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Translation termination in bacteria requires that the stop codon be recognized by release factor RF1 or RF2, leading to hydrolysis of the ester bond between the peptide and tRNA on the ribosome. As a consequence, normal termination cannot proceed if the translated mRNA lacks a stop codon. In Escherichia coli , the ribosome rescue factor ArfA releases the nascent polypeptide from the stalled ribosome with the help of RF2 in a stop codon-independent manner. Interestingly, the reaction does not proceed if RF1 is instead provided, even though the structures of RF1 and RF2 are very similar. Here, we identified the regions of RF2 required for the ArfA-dependent ribosome rescue system. Introduction of hydrophobic residues from RF2 found at the interface between RF2 and ArfA into RF1 allowed RF1 to associate with the ArfA-ribosome complex to a certain extent but failed to promote peptidyl-tRNA hydrolysis, whereas WT RF1 did not associate with the complex. We also identified the key residues required for the process after ribosome binding. Our findings provide a basis for understanding how the ArfA-ribosome complex is specifically recognized by RF2 and how RF2 undergoes a conformational change upon binding to the ArfA-ribosome complex., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Kurita et al.)

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., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

During cellular translation of messenger RNAs by ribosomes, the translation apparatus sometimes pauses or stalls at the elongation and termination steps. With the exception of programmed stalling, which is usually used by cells for regulatory purposes, ribosomes stalled on mRNAs need to be terminated and recycled to maintain adequate translation capacity. Much ribosome stalling originates in aberrant mRNAs that lack a stop codon. Transcriptional errors, misprocessing of primary transcripts, and undesired mRNA cleavage all contribute to the formation of non-stop mRNAs. Ribosomes stalled at the 3' end of non-stop mRNAs do not undergo normal termination owing to the lack of specific stop-codon recognition by canonical peptide release factors at the A-site decoding centre. In bacteria, the transfer-messenger RNA (tmRNA)-SmpB-mediated trans-translation rescue system reroutes stalled ribosomes to the normal elongation cycle and translation termination. Two additional rescue systems, ArfA-RF2 (refs 13, 14, 15, 16) and ArfB (formerly known as YaeJ), are also present in many bacterial species, but their mechanisms are not fully understood. Here, using cryo-electron microscopy, we characterize the structure of the Escherichia coli 70S ribosome bound with ArfA, the release factor RF2, a short non-stop mRNA and a cognate P-site tRNA. The C-terminal loop of ArfA occupies the mRNA entry channel on the 30S subunit, whereas its N terminus is sandwiched between the decoding centre and the switch loop of RF2, leading to marked conformational changes in both the decoding centre and RF2. Despite the distinct conformation of RF2, its conserved catalytic GGQ motif is precisely positioned next to the CCA-end of the P-site tRNA. These data illustrate a stop-codon surrogate mechanism for ArfA in facilitating the termination of non-stop ribosomal complexes by RF2.

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., (© The Authors 2016. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Although trans-translation mediated by tmRNA-SmpB has long been known as the sole system to relieve bacterial stalled ribosomes, ArfA has recently been identified as an alternative factor for ribosome rescue in Escherichia coli. This process requires hydrolysis of nascent peptidyl-tRNA by RF2, which usually acts as a stop codon-specific peptide release factor. It poses a fascinating question of how ArfA and RF2 recognize and rescue the stalled ribosome. Here, we mapped the location of ArfA in the stalled ribosome by directed hydroxyl radical probing. It revealed an ArfA-binding site around the neck region of the 30S subunit in which the N- and C-terminal regions of ArfA are close to the decoding center and the mRNA entry channel, respectively. ArfA and RF2 sequentially enter the ribosome stalled in either the middle or 3' end of mRNA, whereas RF2 induces a productive conformational change of ArfA only when ribosome is stalled at the 3' end of mRNA. On the basis of these results, we propose that ArfA functions as the sensor to recognize the target ribosome after RF2 binding., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Ribosome biogenesis is a tightly regulated, multi-stepped process. The assembly of ribosomal subunits is a central step of the complex biogenesis process, involving nearly 30 protein factors in vivo in bacteria. Although the assembly process has been extensively studied in vitro for over 40 years, very limited information is known for the in vivo process and specific roles of assembly factors. Such an example is ribosome maturation factor M (RimM), a factor involved in the late-stage assembly of the 30S subunit. Here, we combined quantitative mass spectrometry and cryo-electron microscopy to characterize the in vivo 30S assembly intermediates isolated from mutant Escherichia coli strains with genes for assembly factors deleted. Our compositional and structural data show that the assembly of the 3'-domain of the 30S subunit is severely delayed in these intermediates, featured with highly underrepresented 3'-domain proteins and large conformational difference compared with the mature 30S subunit. Further analysis indicates that RimM functions not only to promote the assembly of a few 3'-domain proteins but also to stabilize the rRNA tertiary structure. More importantly, this study reveals intriguing similarities and dissimilarities between the in vitro and the in vivo assembly pathways, suggesting that they are in general similar but with subtle differences.

RsgA is a 30S ribosomal subunit-binding GTPase with an unknown function, shortage of which impairs maturation of the 30S subunit. We identified multiple gain-of-function mutants of Escherichia coli rbfA, the gene for a ribosome-binding factor, that suppress defects in growth and maturation of the 30S subunit of an rsgA-null strain. These mutations promote spontaneous release of RbfA from the 30S subunit, indicating that cellular disorders upon depletion of RsgA are due to prolonged retention of RbfA on the 30S subunit. We also found that RsgA enhances release of RbfA from the mature 30S subunit in a GTP-dependent manner but not from a precursor form of the 30S subunit. These findings indicate that the function of RsgA is to release RbfA from the 30S subunit during a late stage of ribosome biosynthesis. This is the first example of the action of a GTPase on the bacterial ribosome assembly described at the molecular level.

