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Annual Review of Biochemistry Jun 2016Mitochondrial ribosomes (mitoribosomes) perform protein synthesis inside mitochondria, the organelles responsible for energy conversion and adenosine triphosphate... (Review)
Review
Mitochondrial ribosomes (mitoribosomes) perform protein synthesis inside mitochondria, the organelles responsible for energy conversion and adenosine triphosphate production in eukaryotic cells. Throughout evolution, mitoribosomes have become functionally specialized for synthesizing mitochondrial membrane proteins, and this has been accompanied by large changes to their structure and composition. We review recent high-resolution structural data that have provided unprecedented insight into the structure and function of mitoribosomes in mammals and fungi.
Topics: Animals; Anti-Bacterial Agents; Biological Evolution; Carboxylic Ester Hydrolases; DNA, Mitochondrial; Mammals; Mitochondria; Mitochondrial Membranes; Mitochondrial Proteins; Mitochondrial Ribosomes; Models, Molecular; Protein Biosynthesis; RNA, Messenger; RNA, Transfer; Ribosome Subunits; Saccharomyces cerevisiae
PubMed: 27023846
DOI: 10.1146/annurev-biochem-060815-014343 -
Nature Apr 2015Ribosomes are translational machineries that catalyse protein synthesis. Ribosome structures from various species are known at the atomic level, but obtaining the...
Ribosomes are translational machineries that catalyse protein synthesis. Ribosome structures from various species are known at the atomic level, but obtaining the structure of the human ribosome has remained a challenge; efforts to address this would be highly relevant with regard to human diseases. Here we report the near-atomic structure of the human ribosome derived from high-resolution single-particle cryo-electron microscopy and atomic model building. The structure has an average resolution of 3.6 Å, reaching 2.9 Å resolution in the most stable regions. It provides unprecedented insights into ribosomal RNA entities and amino acid side chains, notably of the transfer RNA binding sites and specific molecular interactions with the exit site tRNA. It reveals atomic details of the subunit interface, which is seen to remodel strongly upon rotational movements of the ribosomal subunits. Furthermore, the structure paves the way for analysing antibiotic side effects and diseases associated with deregulated protein synthesis.
Topics: Binding Sites; Cryoelectron Microscopy; Electrons; Humans; Models, Molecular; RNA, Ribosomal; RNA, Transfer; Ribosomal Proteins; Ribosome Subunits; Ribosomes
PubMed: 25901680
DOI: 10.1038/nature14427 -
Journal of Bacteriology Apr 2020When nutrients become scarce, bacteria can enter an extended state of quiescence. A major challenge of this state is how to preserve ribosomes for the return to...
When nutrients become scarce, bacteria can enter an extended state of quiescence. A major challenge of this state is how to preserve ribosomes for the return to favorable conditions. Here, we show that the ribosome dimerization protein hibernation-promoting factor (HPF) functions to protect essential ribosomal proteins. Ribosomes isolated from strains lacking HPF (Δ) or encoding a mutant allele of HPF that binds the ribosome but does not mediate dimerization were substantially depleted of the small subunit proteins S2 and S3. Strikingly, these proteins are located directly at the ribosome dimer interface. We used single-particle cryo-electron microscopy (cryo-EM) to further characterize these ribosomes and observed that a high percentage of ribosomes were missing S2, S3, or both. These data support a model in which the ribosome dimerization activity of HPF evolved to protect labile proteins that are essential for ribosome function. HPF is almost universally conserved in bacteria, and HPF deletions in diverse species exhibit decreased viability during starvation. Our data provide mechanistic insight into this phenotype and establish a mechanism for how HPF protects ribosomes during quiescence. The formation of ribosome dimers during periods of dormancy is widespread among bacteria. Dimerization is typically mediated by a single protein, hibernation-promoting factor (HPF). Bacteria lacking HPF exhibit strong defects in viability and pathogenesis and, in some species, extreme loss of rRNA. The mechanistic basis of these phenotypes has not been determined. Here, we report that HPF from the Gram-positive bacterium preserves ribosomes by preventing the loss of essential ribosomal proteins at the dimer interface. This protection may explain phenotypes associated with the loss of HPF, since ribosome protection would aid survival during nutrient limitation and impart a strong selective advantage when the bacterial cell rapidly reinitiates growth in the presence of sufficient nutrients.
