Chemical and structural characterization of a model Post-Termination Complex (PoTC) for the ribosome recycling reaction: Evidence for the release of the mRNA by RRF and EF-G

Iwakura, Nobuhiro; Yokoyama, Takeshi; Quaglia, Fabio; Mitsuoka, Kaoru; Mio, Kazuhiro; Shigematsu, Hideki; Shirouzu, Mikako; Kaji, Akira; Kaji, Hideko

doi:10.1371/journal.pone.0177972

Cited by 9 publications

(16 citation statements)

References 54 publications

(108 reference statements)

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“…Structural studies of RRF/EF-G·GTP-induced PoTC breakdown demonstrate that the ribosome is in a rotated state relative to a bare 70S ribosome and the deacylated tRNA is bound in a hybrid P/E position ( 29 , 39 – 41 ). A total of 14 bridges between 30S and 50S subunits have been identified within 70S ribosomes ( 39 ).…”

Section: Discussionmentioning

confidence: 99%

The kinetic mechanism of bacterial ribosome recycling

Chen

Kaji

et al. 2017

Nucleic Acids Research

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Bacterial ribosome recycling requires breakdown of the post-termination complex (PoTC), comprising a messenger RNA (mRNA) and an uncharged transfer RNA (tRNA) cognate to the terminal mRNA codon bound to the 70S ribosome. The translation factors, elongation factor G and ribosome recycling factor, are known to be required for recycling, but there is controversy concerning whether these factors act primarily to effect the release of mRNA and tRNA from the ribosome, with the splitting of the ribosome into subunits being somewhat dispensable, or whether their main function is to catalyze the splitting reaction, which necessarily precedes mRNA and tRNA release. Here, we utilize three assays directly measuring the rates of mRNA and tRNA release and of ribosome splitting in several model PoTCs. Our results largely reconcile these previously held views. We demonstrate that, in the absence of an upstream Shine–Dalgarno (SD) sequence, PoTC breakdown proceeds in the order: mRNA release followed by tRNA release and then by 70S splitting. By contrast, in the presence of an SD sequence all three processes proceed with identical apparent rates, with the splitting step likely being rate-determining. Our results are consistent with ribosome profiling results demonstrating the influence of upstream SD-like sequences on ribosome occupancy at or just before the mRNA stop codon.

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Section: Discussionmentioning

confidence: 99%

The kinetic mechanism of bacterial ribosome recycling

Chen

Kaji

et al. 2017

Nucleic Acids Research

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“…Briefly, overnight TSB cultures were washed three times with defined minimal medium 4 (M4), diluted into fresh M4, and adjusted to OD 600 ∼0.1. After growing in a 47°C water bath shaker until OD 600 ∼0.25 (∼135 min), cells were pulse labeled with 30 μCi/mL of Tran 35 S-label (MP Biomedicals) for 3 min and chased with an excess amount of unlabeled methionine/ casamino acid mix at various time points. At each time point, 1 mL of culture was poured over 0.5 mL of ice in a prechilled microtube containing a final concentration of 15 μg/mL lysostaphin (AMBI).…”

Section: Methodsmentioning

confidence: 99%

“…These findings suggest that the dissociation of 100S ribosome involves an active mechanism to dislodge the dimerizing factors from the ribosome. Although factors such as initiation factor 3 (IF3) and ribosome recycling factor (RRF)/elongation factor G (EF-G) have been implicated in counteracting the formation of hibernating 100S and PSRP1-mediated 70S ribosomes in vitro (30,33), the proposed activity of these factors on hibernating ribosomes does not corroborate with their canonical roles in ribosome recycling (34,35) (Discussion, and see Fig. S7).…”

Section: Significancementioning

confidence: 99%

Disassembly of the Staphylococcus aureus hibernating 100S ribosome by an evolutionarily conserved GTPase

Basu

Yap

2017

Proc. Natl. Acad. Sci. U.S.A.