Trans-translation relieves a stalled translation on the bacterial ribosome by transfer-messenger RNA (tmRNA) with the help of SmpB, an essential cofactor of tmRNA. Here, we examined the role of the unstructured C-terminal tail of SmpB using an in vitro trans-translation system. It was found that truncation of the C-terminal tail or substitution of tryptophan residue at 147 in the middle of the C-terminal tail affected the activity in the early stage of trans-translation. Our investigations also revealed that the C-terminal tail is not required for the events until GTP is hydrolyzed by EF-Tu in complex with tmRNA-SmpB. A synthetic peptide corresponding to the C-terminal tail of SmpB inhibited peptidyl-transfer of alanyl-tmRNA and A-site binding of SmpB, but not GTP hydrolysis. These results suggest that the C-terminal tail has a role in the step of accommodation of alanyl-tmRNA-SmpB into the A-site. Directed hydroxyl radical probing indicated that tryptophan residue at 147 is located just downstream of the decoding center in the mRNA path when SmpB is in the A-site.

RsgA is a unique GTP hydrolytic protein in which GTPase activity is significantly enhanced by the small ribosomal subunit. Deletion of RsgA causes slow cell growth as well as defects in subunit assembly of the ribosome and 16S rRNA processing, suggesting its involvement in maturation of the small subunit. In this study, we found that removal of RsgA or inactivation of its ribosome small subunit-dependent GTPase activity provides Escherichia coli cells with resistance to high salt stress. Salt stress suppressed the defects in subunit assembly of the ribosome and processing of 16S rRNA as well as truncation of the 3' end of 16S rRNA in RsgA-deletion cells. In contrast, salt stress transiently impaired subunit assembly of the ribosome and processing of 16S rRNA and induced 3' truncation of 16S rRNA in wild-type cells. These results suggest that the action of RsgA on the ribosome, which usually facilitates maturation of the small subunit, disturbs it under a salt stress condition. Consistently, there was a drastic but transient decrease in the intracellular amount of RsgA after salt shock. Salt shock would make the pathway of maturation of the ribosome small subunit RsgA independent.

RsgA is a unique GTP hydrolytic protein, in which the GTPase activity is significantly enhanced by the small ribosomal subunit. Depletion of RsgA causes slow cell growth as well as defects in the subunit assembly of the ribosome and the 16S rRNA processing, suggesting its involvement in the maturation of the small subunit. Several antibiotics bound to the decoding center of the small subunit inhibited the ribosome-dependent GTPase activity of RsgA. Our recent study using chemical modification indicates that the binding of RsgA induces conformational changes around the A site, P site, and helix 44. These results suggest that RsgA is involved in the maturation step of the decoding center of the small subunit of ribosome. Here, we also show a physiological role of RsgA under stress condition.

RsgA (ribosome-small-subunit-dependent GTPase A, also known as YjeQ) is a unique GTPase in that guanosine triphosphate hydrolytic activity is activated by the small subunit of the ribosome. Disruption of the gene for RsgA from the genome affects the growth of cells, the subunit association of the ribosome, and the maturation of 16S rRNA. To study the interaction of Escherichia coli RsgA with the ribosome, chemical modifications using dimethylsulfate and kethoxal were performed on the small subunit in the presence or in the absence of RsgA. The chemical reactivities at G530, A790, G925, G926, G966, C1054, G1339, G1405, A1413, and A1493 in 16S rRNA were reduced, while those at A532, A923, G1392, A1408, A1468, and A1483 were enhanced, by the addition of RsgA, together with 5'-guanylylimidodiphosphate. Among them, the chemical reactivities at A532, A790, A923, G925, G926, C1054, G1392, A1413, A1468, A1483, and A1493 were not changed when RsgA was added together with GDP. These results indicate that the binding of RsgA induces conformational changes around the A site, P site, and helix 44, and that guanosine triphosphate hydrolysis induces partial conformational restoration, especially in the head, to dissociate RsgA from the small subunit. RsgA has the capacity to coexist with mRNA in the ribosome while it promotes dissociation of tRNA from the ribosome.

RsgA is a unique GTP hydrolytic protein that is widely found in bacteria and plants, and is activated by the small subunit of the ribosome. Disruption of the gene for RsgA from the genome affects the growth of cells, the subunit association of the ribosome in cells and maturation of 16S ribosomal RNA. Here, we investigated the interaction between EscherichiacoliRsgA and the ribosome. Several antibiotics bound to the decoding center of the small subunit inhibited the ribosome-dependent GTPase activity of RsgA, suggesting that RsgA binds to the decoding center. Chemical footprinting was also performed to further investigate the interaction.

The GTPase activity of Escherichia coli YjeQ, here named RsgA (ribosome small subunit-dependent GTPase A), has been shown to be significantly enhanced by ribosome or its small subunit. The enhancement of GTPase activity was inhibited by several aminoglycosides bound at the A site of the small subunit, but not by a P site-specific antibiotic. RsgA stably bound the small subunit in the presence of GDPNP, but not in the presence of GTP or GDP, to dissociate ribosome into subunits. Disruption of the gene for RsgA from the genome affected the growth of the cells, which predominantly contained the dissociated subunits having only a weak activation activity of RsgA. We also found that 17S RNA, a putative precursor of 16S rRNA, was contained in the small subunit of the ribosome from the RsgA-deletion strain. RsgA is a novel GTPase that might provide a new insight into the function of ribosome.