Structure of the Bacillus subtilis 70S ribosome reveals the basis for species-specific stalling

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Although the importance of the nonuniform progression of elongation in translation is well recognized, there have been few attempts to explore this process by directly profiling nascent polypeptides, the relevant intermediates of translation. Such approaches will be essential to complement other approaches, including ribosome profiling, which is extremely powerful but indirect with respect to the actual translation processes. Here, we use the nascent polypeptide's chemical trait of having a covalently attached tRNA moiety to detect translation intermediates. In a case study, Escherichia coli SecA was shown to undergo nascent polypeptide-dependent translational pauses. We then carried out integrated in vivo and in vitro nascent chain profiling (iNP) to characterize 1,038 proteome members of E. coli that were encoded by the first quarter of the chromosome with respect to their propensities to accumulate polypeptidyl-tRNA intermediates. A majority of them indeed undergo single or multiple pauses, some occurring only in vitro, some occurring only in vivo, and some occurring both in vivo and in vitro. Thus, translational pausing can be intrinsically robust, subject to in vivo alleviation, or require in vivo reinforcement. Cytosolic and membrane proteins tend to experience different classes of pauses; membrane proteins often pause multiple times in vivo. We also note that the solubility of cytosolic proteins correlates with certain categories of pausing. Translational pausing is widespread and diverse in nature.translation pausing | nascent polypeptides j Escherichia coli | peptidyl-tRNA

Section: Inp Detection Of Pausing That Is Mediated By Previously Propsupporting

confidence: 86%

mentioning

confidence: 99%

Integrated in vivo and in vitro nascent chain profiling reveals widespread translational pausing

Chadani

Niwa

Chiba

et al. 2016

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“…Furthermore, unlike the elongation arrest in SecM, which requires prolyl-tRNA in the A-site, that of VemP does not require a specific A-site amino acid; the P-site VemP 1-156 -tRNA did not even receive efficient termination reaction when the A-site was programmed by a stop codon. Although the ribosome-tethered VemP 1-156 -tRNA likely interferes with the ribosomal peptidyl transferase activity, the mode of its action is also different from that used by MifM, which induces multisite stalling of the B. subtilis ribosome, but not the E. coli ribosome (24,44). We showed that VemP can stall the ribosomes from V. alginolyticus and E. coli, both being gram-negative, equally well.…”

Section: Discussionmentioning

confidence: 76%

Nascent chain-monitored remodeling of the Sec machinery for salinity adaptation of marine bacteria

Ishii

Chiba

Hashimoto

et al. 2015

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112

(A) Predicted secondary structure of mRNA at the vemP-secD2 VA intergenic region. The RNA sequence from the fifth last codon of vemP to the third codon of secD2 VA are shown with the secondary structure predicted by CentroidHomfold. The putative SD sequence and the start codon of secD2 VA are indicated by box and underline, respectively. The P site and the A site of the VemP-stalled ribosome (Fig. 4C) are shown schematically. To separate the VemP arrest point and the secondary structure-forming region, we inserted one or two copies of the 18 nucleotides encoding the last five amino acid residues of VemP followed by the termination codon, shown at the bottom. (B) Separation of the arrest point and the stem-loop-forming region impairs the regulation. E. coli cells carrying indicated vemP-secDF2 VA plasmids (with or without the insertion mutations shown in A) were induced with IPTG for 15 min at 37°C and treated with (+) or without (−) 3 mM NaN 3 for 5 min. Cells were then pulse-labeled for 1 min. Cell extracts of equivalent radioactivities were subjected to anti-VemP (upper gel) and anti-V. SecD2 (lower gel) immunoprecipitation. In the upper gel, "p" indicates the signal peptide unprocessed form of VemP, which included the polypeptide part of the elongation arrested VemP, whereas "m" indicates the signal peptide-processed mature form. Relative intensities of V.SecD2 are shown in the bottom graph, taking the value of lane 1 as unity (n = 3). Gray and black bars represent results obtained without and with NaN 3 treatment, respectively. (C) The insertion mutations do not affect translation arrest of VemP. E. coli cells carrying pBAD24-Δss-vemP-V.secDF2 with or without the insertion mutations were induced with 0.02% arabinose for 1 h at 37°C. The same amounts of proteins were separated by 10% wide-range gel electrophoresis, followed by immunodetection of VemP. (D) The secD2 VA -secF2 VA interval. The nucleotide sequence from the fourth last codon of secD2 VA to the fifth codon of secF2 VA is shown. The predicted SD sequence of secF2 VA is underlined.

“…Sequence alignments (Fig. 2D), as well as comparison with the structures of the B. subtilis 70S ribosome (29) and S. aureus 50S subunit (30) (Fig. S2) Interaction of EVN and AVI with H89 and H91 of the 23S rRNA.…”

Section: Resultsmentioning

confidence: 99%

Structures of the orthosomycin antibiotics avilamycin and evernimicin in complex with the bacterial 70S ribosome

Arenz¹,

Juette

Graf³

et al. 2016

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The ribosome is one of the major targets for therapeutic antibiotics; however, the rise in multidrug resistance is a growing threat to the utility of our current arsenal. The orthosomycin antibiotics evernimicin (EVN) and avilamycin (AVI) target the ribosome and do not display cross-resistance with any other classes of antibiotics, suggesting that they bind to a unique site on the ribosome and may therefore represent an avenue for development of new antimicrobial agents. Here we present cryo-EM structures of EVN and AVI in complex with the Escherichia coli ribosome at 3.6- to 3.9-Å resolution. The structures reveal that EVN and AVI bind to a single site on the large subunit that is distinct from other known antibiotic binding sites on the ribosome. Both antibiotics adopt an extended conformation spanning the minor grooves of helices 89 and 91 of the 23S rRNA and interacting with arginine residues of ribosomal protein L16. This binding site overlaps with the elbow region of A-site bound tRNA. Consistent with this finding, single-molecule FRET (smFRET) experiments show that both antibiotics interfere with late steps in the accommodation process, wherein aminoacyl-tRNA enters the peptidyltransferase center of the large ribosomal subunit. These data provide a structural and mechanistic rationale for how these antibiotics inhibit the elongation phase of protein synthesis.