Faculty Opinions recommendation of Reverse genetics-based biochemical studies of the ribosomal exit tunnel constriction region in eukaryotic ribosome stalling: spatial allocation of the regulatory nascent peptide at the constriction.

Abstract A number of regulatory nascent peptides have been shown to regulate gene expression by causing programmed ribosome stalling during translation. Nascent peptide emerges from the ribosome through the exit tunnel, and one-third of the way along which β-loop structures of ribosomal proteins uL4 and uL22 protrude into the tunnel to form the constriction region. Structural studies have shown interactions between nascent peptides and the exit tunnel components including the constriction region. In eukaryotes, however, there is a lack of genetic studies for the involvement of the constriction region in ribosome stalling. Here, we established transgenic Arabidopsis lines that carry mutations in the β-loop structure of uL4. Translation analyses using a cell-free translation system derived from the transgenic Arabidopsis carrying the mutant ribosome showed that the uL4 mutations reduced the ribosome stalling of four eukaryotic stalling systems, including those for which stalled structures have been solved. Our data, which showed differential effects of the uL4 mutations depending on the stalling systems, explained the spatial allocations of the nascent peptides at the constriction that were deduced by structural studies. Conversely, our data may predict allocation of the nascent peptide at the constriction of stalling systems for which structural studies are not done.

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Context-specific action of macrolide antibiotics on the eukaryotic ribosome

Nature Communications ◽

10.1038/s41467-021-23068-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Maxim S. Svetlov ◽

Timm O. Koller ◽

Sezen Meydan ◽

Vaishnavi Shankar ◽

Dorota Klepacki ◽

...

Keyword(s):

Amino Acid Sequences ◽

Macrolide Antibiotics ◽

Single Mutation ◽

Sequence Motifs ◽

Eukaryotic Translation ◽

Eukaryotic Ribosome ◽

Cellular Translation ◽

Distinct Sequence ◽

Exit Tunnel ◽

Context Specific

AbstractMacrolide antibiotics bind in the nascent peptide exit tunnel of the bacterial ribosome and prevent polymerization of specific amino acid sequences, selectively inhibiting translation of a subset of proteins. Because preventing translation of individual proteins could be beneficial for the treatment of human diseases, we asked whether macrolides, if bound to the eukaryotic ribosome, would retain their context- and protein-specific action. By introducing a single mutation in rRNA, we rendered yeast Saccharomyces cerevisiae cells sensitive to macrolides. Cryo-EM structural analysis showed that the macrolide telithromycin binds in the tunnel of the engineered eukaryotic ribosome. Genome-wide analysis of cellular translation and biochemical studies demonstrated that the drug inhibits eukaryotic translation by preferentially stalling ribosomes at distinct sequence motifs. Context-specific action markedly depends on the macrolide structure. Eliminating macrolide-arrest motifs from a protein renders its translation macrolide-tolerant. Our data illuminate the prospects of adapting macrolides for protein-selective translation inhibition in eukaryotic cells.

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Gradual compaction of the nascent peptide during cotranslational folding on the ribosome

eLife ◽

10.7554/elife.60895 ◽

2020 ◽

Vol 9 ◽

Author(s):

Marija Liutkute ◽

Manisankar Maiti ◽

Ekaterina Samatova ◽

Jörg Enderlein ◽

Marina V Rodnina

Keyword(s):

Profile Analysis ◽

Dynamic Properties ◽

Correlation Spectroscopy ◽

Hydrophobic Core ◽

Fluorescence Correlation ◽

Nascent Peptide ◽

Nascent Chain ◽

Force Profile ◽

Exit Tunnel ◽

Domain Stabilization

Nascent polypeptides begin to fold in the constrained space of the ribosomal peptide exit tunnel. Here we use force-profile analysis (FPA) and photo-induced energy-transfer fluorescence correlation spectroscopy (PET-FCS) to show how a small α-helical domain, the N-terminal domain of HemK, folds cotranslationally. Compaction starts vectorially as soon as the first α-helical segments are synthesized. As nascent chain grows, emerging helical segments dock onto each other and continue to rearrange at the vicinity of the ribosome. Inside or in the proximity of the ribosome, the nascent peptide undergoes structural fluctuations on the µs time scale. The fluctuations slow down as the domain moves away from the ribosome. Mutations that destabilize the packing of the domain’s hydrophobic core have little effect on folding within the exit tunnel, but abolish the final domain stabilization. The results show the power of FPA and PET-FCS in solving the trajectory of cotranslational protein folding and in characterizing the dynamic properties of folding intermediates.

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The GTPase Nog1 co-ordinates the assembly, maturation and quality control of distant ribosomal functional centers

eLife ◽

10.7554/elife.52474 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 7

Author(s):

Purnima Klingauf-Nerurkar ◽

Ludovic C Gillet ◽

Daniela Portugal-Calisto ◽

Michaela Oborská-Oplová ◽

Martin Jäger ◽

...

