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Nucleic Acids Research Jan 2018Human translation initiation relies on the combined activities of numerous ribosome-associated eukaryotic initiation factors (eIFs). The largest factor, eIF3, is an...
Human translation initiation relies on the combined activities of numerous ribosome-associated eukaryotic initiation factors (eIFs). The largest factor, eIF3, is an ∼800 kDa multiprotein complex that orchestrates a network of interactions with the small 40S ribosomal subunit, other eIFs, and mRNA, while participating in nearly every step of initiation. How these interactions take place during the time course of translation initiation remains unclear. Here, we describe a method for the expression and affinity purification of a fluorescently-tagged eIF3 from human cells. The tagged eIF3 dodecamer is structurally intact, functions in cell-based assays, and interacts with the HCV IRES mRNA and the 40S-IRES complex in vitro. By tracking the binding of single eIF3 molecules to the HCV IRES RNA with a zero-mode waveguides-based instrument, we show that eIF3 samples both wild-type IRES and an IRES that lacks the eIF3-binding region, and that the high-affinity eIF3-IRES interaction is largely determined by slow dissociation kinetics. The application of single-molecule methods to more complex systems involving eIF3 may unveil dynamics underlying mRNA selection and ribosome loading during human translation initiation.
Topics: Eukaryotic Initiation Factor-3; Fluorescent Dyes; Hepacivirus; Humans; Internal Ribosome Entry Sites; Protein Biosynthesis; RNA, Messenger; RNA, Viral; Reproducibility of Results; Ribosome Subunits, Small, Eukaryotic; Single Molecule Imaging; Spectrum Analysis
PubMed: 29136179
DOI: 10.1093/nar/gkx1050 -
International Journal of Molecular... Dec 2023Ribosome is a major part of the protein synthesis machinery, and analysis of its structure is of paramount importance. However, the structure of ribosomes from only a...
Ribosome is a major part of the protein synthesis machinery, and analysis of its structure is of paramount importance. However, the structure of ribosomes from only a limited number of organisms has been resolved to date; it especially concerns plant ribosomes and ribosomal subunits. Here, we report a high-resolution cryo-electron microscopy reconstruction of the small subunit of the (common wheat) cytoplasmic ribosome. A detailed atomic model was built that includes the majority of the rRNA and some of the protein modifications. The analysis of the obtained data revealed structural peculiarities of the 40S subunit in the monocot plant ribosome. We applied the 3D Flexible Refinement approach to analyze the internal mobility of the 40S subunit and succeeded in decomposing it into four major motions, describing rotations of the head domain and a shift in the massive rRNA expansion segment. It was shown that these motions are almost uncorrelated and that the 40S subunit is flexible enough to spontaneously adopt any conformation it takes as a part of a translating ribosome or ribosomal complex. Here, we introduce the first high-resolution structure of an isolated plant 40S subunit and the first quantitative analysis of the flexibility of small ribosomal subunits, hoping that it will help in studying various aspects of ribosome functioning.
Topics: Ribosome Subunits, Small, Eukaryotic; Cryoelectron Microscopy; Ribosomes; RNA, Ribosomal; Protein Biosynthesis; Ribosomal Proteins
PubMed: 38139282
DOI: 10.3390/ijms242417453 -
Biochimie Jul 2015The accuracy of start codon selection is determined by the translation initiation process. In prokaryotes the initiation step on most mRNAs relies on recruitment of the... (Review)
Review
The accuracy of start codon selection is determined by the translation initiation process. In prokaryotes the initiation step on most mRNAs relies on recruitment of the small ribosomal subunit onto the initiation codon by base pairing between the mRNA and the 16S rRNA. Eukaryotes have evolved a complex molecular machinery involving at least 11 initiation factors, and mRNAs do not directly recruit the small ribosomal subunit. Instead the initiation complex is recruited to the 5' end of the mRNA through a complex protein network including eIF4E that interacts with the 5' cap structure and poly-A binding protein that interacts with the 3'end. However, some viral and cellular mRNAs are able to escape this pathway by internal recruitment of one or several components of the translation machinery. Here we review those eukaryotic mRNAs that have been reported to directly recruit the 40S ribosomal subunit internally. In the well characterized cases of viral IRESes, a specific RNA structure is involved in this process, and in addition to recruitment of the ribosome, the mRNA also manipulates the ribosome structure to stimulate the first translocation step. We also review recently described IRES/ribosome interactions in cases where the molecular mechanism leading to translation initiation has yet to be described. Finally we evaluate the possibility that mRNA may recruit the 40S ribosomal subunit through base pairing with the 18S rRNA.
