Hyouta Himeno

Translating ribosomes can stall on mRNA for various reasons, including nuclease cleavage, arrest peptide sequences or ribosome collisions. In Escherichia coli , several ribosome rescue factors act to release ribosomes stalled at the 3′ end of mRNA. Among these factors, ArfB can rescue stalled ribosomes without the help of other factors. ArfB consists of an N-terminal domain containing the catalytic GGQ motif, which mediates peptidyl-tRNA hydrolysis, and a C-terminal extension that functions as a sensor for recognizing stalled ribosomes. However, how these two regions coordinate to resolve ribosome stalling remains unclear. Here, using a reconstituted translation system, we found the functional importance of histidine residues in the N-terminal domain of ArfB. In particular, histidine at position 62 in E. coli ArfB was required for stop codon–independent fMet-tRNA hydrolysis, whereas its substitution did not affect affinity for the ribosome. Furthermore, directed hydroxyl radical probing revealed that H62A mutation did not significantly alter the overall positioning of either the N-terminal domain or the C-terminal extension of ArfB on the ribosome. These findings suggest that H62 contributes to ArfB function during a step following initial ribosome binding, thereby facilitating peptidyl-tRNA hydrolysis.

Cells are exposed to various kinds of stress, obliging them to cope with the challenges they encounter. Upon subjecting E. coli cells to oxidative stress induced by elevated concentrations of hydrogen peroxide or plumbagin, as well as osmotic stress by elevated NaCl concentrations, cell growth stopped immediately after stress and it resumed after a period of time. In the present study, we found that the stress-induced growth arrest disappeared when the gene for cAMP synthesis (cyaA) or its receptor protein (crp) was deleted, whereas it reappeared when cAMP is exogenously added to ∆cyaA cells. With increasing stress intensity, the period of growth arrest increased in the wild-type cells, while ∆cyaA or ∆crp cells continued to grow without arrest, although their growth rates were reduced. These results indicate that E. coli cell has a novel cAMP-CRP-dependent mechanism to stop cell growth temporarily for adaptation to new environment when subjected to a strong stress, whether oxidative or osmotic stress.

Escherichia coli possesses three stalled-ribosome rescue factors, tmRNA·SmpB (primary factor), ArfA (alternative factor to tmRNA·SmpB), and ArfB. Here, we examined the susceptibility of rescue factor-deficient strains from E. coli SE15 to various ribosome-targeting antibiotics. Aminoglycosides specifically decreased the growth of the ΔssrA (tmRNA gene) strain, in which the levels of reactive oxygen species were elevated. The decrease in growth of ΔssrA could not be complemented by plasmid-borne expression of arfA, arfB, or ssrA^{AA to DD} mutant gene possessing a proteolysis-resistant tag sequence. These results highlight the significance of tmRNA·SmpB-mediated proteolysis during growth under aminoglycoside stress. In contrast, tetracyclines or amphenicols decreased the growth of the ΔarfA strain despite the presence of tmRNA·SmpB. Quantitative RT-PCR revealed that tetracyclines and amphenicols, but not aminoglycosides, considerably induced mRNA expression of arfA. These findings indicate that tmRNA·SmpB, and ArfA exert differing functions during stalled-ribosome rescue depending on the type of ribosome-targeting antibiotic.

To maintain proteostasis, the cell employs multiple ribosome rescue systems to relieve the stalled ribosome on problematic mRNA. One example of problematic mRNA is non-stop mRNA that lacks an in-frame stop codon produced by endonucleolytic cleavage or transcription error. In Escherichia coli, there are at least three ribosome rescue systems that deal with the ribosome stalled on non-stop mRNA. According to one estimation, 2–4% of translation is the target of ribosome rescue systems even under normal growth conditions. In the present review, we discuss the recent findings of ribosome rescue systems in bacteria.

During 30S ribosomal subunit biogenesis, assembly factors are believed to prevent accumulation of misfolded intermediate states of low free energy that slowly convert into mature 30S subunits, namely, kinetically trapped particles. Among the assembly factors, the circularly permuted GTPase, RsgA, plays a crucial role in the maturation of the 30S decoding center. Here, directed hydroxyl radical probing and single particle cryo-EM are employed to elucidate RsgA΄s mechanism of action. Our results show that RsgA destabilizes the 30S structure, including late binding r-proteins, providing a structural basis for avoiding kinetically trapped assembly intermediates. Moreover, RsgA exploits its distinct GTPase pocket and specific interactions with the 30S to coordinate GTPase activation with the maturation state of the 30S subunit. This coordination validates the architecture of the decoding center and facilitates the timely release of RsgA to control the progression of 30S biogenesis.

Addition of some kinds of translation inhibitors targeting the ribosome such as kasugamycin to the culture medium as well as removal of a ribosome maturation factor or a ribosomal protein provides Escherichia coli cells with tolerance to high salt stress. Here, we found that another kind of translation inhibitor, serine hydroxamate (SHX), which induces amino acid starvation leading to (p)ppGpp production, also has a similar effect, but via a different pathway. Unlike kasugamycin, SHX was not effective in (p)ppGpp-null mutant cells. SHX and depletion of RsgA, a ribosome maturation factor, had an additive effect on salt tolerance, while kasugamycin or depletion of RsgA did not. These results indicate the presence of two distinct pathways, (p)ppGpp-dependent and -independent pathways, for salt tolerance of E. coli cell. Both pathways operate even in the absence of σ^S, an alternative sigma factor involved in the stationary phase or stress response. Hastened activation of the exocytoplasmic stress-specific sigma factor, σ^E, after salt shock was observed in the cells treated with SHX, as has been observed in the cells treated with a translation inhibitor or depleted of a ribosome maturation factor.

リボソーム上のタンパク質合成は開始コドンに始まり，遺伝情報に基づいたペプチド伸長サイクルの繰り返しを経て終止コドンで終了する．翻訳の終了にあたっては，ペプチド解離因子（RF1またはRF2）が合成されたポリペプチドをtRNAから切り離す．しかしながら，さまざまな原因により（場合によっては計画的に）タンパク質合成を途中で中断せざるをえない状況に追い込まれることがある．たとえば，mRNAが翻訳中に切断を受けて3′側を失うと，リボソームは終止コドンに出会うことなしにmRNAの3′末端に到達してしまう．この場合，ペプチドの解離が行われないため，リボソームはそこで立ち往生することになる．こうした状況を解消すべく，細胞はリボソームレスキュー機構（翻訳停滞解消機構）を備えている．細菌のリボソームレスキュー機構として最初に見つかったのはtmRNAとSmpBによるトランストランスレーションである．ほぼすべての細菌はトランストランスレーション機構を必ずもっているが，それ以外にも2つのリボソームレスキュー機構が存在することが明らかになってきた．本稿では，これら3種類を中心に細菌のリボソームレスキュー機構について概説する（図1）．