Protein synthesis by ribosomes with tethered subunits

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...

Section: Resultsmentioning

confidence: 99%

Disassembly of theStaphylococcus aureushibernating 100S ribosome by an evolutionarily conserved GTPase

Basu

Yap

2017

“…The nonspecific cross-linking procedure likely establishes several cross-links between the subunits of one ribosome; thus, X-70S ribosomes should not be able to open the mRNA tunnel required for dissociation from an mRNA. However, a single cross-link per 70S ribosome would allow for separation at the subunit interface, possibly opening the tunnel, as shown with active 70S ribosomes containing covalently tethered 16S and 23S rRNAs (41).…”

Section: If1 If3mentioning

confidence: 99%

70S-scanning initiation is a novel and frequent initiation mode of ribosomal translation in bacteria

Yamamoto

Wittek

Gupta

et al. 2016

106

According to the standard model of bacterial translation initiation, the small ribosomal 30S subunit binds to the initiation site of an mRNA with the help of three initiation factors (IF1-IF3). Here, we describe a novel type of initiation termed "70S-scanning initiation," where the 70S ribosome does not necessarily dissociate after translation of a cistron, but rather scans to the initiation site of the downstream cistron. We detailed the mechanism of 70S-scanning initiation by designing unique monocistronic and polycistronic mRNAs harboring translation reporters, and by reconstituting systems to characterize each distinct mode of initiation. Results show that 70S scanning is triggered by fMet-tRNA and does not require energy; the Shine-Dalgarno sequence is an essential recognition element of the initiation site. IF1 and IF3 requirements for the various initiation modes were assessed by the formation of productive initiation complexes leading to synthesis of active proteins. IF3 is essential and IF1 is highly stimulating for the 70S-scanning mode. The task of IF1 appears to be the prevention of untimely interference by ternary aminoacyl (aa)-tRNA•elongation factor thermo unstable (EF-Tu)•GTP complexes. Evidence indicates that at least 50% of bacterial initiation events use the 70S-scanning mode, underscoring the relative importance of this translation initiation mechanism.protein synthesis | ribosomal functions | translational initiation | 30S-binding initiation | 70S-scanning initiation I t is textbook knowledge that 30S subunits initiate protein synthesis in bacteria; they recognize the initiation site of the mRNA composed of the Shine-Dalgarno (SD) sequence, the AUG codon, and fMet-tRNA, together with three initiation factors (IFs) forming the 30S initiation complex (30SIC). Association of the large 50S subunit triggers the release of the IFs, leading to the 70S initiation complex (70SIC) that enters the elongation phase of translation (reviewed in 1). We term this initiation path the "30S-binding mode" of bacterial initiation. After elongation and termination, it is thought that the ribosome dissociates into its subunits, thus providing 30S subunits for the next round of initiation.The functional role of IF2 is well defined. It can bind directly to the 30S, providing a docking site for fMet-tRNA (2), but it can also enter the 30S subunit as ternary complex fMet-tRNA•IF2•GTP (3). Both IF2 and IF3 are essential for viability. IF3 has a binding site at the 30S interface (4), which explains its antiassociation effect (5, 6), as well as its role in dissociation of the terminating 70S ribosome (7). However, the in vivo concentration of IF3 is 100-fold less (8) than required for full dissociation of 70S in vitro (4). Evidence for the presence of IF3 on 70S ribosomes was reported (9), indicating that the functional spectrum of IF3 is possibly not restricted to an antiassociation effect. Both IF3 and IF2 are also responsible for the fidelity of decoding the initiation AUG by fMet-tRNA Met f at the P site of 30S subun...

“…Early as well as current studies show that avi largely inhibits tRNA binding (12). Evn shows sequence-dependent inhibition (14) and EF4 back translocation (4), indicating translation elongation inhibition. Avi and evn do not inhibit Gram-negative bacteria (12) or eukaryotic cells (4).…”

mentioning

confidence: 99%

Avilamycin and evernimicin induce structural changes in rProteins uL16 and CTC that enhance the inhibition of A-site tRNA binding

Krupkin

Wekselman

Matzov

et al. 2016

Two structurally unique ribosomal antibiotics belonging to the orthosomycin family, avilamycin and evernimicin, possess activity against Enterococci, Staphylococci, and Streptococci, and other Gram-positive bacteria. Here, we describe the high-resolution crystal structures of the eubacterial large ribosomal subunit in complex with them. Their extended binding sites span the A-tRNA entrance corridor, thus inhibiting protein biosynthesis by blocking the binding site of the A-tRNA elbow, a mechanism not shared with other known antibiotics. Along with using the ribosomal components that bind and discriminate the A-tRNA-namely, ribosomal RNA (rRNA) helices H89, H91, and ribosomal proteins (rProtein) uL16-these structures revealed novel interactions with domain 2 of the CTC protein, a feature typical to various Gram-positive bacteria. Furthermore, analysis of these structures explained how single nucleotide mutations and methylations in helices H89 and H91 confer resistance to orthosomycins and revealed the sequence variations in 23S rRNA nucleotides alongside the difference in the lengths of the eukaryotic and prokaryotic α1 helix of protein uL16 that play a key role in the selectivity of those drugs. The accurate interpretation of the crystal structures that could be performed beyond that recently reported in cryo-EM models provide structural insights that may be useful for the design of novel pathogen-specific antibiotics, and for improving the potency of orthosomycins. Because both drugs are extensively metabolized in vivo, their environmental toxicity is very low, thus placing them at the frontline of drugs with reduced ecological hazards. degradable antibiotics | ribosomes | resistance | species-specific antibiotics | Gram-positive