Topics: Animals; Binding Sites; Humans; Protein Biosynthesis; RNA Transport; RNA, Messenger; RNA, Transfer; Ribosome Subunits, Large, Eukaryotic; Ribosome Subunits, Small, Eukaryotic
PubMed: 25530261
DOI: 10.1016/j.biochi.2014.12.008 -
Nature Structural & Molecular Biology Jan 2020The bacterial ribosome is recycled into subunits by two conserved proteins, elongation factor G (EF-G) and the ribosome recycling factor (RRF). The molecular basis for...
The bacterial ribosome is recycled into subunits by two conserved proteins, elongation factor G (EF-G) and the ribosome recycling factor (RRF). The molecular basis for ribosome recycling by RRF and EF-G remains unclear. Here, we report the crystal structure of a posttermination Thermus thermophilus 70S ribosome complexed with EF-G, RRF and two transfer RNAs at a resolution of 3.5 Å. The deacylated tRNA in the peptidyl (P) site moves into a previously unsuspected state of binding (peptidyl/recycling, p/R) that is analogous to that seen during initiation. The terminal end of the p/R-tRNA forms nonfavorable contacts with the 50S subunit while RRF wedges next to central inter-subunit bridges, illuminating the active roles of tRNA and RRF in dissociation of ribosomal subunits. The structure uncovers a missing snapshot of tRNA as it transits between the P and exit (E) sites, providing insights into the mechanisms of ribosome recycling and tRNA translocation.
Topics: Bacterial Proteins; Crystallography, X-Ray; Models, Molecular; Peptide Elongation Factor G; Protein Conformation; RNA, Transfer; Ribosomal Proteins; Ribosome Subunits, Large, Bacterial; Ribosomes; Thermus thermophilus
PubMed: 31873307
DOI: 10.1038/s41594-019-0350-7 -
Proceedings of the National Academy of... Dec 2017Aminoglycosides are chemically diverse, broad-spectrum antibiotics that target functional centers within the bacterial ribosome to impact all four principle stages...
Aminoglycosides are chemically diverse, broad-spectrum antibiotics that target functional centers within the bacterial ribosome to impact all four principle stages (initiation, elongation, termination, and recycling) of the translation mechanism. The propensity of aminoglycosides to induce miscoding errors that suppress the termination of protein synthesis supports their potential as therapeutic interventions in human diseases associated with premature termination codons (PTCs). However, the sites of interaction of aminoglycosides with the eukaryotic ribosome and their modes of action in eukaryotic translation remain largely unexplored. Here, we use the combination of X-ray crystallography and single-molecule FRET analysis to reveal the interactions of distinct classes of aminoglycosides with the 80S eukaryotic ribosome. Crystal structures of the 80S ribosome in complex with paromomycin, geneticin (G418), gentamicin, and TC007, solved at 3.3- to 3.7-Å resolution, reveal multiple aminoglycoside-binding sites within the large and small subunits, wherein the 6'-hydroxyl substituent in ring I serves as a key determinant of binding to the canonical eukaryotic ribosomal decoding center. Multivalent binding interactions with the human ribosome are also evidenced through their capacity to affect large-scale conformational dynamics within the pretranslocation complex that contribute to multiple aspects of the translation mechanism. The distinct impacts of the aminoglycosides examined suggest that their chemical composition and distinct modes of interaction with the ribosome influence PTC read-through efficiency. These findings provide structural and functional insights into aminoglycoside-induced impacts on the eukaryotic ribosome and implicate pleiotropic mechanisms of action beyond decoding.
Topics: Aminoglycosides; Bacteria; Binding Sites; Eukaryota; Humans; Models, Molecular; Molecular Conformation; Protein Binding; Ribosome Subunits; Ribosomes
PubMed: 29208708
DOI: 10.1073/pnas.1715501114 -
Nature Communications Feb 2019Ribo-T is an engineered ribosome whose small and large subunits are tethered together by linking 16S rRNA and 23S rRNA in a single molecule. Although Ribo-T can support...
