNA. The complex comprised of aminoacylated tmRNA and SmpB enters into the A site of the stalled ribosome and then the mRNA on the ribosome is switched to the open reading frame (ORF) of tmRNA, directing ribosome to translate a specific peptide sequence encoded on the ORF. In addition to SmpB, some other factors are thought to play some roles in the trans-translation system. Ribosomal protein S1 is one of these factors. Ribosomal protein S1 is known as an RNA binding protein. Some studies had reported that S1 has significantly high affinity for the SsrA RNA. However, the detailed function S1 is unclear. I investigated the function of S1 by constructing the assay system utilizing the PURE system.

1.1. Trans-translation in the PURE system

The trans-translation system was reconstructed in the PURE system using purified SsrA RNA and SmpB. SsrA RNA and SmpB were two essential factors in the trans-translation system. A stalled ribosome complex was formed using a template without a stop codon.By the addition of SsrA RNA and SmpB to this stalled ribosome complex, the tag-peptide that is encoded on SsrA RNA was found to be attached to the nascent polypeptide.

1.2. Ribosomal protein S1 is not essential for the trans-translation machinery

An S1-free ribosome was prepared by using a poly (U) column and the S1-free trans-translation detection system was constructed by utilizing the PURE system.By using this system, the effect of the presence or the absence of S1 on the transtranslation was investigated.The results showed that the trans-translation reaction still proceeded even in the absence S1. Furthermore,the addition of the purified S1 did not affect the initial rate of the trans-translation reaction. Therefore, I concluded that the ribosomal protein S1 is not essential for the trans-translation machinery.

2. Exploring a novel pathway for rescuing the stalled ribosome complex

In E. coli, it is known that some specific sequences encoded on mRNA arrest the translation process. In such a case, unlikely to the case of mRNA without a stop codon,the ribosome is stalled at the middle of mRNA and the 3'-terminus of mRNA is left on the ribosome. Several reports showed that such a stalled ribosome complex is resistant to the SsrA RNA mediated trans-translation and mRNA cleavage at the A site of the ribosome is necessary prior to the SsrA RNA entry into the ribosome. Furthermore, SsrA RNA is not an essential gene in E. coli. Cells without a gene for the SsrA RNA showed normal growth under the normal condition. Thus, these studies suggest that there are some other processes that can assist with trans-translation or are alternative to the transtranslation system. According to this aspect, I explored another pathway that rescues and releases the stalled ribosome complex. Particularly, several recent reports suggested the involvement of peptidyl-tRNA hydrolase (Pth) in rescuing such complex, although clear evidence is not proposed. Therefore, I investigated the effect of Pth on the stalled ribosome complex and found a novel activity of Pth on the stalled ribosome complex.

2.1. In vitro analysis of the stalled ribosome complex

The nucleotide sequence that induces translation arrest was inserted into the 3'-terminous of GFP (green fluorescent protein) template for the PURE system. By using this template, the stalled ribosome complex comprised of ribosome, mRNA, and peptidyltRNA that carries GFP, was formed. The stalled ribosome complex formation was analyzed by sucrose density gradient centrifugation. The results showed that GFP,mRNA, and the ribosome were detected in the same fraction suggesting that the stalled ribosome complex was successfully formed. Same analysis was also examined by the gel filtration assay. The results showed that almost all the synthesized GFP was detected in the same fraction with the ribosome, suggesting that the complex formed in the PURE system is extremely stable.

2.2. Identification of the novel activity of peptidyl-tRNA hydrolase on the stalled ribosome rescue process

An experiment was constructed to analyze Pth activity on the stalled ribosome complex using the complex formed in the PURE system. After the complex was treated with Pth,the complex was subjected to a gel filtration chromatography to analyze the peptide release from the stalled ribosome. The results showed that the treatment by Pth released specific amount of GFP from the ribosome complex, suggesting Pth promoted the release of translation product from the stalled. ribosome.

2.3. Mechanism analysis of Pth in the stalled ribosome rescue process

SDS-PAGE analysis of the stalled ribosome complex showed that the translation product arrested in the ribosome was kept as peptidyl-tRNA. This indicates that the stalled ribosome is in the elongation phase and the peptidyl-tRNA is protected from the hydrolysis attack stimulated by the interaction between the ribosome and the translation release factors. On the contrary, the treatment of the stalled ribosome by Pth released the peptide from the ribosome. SDS-PAGE analysis showed that this released peptide is no longer attached to the tRNA. As described above, the stalled ribosome complex is extremely stable and therefore,the results suggest that Pth hydrolyzed the peptidyl-tRNA on the ribosome and this hydrolysis is not originated from the drop-off of the peptidyltRNA from the ribosome.

To elucidate how Pth accesses to the peptidyl-tRNA on the ribosome,the dependency of magnesium ion concentration on the Pth activity was examined.The results indicated that the lower concentration of magnesium showed higher activity of Pth on the stalled ribosome.Since the magnesium ion is known to affect the affinity between the large and small subunit of the ribosome, this suggests that the Pth cannot access to the peptidyltRNA on the 70S ribosome but can access to the one on the 50S subunit.