Topics: Bacillus subtilis; Bacterial Proteins; Cryoelectron Microscopy; Dimerization; Ribosome Subunits, Small; Ribosomes
PubMed: 32123037
DOI: 10.1128/JB.00009-20 -
Science (New York, N.Y.) Sep 2020Production of small ribosomal subunits initially requires the formation of a 90 precursor followed by an enigmatic process of restructuring into the primordial pre-40...
Production of small ribosomal subunits initially requires the formation of a 90 precursor followed by an enigmatic process of restructuring into the primordial pre-40 subunit. We elucidate this process by biochemical and cryo-electron microscopy analysis of intermediates along this pathway in yeast. First, the remodeling RNA helicase Dhr1 engages the 90 pre-ribosome, followed by Utp24 endonuclease-driven RNA cleavage at site A, thereby separating the 5'-external transcribed spacer (ETS) from 18 ribosomal RNA. Next, the 5'-ETS and 90 assembly factors become dislodged, but this occurs sequentially, not en bloc. Eventually, the primordial pre-40 emerges, still retaining some 90 factors including Dhr1, now ready to unwind the final small nucleolar U3-18 RNA hybrid. Our data shed light on the elusive 90 to pre-40 transition and clarify the principles of assembly and remodeling of large ribonucleoproteins.
Topics: Cryoelectron Microscopy; DEAD-box RNA Helicases; Nuclear Proteins; Protein Conformation; RNA Cleavage; RNA, Ribosomal, 18S; Ribosomal Proteins; Ribosome Subunits, Large, Eukaryotic; Ribosome Subunits, Small, Eukaryotic; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 32943521
DOI: 10.1126/science.abb4119 -
Nature Reviews. Molecular Cell Biology Feb 2019In the past 25 years, genetic and biochemical analyses of ribosome assembly in yeast have identified most of the factors that participate in this complex pathway and... (Review)
Review
In the past 25 years, genetic and biochemical analyses of ribosome assembly in yeast have identified most of the factors that participate in this complex pathway and have generated models for the mechanisms driving the assembly. More recently, the publication of numerous cryo-electron microscopy structures of yeast ribosome assembly intermediates has provided near-atomic resolution snapshots of ribosome precursor particles. Satisfyingly, these structural data support the genetic and biochemical models and provide additional mechanistic insight into ribosome assembly. In this Review, we discuss the mechanisms of assembly of the yeast small ribosomal subunit and large ribosomal subunit in the nucleolus, nucleus and cytoplasm. Particular emphasis is placed on concepts such as the mechanisms of RNA compaction, the functions of molecular switches and molecular mimicry, the irreversibility of assembly checkpoints and the roles of structural and functional proofreading of pre-ribosomal particles.
Topics: Animals; Cell Nucleus; Cryoelectron Microscopy; Cytoplasm; Humans; RNA; Ribosome Subunits
PubMed: 30467428
DOI: 10.1038/s41580-018-0078-y -
Structure (London, England : 1993) Jun 2015In this issue of Structure, Chen et al. (2015) report the use of a mixing-spraying method of time-resolved cryogenic electron microscopy, which allowed the progression...
In this issue of Structure, Chen et al. (2015) report the use of a mixing-spraying method of time-resolved cryogenic electron microscopy, which allowed the progression of ribosomal subunit association to be visualized on the millisecond timescale.
Topics: Cryoelectron Microscopy; Escherichia coli; Models, Molecular; Ribosome Subunits
PubMed: 26039346
DOI: 10.1016/j.str.2015.05.007 -
Nature Structural & Molecular Biology May 2023During transcription of eukaryotic ribosomal DNA in the nucleolus, assembly checkpoints exist that guarantee the formation of stable precursors of small and large...
During transcription of eukaryotic ribosomal DNA in the nucleolus, assembly checkpoints exist that guarantee the formation of stable precursors of small and large ribosomal subunits. While the formation of an early large subunit assembly checkpoint precedes the separation of small and large subunit maturation, its mechanism of action and function remain unknown. Here, we report the cryo-electron microscopy structure of the yeast co-transcriptional large ribosomal subunit assembly intermediate that serves as a checkpoint. The structure provides the mechanistic basis for how quality-control pathways are established through co-transcriptional ribosome assembly factors, that structurally interrogate, remodel and, together with ribosomal proteins, cooperatively stabilize correctly folded pre-ribosomal RNA. Our findings thus provide a molecular explanation for quality control during eukaryotic ribosome assembly in the nucleolus.