Keyword(s):

Quality Control ◽

Protein Folding ◽

Atpase Activity ◽

Ribosomal Protein ◽

Peptidyl Transferase ◽

Peptidyl Transferase Center ◽

Eukaryotic Ribosome ◽

Ribosomal Stalk ◽

Exit Tunnel ◽

Ribosome Formation

Eukaryotic ribosome precursors acquire translation competence in the cytoplasm through stepwise release of bound assembly factors, and proofreading of their functional centers. In case of the pre-60S, these steps include removal of placeholders Rlp24, Arx1 and Mrt4 that prevent premature loading of the ribosomal protein eL24, the protein-folding machinery at the polypeptide exit tunnel (PET), and the ribosomal stalk, respectively. Here, we reveal that sequential ATPase and GTPase activities license release factors Rei1 and Yvh1 to trigger Arx1 and Mrt4 removal. Drg1-ATPase activity removes Rlp24 from the GTPase Nog1 on the pre-60S; consequently, the C-terminal tail of Nog1 is extracted from the PET. These events enable Rei1 to probe PET integrity and catalyze Arx1 release. Concomitantly, Nog1 eviction from the pre-60S permits peptidyl transferase center maturation, and allows Yvh1 to mediate Mrt4 release for stalk assembly. Thus, Nog1 co-ordinates the assembly, maturation and quality control of distant functional centers during ribosome formation.

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Gradual Compaction of the Nascent Peptide During Cotranslational Folding on the Ribosome

10.1101/2020.07.03.185876 ◽

2020 ◽

Author(s):

Marija Liutkute ◽

Manisankar Maiti ◽

Ekaterina Samatova ◽

Jörg Enderlein ◽

Marina V. Rodnina

Keyword(s):

Profile Analysis ◽

Dynamic Properties ◽

Correlation Spectroscopy ◽

Fluorescence Correlation ◽

Cotranslational Folding ◽

Nascent Peptide ◽

Nascent Chain ◽

Force Profile ◽

Exit Tunnel ◽

Domain Stabilization

ABSTRACTNascent polypeptides begin to fold in the constrained space of the ribosomal peptide exit tunnel. Here we use force profile analysis (FPA) and photo-induced energy-transfer fluorescence correlation spectroscopy (PET-FCS) to show how a small α-helical domain, the N-terminal domain of HemK, folds cotranslationally. Compaction starts vectorially as soon as the first α-helical segments are synthesized. As nascent chain grows, emerging helical segments dock onto each other and continue to rearrange at the vicinity of the ribosome. Inside or in the proximity of the ribosome, the nascent peptide undergoes structural fluctuations on the μs time scale. The fluctuations slow down as the domain moves away from the ribosome. Folding mutations have little effect on folding within the exit tunnel, but abolish the final domain stabilization. The results show the power of FPA and PET-FCS in solving the trajectory of cotranslational protein folding and in characterizing the dynamic properties of folding intermediates.

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Ribosome profiling in Streptococcus pneumoniae reveals the role of methylation of 23S rRNA nucleotide G748 on ribosome stalling

10.1101/2020.12.21.423859 ◽

2020 ◽

Author(s):

Tatsuma Shoji ◽

Akiko Takaya ◽

Yoko Kusuya ◽

Hiroki Takahashi ◽

Hiroto Kawashima

Keyword(s):

Streptococcus Pneumoniae ◽

Ribosome Profiling ◽

23S Rrna ◽

Ribosome Stalling ◽

70S Ribosome ◽

Exit Tunnel ◽

Species Specific ◽

Peptidyltransferase Center ◽

First Time

2.Abstract(1) BackgroundMany nucleotides in 23S rRNA are methylated post-transcriptionally by methyltransferases and cluster around the peptidyltransferase center (PTC) and the nascent peptidyl exit tunnel (NPET) located in 50S subunit of 70S ribosome. Biochemical interactions between a nascent peptide and the tunnel may stall ribosome movement and affect expression levels of the protein. However, no studies have shown a role for NPET on ribosome stalling using an NPET mutant.(2) ResultsA ribosome profiling assay in Streptococcus pneumoniae demonstrates for the first time that an NPET mutant exhibits completely different ribosome occupancy compared to wild-type. We demonstrate, using RNA footprinting, that changes in ribosome occupancy correlate with changes in ribosome stalling. Further, statistical analysis shows that short peptide sequences that cause ribosome stalling are species-specific and evolutionarily selected. NPET structure is required to realize these specie-specific ribosome stalling.(3) ConclusionsResults support the role of NPET on ribosome stalling. NPET structure is required to realize the species-specific and evolutionary conserved ribosome stalling. These findings clarify the role of NPET structure on the translation process.

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