To verify this point more clearly, the stalled ribosome complex was analyzed by the sucrose density gradient centrifugation analysis. The results showed that under low concentration of magnesium ion, a specific proportion of the stalled ribosome is dissociated into two subunits. Furthermore, Pth treatment released almost all peptide from 50s ribosome subunit. These results suggest that Pth can promote hydrolysis of peptidyl-tRNA and release it from 50s ribosome subunit following a stalled complex dissociation i nto two subunits.

Figure 1: Time course of trans-translation with additon of S1

Figure 2: Translation product GFP detected in Gel filtration assay

本論文は3章からなり、第1章は無細胞蛋白質翻訳合成系(PUREシステム)を用い、原核生物E,coliの蛋白質翻訳中に異常停止したリボソーム複合体の調製、第2章はE.coliの翻訳異常停止したリボソーム複合体をレスキューするtrans-ranslationにおけるリボソーム蛋白質S1の機能解析、第3章はtrans-translationを非依存する翻訳異常停止をレスキューする過程の解析について述べられている。

大腸菌の生育は不完全mRNA翻訳の異常停止によるリボソーム複合体の形成に阻害されている。翻訳異常停止をレスキューする反応過程は大腸菌が環境に対応し、生育していく上で重要である。本論文の第1章は無細胞蛋白質合成系(PUREシステム)を用い、E.coliの蛋白質翻訳異常停止したリボソーム複合体の調製について述べられている。本論文の実験で基盤技術として使用された無細胞蛋白質合成系(PUREシステム)は原核生物E.coliの蛋白質翻訳過程に関わる必須のすべての因子を精製し、試験管で完全再構築された無細胞蛋白質翻訳システムである。このシステムは必須な最小因子群からなるため、反応メカニズム解析実験において、未知因子関与を最大限に排除することが出来る。論文提出者は終止コドンを持たない不完全のnonstop mRNAを用い、PUREシステムで、nonstop mRNAの翻訳系を構築し、無細胞蛋白質合成系で翻訳の異常停止を再現し、リボソームの複合体を調製した。その結果、安定する翻訳異常停止リボソーム複合体が結成することを示した。

E.coliでは、翻訳異常停止のレスキュー過程としてtrans-translation反応が知られている。論文提出者はこのtrans-translation反応においてリボソーム蛋白質S1の機能に関して、in vivoとin vitroの実験報告に矛盾があることに注目した。本論文の第2章は論文提出者がPUREシステムで確立したnonstop mRNAの異常翻訳停止システムを使い、trans-translationにおいてリボソーム蛋白質S1の機能解析を行った。E.coliにはリボソーム蛋白質S1が必須であり、in vivoではリボソーム蛋白質S1の関与を明らかにできない。論文提出者は精製したE.coliのリボソームから蛋白質S1を除き、S1除去したリボソームを調製し、PUREシステムでS1フリーの翻訳システムを構築した。論文提出者はこの蛋白質S1フリー翻訳システムを用い、S1フリー条件でtrans-translation反応を行い、E.coli蛋白質S1が翻訳異常停止レスキュー反応に必須因子でないことを初めて証明した。さらに精製した蛋白質S1の添加で、trans-translationのnonstop mRNAの翻訳異常停止複合体をレスキューする反応速度を測定したところ、顕著な影響が見い出せなかった。その結果、論文提出者は初めてリボソーム蛋白質S1フリーな翻訳システムを構築し、蛋白質S1が翻訳中に異常停止したリボソーム上でtrans-translation反応に関与しないことを証明した。

E.coliではtrans-translationを非依存し、翻訳異常停止をレスキューする反応過程が幾つのin vivoでの研究によって提唱されている。本論文の第3章はE.coliに、trans-translationを非依存する翻訳異常停止のレスキュー反応過程の解析を行った。論文提出者はラージペプチドを有する翻訳異常停止リボソーム複合体のレスキューを注目し、簡易かつ迅速に翻訳異常停止リボソーム複合体の解析システムを構築した。最小限無細胞蛋白質合成システムを用い、nonstop mRNAの翻訳による翻訳異常停止のリボソーム複合体から長鎖のポリペプチドが自発的に解離できないことを証明し、さらに、E.coliの必須遺伝子Pth(peptidyl-tRNA hydrolase)がこの安定な翻訳異常停止複合体を基質に長鎖のポリペプチドの解離を促進することを見いだした。または、Pthのこの新しい活性はより低いマグネシウムイオン濃度やRRF(ribosome recycling factor)の添加などの条件で促進された。その結果 、論文提出者は長鎖のポリペプチドを有する翻訳異常停止複合体がRRFなど翻訳因子によって構造変化した後、Pthによってリボソーム上でpeptidyl-tRNAの加水分解、解離が促進されるモデルを提示した。

なお、本論文は上田卓也氏、清水義宏氏との共同研究であるが、論文提出者が主体となって解析及び検証を行ったもので、論文提出者の寄与が十分であると判断する。

したがって、博士(生命科学)の学位を授与できると認める。