High-resolution structure of the Escherichia coli ribosome

Ribosome biogenesis requires multiple assembly factors. In Escherichia coli, deletion of RlmE, the methyltransferase responsible for the 2′-O-methyluridine modification at position 2552 (Um2552) in helix 92 of the 23S rRNA, results in slow growth and accumulation of the 45S particle. We demonstrate that the 45S particle that accumulates in ΔrlmE is a genuine precursor that can be assembled into the 50S subunit. Indeed, 50S formation from the 45S precursor could be promoted by RlmE-mediated Um2552 formation in vitro. Ribosomal protein L36 (encoded by rpmJ) was completely absent from the 45S precursor in ΔrlmE, and we observed a strong genetic interaction between rlmE and rpmJ. Structural probing of 23S rRNA and high-salt stripping of 45S components revealed that RlmE-mediated methylation promotes interdomain interactions via the association between helices 92 and 71, stabilized by the single 2′-O-methylation of Um2552, in concert with the incorporation of L36, triggering late steps of 50S subunit assembly.R ibosomes are essential ribonucleoprotein complexes that translate the genetic information encoded by mRNA into protein. Intact bacterial ribosomes, which have a sedimentation coefficient of 70S, consist of large (50S) and small (30S) subunits. Each subunit can be reconstituted in vitro from its individual component ribosomal RNAs and proteins (1-5), indicating that these components contain all of the information necessary to automatically assemble into functional subunits. Because the intermediate particles observed in an in vitro reconstitution of the subunits are similar to those observed in vivo, assembly maps that describe the order of ribosomal protein binding in vitro are useful tools for understanding ribosome biogenesis in the cell (6). However, assembly is slower in vitro than in vivo, and nonphysiological conditions, such as high-salt concentration and high temperature, are required for in vitro assembly.In the cell, ribosome assembly is coordinated with the transcription and processing of large precursor rRNAs encoded by the rrn operon (3, 7). Once transcription starts, nascent transcripts rapidly form local secondary and tertiary structures that serve as binding sites for the primary ribosomal proteins, thereby initiating ribosome assembly. However, hierarchical assembly starting at the 5′-terminal domains of the 16S and 23S rRNAs is not essential for ribosome formation in the cell (8) because nonribosomal factors, termed "assembly factors," facilitate rapid and efficient assembly of ribosomal particles. Ribosome biogenesis is an energy-consuming process in which a number of assembly factors, including RNA helicases, GTPases, protein chaperones, and rRNA-modifying enzymes, support efficient assembly of each subunit (6, 9, 10). Individual mutation or deletion of several assembly factors causes accumulation of immature 50S subunits that sediment at 40S or 45S. For example, the RNA helicase SrmB participates in the early stage of 50S assembly (11,12); deletion of srmB results in accumulation of a 40S ...

“…1B; and see Fig. 6A) (24)(25)(26). The C2 carbonyl group of Um2552 is involved in this interaction (see Fig.…”

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Single methylation of 23S rRNA triggers late steps of 50S ribosomal subunit assembly

Arai

Ishiguro

Kimura

et al. 2015

“…Despite the high sequence conservation, local variability was detected. To highlight the unique features of SA50S, we compared it with the available eubacterial ribosome structures D50S (40) and the large ribosomal subunits in the T70S (6) and E70S (7) ribosomes (29,41).…”

Section: Resultsmentioning

confidence: 99%

Structural insights into species-specific features of the ribosome from the pathogen Staphylococcus aureus

Eyal

Matzov

Krupkin

et al. 2015

119

151

The emergence of bacterial multidrug resistance to antibiotics threatens to cause regression to the preantibiotic era. Here we present the crystal structure of the large ribosomal subunit from Staphylococcus aureus, a versatile Gram-positive aggressive pathogen, and its complexes with the known antibiotics linezolid and telithromycin, as well as with a new, highly potent pleuromutilin derivative, BC-3205. These crystal structures shed light on specific structural motifs of the S. aureus ribosome and the binding modes of the aforementioned antibiotics. Moreover, by analyzing the ribosome structure and comparing it with those of nonpathogenic bacterial models, we identified some unique internal and peripheral structural motifs that may be potential candidates for improving known antibiotics and for use in the design of selective antibiotic drugs against S. aureus.

“…The 70S ribosomes with no nascent chain were a gift from Jonas Noeske, University of California, Berkeley, and prepared as described previously (39). For RNCs, IVT reactions were quenched with chloramphenicol to a final concentration of 2 mM after preparation as described above, diluted to 100 μL to the desired urea concentrations, and incubated for at least 12 h at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Quantitative determination of ribosome nascent chain stability

Samelson

Jensen

Soto

et al. 2016

133

Accurate protein folding is essential for proper cellular and organismal function. In the cell, protein folding is carefully regulated; changes in folding homeostasis (proteostasis) can disrupt many cellular processes and have been implicated in various neurodegenerative diseases and other pathologies. For many proteins, the initial folding process begins during translation while the protein is still tethered to the ribosome; however, most biophysical studies of a protein's energy landscape are carried out in isolation under idealized, dilute conditions and may not accurately report on the energy landscape in vivo. Thus, the energy landscape of ribosome nascent chains and the effect of the tethered ribosome on nascent chain folding remain unclear. Here we have developed a general assay for quantitatively measuring the folding stability of ribosome nascent chains, and find that the ribosome exerts a destabilizing effect on the polypeptide chain. This destabilization decreases as a function of the distance away from the peptidyl transferase center. Thus, the ribosome may add an additional layer of robustness to the protein-folding process by avoiding the formation of stable partially folded states before the protein has completely emerged from the ribosome.protein folding | cotranslational folding | pulse proteolysis | protein stability P roper protein folding is necessary for the function of all cells, and changes in cellular protein folding capacity can lead to cell death and disease (1). For more than 50 years, experimental studies have probed the physical properties of proteins, paving the way for incredible advances in protein design and protein structure prediction, as well as for understanding the first principles of protein folding (2, 3). These in vitro studies, however, do not necessarily recapitulate the folding process in vivo, including the constraints that the ribosome imposes on the emerging nascent chain during translation (4).In the cell, proteins are synthesized by the ribosome one amino acid at a time on a time scale that is slower than most in vitro protein folding rates (5). Thus, the emerging chain has time to explore conformational space and adopt structured conformations before its entire sequence has been synthesized (6). This vectorial process, and the proximity of the ribosome itself, can modulate the emerging chain's energy landscape in ways that are just beginning to be appreciated. Translation can affect folding efficiency, enable the population of intermediates that are not revealed during offribosome studies, and even determine the final conformational fate of the nascent protein (6-10). To understand the folding process in vivo, general rules and biophysical mechanisms for these modulations of the emerging chain's energy landscape need to be elucidated.To unravel the in vivo folding landscape, we need to interrogate the energetics and dynamics of ribosome-bound nascent chains in a manner analogous to studies of the in vitro folding process. The challenge, however, is that standard ...