Ribo-T is an engineered ribosome whose small and large subunits are tethered together by linking 16S rRNA and 23S rRNA in a single molecule. Although Ribo-T can support cell proliferation in the absence of wild type ribosomes, Ribo-T cells grow slower than those with wild type ribosomes. Here, we show that cell growth defect is likely explained primarily by slow Ribo-T assembly rather than its imperfect functionality. Ribo-T maturation is stalled at a late assembly stage. Several post-transcriptional rRNA modifications and some ribosomal proteins are underrepresented in the accumulated assembly intermediates and rRNA ends are incompletely trimmed. Ribosome profiling of Ribo-T cells shows no defects in translation elongation but reveals somewhat higher occupancy by Ribo-T of the start codons and to a lesser extent stop codons, suggesting that subunit tethering mildly affects the initiation and termination stages of translation. Understanding limitations of Ribo-T system offers ways for its future development.
Topics: Codon, Initiator; Escherichia coli; Protein Biosynthesis; RNA Processing, Post-Transcriptional; RNA, Bacterial; RNA, Ribosomal, 16S; RNA, Ribosomal, 23S; Ribosomal Proteins; Ribosome Subunits
PubMed: 30804338
DOI: 10.1038/s41467-019-08892-w -
Microbiology and Molecular Biology... Sep 2011The correct translation of mRNA depends critically on the ability to initiate at the right AUG codon. For most mRNAs in eukaryotic cells, this is accomplished by the... (Review)
Review
The correct translation of mRNA depends critically on the ability to initiate at the right AUG codon. For most mRNAs in eukaryotic cells, this is accomplished by the scanning mechanism, wherein the small (40S) ribosomal subunit attaches to the 5' end of the mRNA and then inspects the leader base by base for an AUG in a suitable context, using complementarity with the anticodon of methionyl initiator tRNA (Met-tRNAiMet) as the key means of identifying AUG. Over the past decade, a combination of yeast genetics, biochemical analysis in reconstituted systems, and structural biology has enabled great progress in deciphering the mechanism of ribosomal scanning. A robust molecular model now exists, describing the roles of initiation factors, notably eukaryotic initiation factor 1 (eIF1) and eIF1A, in stabilizing an "open" conformation of the 40S subunit with Met-tRNAiMet bound in a low-affinity state conducive to scanning and in triggering rearrangement into a "closed" conformation incompatible with scanning, which features Met-tRNAiMet more tightly bound to the "P" site and base paired with AUG. It has also emerged that multiple DEAD-box RNA helicases participate in producing a single-stranded "landing pad" for the 40S subunit and in removing the secondary structure to enable the mRNA to traverse the 40S mRNA-binding channel in the single-stranded form for base-by-base inspection in the P site.
Topics: Codon, Initiator; Eukaryota; Eukaryotic Initiation Factors; Guanosine Triphosphate; Hydrolysis; Peptide Chain Initiation, Translational; Protein Binding; Protein Biosynthesis; RNA Helicases; RNA, Ribosomal, 18S; RNA, Transfer, Met; Ribosome Subunits
PubMed: 21885680
DOI: 10.1128/MMBR.00008-11 -
BMC Molecular Biology Feb 2015Ribosomes and functional complexes of them have been analyzed at the atomic level. Far less is known about the dynamic assembly and degradation events that define the...
BACKGROUND
Ribosomes and functional complexes of them have been analyzed at the atomic level. Far less is known about the dynamic assembly and degradation events that define the half-life of ribosomes and guarantee their quality control.
RESULTS
We developed a system that allows visualization of intact ribosomal subunits and assembly intermediates (i.e. assembly landscapes) by convenient fluorescence-based analysis. To this end, we labeled the early assembly ribosomal proteins L1 and S15 with the fluorescent proteins mAzami green and mCherry, respectively, using chromosomal gene insertion. The reporter strain harbors fluorescently labeled ribosomal subunits that operate wild type-like, as shown by biochemical and growth assays. Using genetic and chemical perturbations by depleting genes encoding the ribosomal proteins L3 and S17, respectively, or using ribosome-targeting antibiotics, we provoked ribosomal subunit assembly defects. These defects were readily identified by fluorometric analysis after sucrose density centrifugation in unprecedented resolution.