Topics: Cryoelectron Microscopy; RNA, Ribosomal; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ribosomal Proteins; Ribosome Subunits, Large; Ribosome Subunits, Large, Eukaryotic; Ribosome Subunits, Small, Eukaryotic
PubMed: 37037974
DOI: 10.1038/s41594-023-00947-3 -
Science (New York, N.Y.) Jul 2023During the early stages of human large ribosomal subunit (60) biogenesis, an ensemble of assembly factors establishes and fine-tunes the essential RNA functional centers...
During the early stages of human large ribosomal subunit (60) biogenesis, an ensemble of assembly factors establishes and fine-tunes the essential RNA functional centers of pre-60 particles by an unknown mechanism. Here, we report a series of cryo-electron microscopy structures of human nucleolar and nuclear pre-60 assembly intermediates at resolutions of 2.5 to 3.2 angstroms. These structures show how protein interaction hubs tether assembly factor complexes to nucleolar particles and how guanosine triphosphatases and adenosine triphosphatase couple irreversible nucleotide hydrolysis steps to the installation of functional centers. Nuclear stages highlight how a conserved RNA-processing complex, the rixosome, couples large-scale RNA conformational changes with pre-ribosomal RNA processing by the RNA degradation machinery. Our ensemble of human pre-60 particles provides a rich foundation with which to elucidate the molecular principles of ribosome formation.
Topics: Humans; Cell Nucleus; Cryoelectron Microscopy; Ribosomal Proteins; Ribosome Subunits, Large, Eukaryotic; RNA, Ribosomal; Saccharomyces cerevisiae; Protein Conformation
PubMed: 37410842
DOI: 10.1126/science.adh3892 -
Journal of Molecular Biology Aug 2008RsgA (ribosome-small-subunit-dependent GTPase A, also known as YjeQ) is a unique GTPase in that guanosine triphosphate hydrolytic activity is activated by the small...
RsgA (ribosome-small-subunit-dependent GTPase A, also known as YjeQ) is a unique GTPase in that guanosine triphosphate hydrolytic activity is activated by the small subunit of the ribosome. Disruption of the gene for RsgA from the genome affects the growth of cells, the subunit association of the ribosome, and the maturation of 16S rRNA. To study the interaction of Escherichia coli RsgA with the ribosome, chemical modifications using dimethylsulfate and kethoxal were performed on the small subunit in the presence or in the absence of RsgA. The chemical reactivities at G530, A790, G925, G926, G966, C1054, G1339, G1405, A1413, and A1493 in 16S rRNA were reduced, while those at A532, A923, G1392, A1408, A1468, and A1483 were enhanced, by the addition of RsgA, together with 5'-guanylylimidodiphosphate. Among them, the chemical reactivities at A532, A790, A923, G925, G926, C1054, G1392, A1413, A1468, A1483, and A1493 were not changed when RsgA was added together with GDP. These results indicate that the binding of RsgA induces conformational changes around the A site, P site, and helix 44, and that guanosine triphosphate hydrolysis induces partial conformational restoration, especially in the head, to dissociate RsgA from the small subunit. RsgA has the capacity to coexist with mRNA in the ribosome while it promotes dissociation of tRNA from the ribosome.
Topics: Aldehydes; Binding Sites; Butanones; Escherichia coli Proteins; GTP Phosphohydrolases; Guanosine Triphosphate; Hygromycin B; Protein Binding; RNA, Messenger; RNA, Transfer; Ribosome Subunits; Sulfuric Acid Esters
PubMed: 18588897
DOI: 10.1016/j.jmb.2008.06.023 -
The Journal of Antimicrobial... Apr 2020This article describes 20 years of research that investigated a second novel target for ribosomal antibiotics, the biogenesis of the two subunits. Over that period, we... (Review)
Review
This article describes 20 years of research that investigated a second novel target for ribosomal antibiotics, the biogenesis of the two subunits. Over that period, we have examined the effect of 52 different antibiotics on ribosomal subunit formation in six different microorganisms. Most of the antimicrobials we have studied are specific, preventing the formation of only the subunit to which they bind. A few interesting exceptions have also been observed. Forty-one research publications and a book chapter have resulted from this investigation. This review will describe the methodology we used and the fit of our results to a hypothetical model. The model predicts that inhibition of subunit assembly and translation are equivalent targets for most of the antibiotics we have investigated.
Topics: Anti-Bacterial Agents; Bacteria; Protein Biosynthesis; Ribosomal Proteins; Ribosome Subunits; Ribosomes
PubMed: 31942624
DOI: 10.1093/jac/dkz544