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The bacterial hibernating 100S ribosome is a poorly understood form of the dimeric 70S particle that has been linked to pathogenesis, translational repression, starvation responses, and ribosome turnover. In the opportunistic pathogen Staphylococcus aureus and most other bacteria, hibernation-promoting factor (HPF) homodimerizes the 70S ribosomes to form a translationally silent 100S complex. Conversely, the 100S ribosomes dissociate into subunits and are presumably recycled for new rounds of translation. The regulation and disassembly of the 100S ribosome are largely unknown because the temporal abundance of the 100S ribosome varies considerably among different bacterial phyla. Here, we identify a universally conserved GTPase (HflX) as a bona fide dissociation factor of the S. aureus 100S ribosome. The expression levels hpf and hflX are coregulated by general stress and stringent responses in a temperature-dependent manner. While all tested guanosine analogs stimulate the splitting activity of HflX on the 70S ribosome, only GTP can completely dissociate the 100S ribosome. Our results reveal the antagonistic relationship of HPF and HflX and uncover the key regulators of 70S and 100S ribosome homeostasis that are intimately associated with bacterial survival.T he biogenesis and function of bacterial 30S and 50S ribosomal subunits and the 70S complex have been studied extensively, but the significance of the 100S ribosome (homodimeric 70S) has only begun to emerge in recent years (1). The 100S ribosome is ubiquitously found in all bacterial phyla and is important for bacterial survival during nutrient limitation (2-6), antibiotic stress (7), host colonization (8), dark adaptation (9), and biofilm formation (10, 11). A common feature of these biological processes is that cells generally conserve energy by undergoing metabolic and translational dormancy because protein synthesis accounts for >50% of energy costs (12, 13). The dimerization of 70S ribosomes has been shown to down-regulate translational efficiency in vivo (3) and in vitro (3,14), and bacteria lacking 100S ribosomes are prone to early cell death concomitant with rapid ribosome degradation (3,10,15,16). These studies lead to a model whereby the formation of the 100S complex sequesters the ribosome pool away from active translation, and 70S self-dimerization prevents ribosome degradation by an unknown pathway (3,17). During the stationary phase, the 100S ribosomes are presumably dissociated and reused for new cycles of translation, thereby maintaining cell viability (1,3,16,18). The process and dissociation factors involved in the reversible transition of silent 100S to a translationally competent 70S ribosome remain poorly understood.By contrast, the 70S dimerizing factor has been characterized in many bacterial species (1,2,4,14). In Firmicutes (such as Staphylococcus aureus and Bacillus subtilis), a single long form of hibernation-promoting factor (HPF) provides the binding platform to conjoin the 30S subunits of the two 70S monomers via a direct inter...

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“…The bacterial ribosome recycling factor (RRF) and the GTPase elongation factor-G (EF-G) are known to break down the PoTc consisting of an mRNA and an uncharged P/E-site tRNA on a fully rotated 70S complex (34). The exact order of mRNA and tRNA release and 70S splitting remains controversial (35)(36)(37)(38)(39). In addition, PSRP1induced hibernating ribosomes in chloroplasts, which are not dimerized and remain as 70S monomers, are bound with Chl-RRF at the intersubunit junction (40).…”

Section: Introductionmentioning

confidence: 99%

The hibernating 100S complex is a target of ribosome-recycling factor and elongation factor G in Staphylococcus aureus

Basu

Shields

Yap

2020

Journal of Biological Chemistry

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The formation of translationally inactive 70S dimers (called 100S ribosomes) by hibernation-promoting factor is a widespread survival strategy among bacteria. Ribosome dimerization is thought to be reversible, with the dissociation of the 100S complexes enabling ribosome recycling for participation in new rounds of translation. The precise pathway of 100S ribosome recycling has been unclear. We previously found that the heat-shock GTPase HflX in the human pathogen Staphylococcus aureus is a minor disassembly factor. Cells lacking hflX do not accumulate 100S ribosomes unless they are subjected to heat exposure, suggesting the existence of an alternative pathway during nonstressed conditions. Here, we provide biochemical and genetic evidence that two essential translation factors, ribosome-recycling factor (RRF) and GTPase elongation factor G (EF-G), synergistically split 100S ribosomes in a GTP-dependent but tRNA translocation-independent manner. We found that although HflX and the RRF/EF-G pair are functionally interchangeable, HflX is expressed at low levels and is dispensable under normal growth conditions. The bacterial RRF/EF-G pair was previously known to target only the post-termination 70S complexes; our results reveal a new role in the reversal of ribosome hibernation that is intimately linked to bacterial pathogenesis, persister formation, stress responses, and ribosome integrity.

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Chemical and structural characterization of a model Post-Termination Complex (PoTC) for the ribosome recycling reaction: Evidence for the release of the mRNA by RRF and EF-G

Cited by 9 publications

References 54 publications

The kinetic mechanism of bacterial ribosome recycling

The kinetic mechanism of bacterial ribosome recycling

Disassembly of the Staphylococcus aureus hibernating 100S ribosome by an evolutionarily conserved GTPase

The hibernating 100S complex is a target of ribosome-recycling factor and elongation factor G in Staphylococcus aureus

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