CONCLUSION
This strategy is useful to monitor and characterize subunit specific assembly defects caused by ribosome-targeting drugs that are currently used and to characterize new molecules that affect ribosome assembly and thereby constitute new classes of antibacterial agents.
Topics: Anti-Bacterial Agents; Escherichia coli; Escherichia coli Proteins; Fluorometry; Gene Knockout Techniques; Green Fluorescent Proteins; Luminescent Proteins; Mutagenesis, Insertional; Protein Multimerization; Ribosomal Proteins; Ribosome Subunits, Large, Bacterial; Ribosome Subunits, Small, Bacterial; Ribosomes; Red Fluorescent Protein
PubMed: 25884162
DOI: 10.1186/s12867-015-0031-y -
Nature Mar 2024Stalled ribosomes at the endoplasmic reticulum (ER) are covalently modified with the ubiquitin-like protein UFM1 on the 60S ribosomal subunit protein RPL26 (also known...
Stalled ribosomes at the endoplasmic reticulum (ER) are covalently modified with the ubiquitin-like protein UFM1 on the 60S ribosomal subunit protein RPL26 (also known as uL24). This modification, which is known as UFMylation, is orchestrated by the UFM1 ribosome E3 ligase (UREL) complex, comprising UFL1, UFBP1 and CDK5RAP3 (ref. ). However, the catalytic mechanism of UREL and the functional consequences of UFMylation are unclear. Here we present cryo-electron microscopy structures of UREL bound to 60S ribosomes, revealing the basis of its substrate specificity. UREL wraps around the 60S subunit to form a C-shaped clamp architecture that blocks the tRNA-binding sites at one end, and the peptide exit tunnel at the other. A UFL1 loop inserts into and remodels the peptidyl transferase centre. These features of UREL suggest a crucial function for UFMylation in the release and recycling of stalled or terminated ribosomes from the ER membrane. In the absence of functional UREL, 60S-SEC61 translocon complexes accumulate at the ER membrane, demonstrating that UFMylation is necessary for releasing SEC61 from 60S subunits. Notably, this release is facilitated by a functional switch of UREL from a 'writer' to a 'reader' module that recognizes its product-UFMylated 60S ribosomes. Collectively, we identify a fundamental role for UREL in dissociating 60S subunits from the SEC61 translocon and the basis for UFMylation in regulating protein homeostasis at the ER.
Topics: Adaptor Proteins, Signal Transducing; Binding Sites; Cell Cycle Proteins; Cryoelectron Microscopy; Endoplasmic Reticulum; Homeostasis; Intracellular Membranes; Peptidyl Transferases; Protein Processing, Post-Translational; Ribosomal Proteins; RNA, Transfer; SEC Translocation Channels; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases; Ribosome Subunits, Large, Eukaryotic
PubMed: 38383789
DOI: 10.1038/s41586-024-07093-w -
Current Opinion in Structural Biology Apr 2018The small subunit processome is the first precursor of the small eukaryotic ribosomal subunit. During its assembly in the nucleolus, many ribosome biogenesis factors, an... (Review)
Review
The small subunit processome is the first precursor of the small eukaryotic ribosomal subunit. During its assembly in the nucleolus, many ribosome biogenesis factors, an RNA chaperone, and ribosomal proteins associate with the nascent pre-rRNA. Biochemical studies have elucidated the rRNA-subdomain dependent recruitment of these factors during SSU processome assembly and have been complemented by structural studies of the assembled particle. Ribosome biogenesis factors encapsulate and guide subdomains of pre-ribosomal RNA in distinct compartments. This prevents uncoordinated maturation and enables processing of regions not accessible in the mature subunit. By sequentially reducing conformational freedom, flexible proteins facilitate the incorporation of dynamic subcomplexes into a globular particle. Large rearrangements within the SSU processome are required for compaction into the mature small ribosomal subunit.
Topics: Animals; Humans; Models, Molecular; Nucleic Acid Conformation; Protein Conformation; RNA Precursors; RNA, Ribosomal; Ribosomal Proteins; Ribosome Subunits, Small
PubMed: 29414516
DOI: 10.1016/j.sbi.2018.01.008