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Biological Chemistry Jul 2023During their biogenesis, the ribosomal subunits undergo numerous structural and compositional changes to achieve their final architecture. RNA helicases are a key... (Review)
Review
During their biogenesis, the ribosomal subunits undergo numerous structural and compositional changes to achieve their final architecture. RNA helicases are a key driving force of such remodelling events but deciphering their particular functions has long been challenging due to lack of knowledge of their molecular functions and RNA substrates. Advances in the biochemical characterisation of RNA helicase activities together with new insights into RNA helicase binding sites on pre-ribosomes and structural snapshots of pre-ribosomal complexes containing RNA helicases now open the door to a deeper understanding of precisely how different RNA helicases contribute to ribosomal subunit maturation.
Topics: RNA Helicases; Ribosomes; Ribosome Subunits; RNA; Binding Sites; RNA, Ribosomal; Saccharomyces cerevisiae Proteins
PubMed: 37233600
DOI: 10.1515/hsz-2023-0135 -
Nature Jan 2023CRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargos using an RNA-guided mechanism. CAST elements contain multiple...
CRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargos using an RNA-guided mechanism. CAST elements contain multiple conserved proteins: a CRISPR effector (Cas12k or Cascade), a AAA+ regulator (TnsC), a transposase (TnsA-TnsB) and a target-site-associated factor (TniQ). These components are thought to cooperatively integrate DNA via formation of a multisubunit transposition integration complex (transpososome). Here we reconstituted the approximately 1 MDa type V-K CAST transpososome from Scytonema hofmannii (ShCAST) and determined its structure using single-particle cryo-electon microscopy. The architecture of this transpososome reveals modular association between the components. Cas12k forms a complex with ribosomal subunit S15 and TniQ, stabilizing formation of a full R-loop. TnsC has dedicated interaction interfaces with TniQ and TnsB. Of note, we observe TnsC-TnsB interactions at the C-terminal face of TnsC, which contribute to the stimulation of ATPase activity. Although the TnsC oligomeric assembly deviates slightly from the helical configuration found in isolation, the TnsC-bound target DNA conformation differs markedly in the transpososome. As a consequence, TnsC makes new protein-DNA interactions throughout the transpososome that are important for transposition activity. Finally, we identify two distinct transpososome populations that differ in their DNA contacts near TniQ. This suggests that associations with the CRISPR effector can be flexible. This ShCAST transpososome structure enhances our understanding of CAST transposition systems and suggests ways to improve CAST transposition for precision genome-editing applications.
Topics: Clustered Regularly Interspaced Short Palindromic Repeats; DNA Transposable Elements; DNA-Binding Proteins; Gene Editing; Transposases; RNA, Guide, CRISPR-Cas Systems; CRISPR-Cas Systems; Holoenzymes; Multiprotein Complexes; Cryoelectron Microscopy; Ribosome Subunits; Bacterial Proteins
PubMed: 36442503
DOI: 10.1038/s41586-022-05573-5 -
Protein & Cell Nov 2017The lipid droplet (LD) is a unique multi-functional organelle that contains a neutral lipid core covered with a phospholipid monolayer membrane. The LDs have been found... (Review)
Review
The lipid droplet (LD) is a unique multi-functional organelle that contains a neutral lipid core covered with a phospholipid monolayer membrane. The LDs have been found in almost all organisms from bacteria to humans with similar shape. Several conserved functions of LDs have been revealed by recent studies, including lipid metabolism and trafficking, as well as nucleic acid binding and protection. We summarized these findings and proposed a hypothesis that the LD is a conserved organelle.
Topics: Animals; Bacteria; Biological Evolution; Cholesterol Esters; Humans; Lipid Droplets; Lipid Metabolism; Nucleic Acids; Peptide Initiation Factors; Protein Binding; RNA-Binding Proteins; Ribosome Subunits; Triglycerides
PubMed: 28913786
DOI: 10.1007/s13238-017-0467-6 -
Scientific Reports Jul 2018In eukaryotic translation the 60S ribosome subunit has not been proposed to interact with mRNA or closed-loop factors eIF4E, eIF4G, and PAB1. Using analytical...
In eukaryotic translation the 60S ribosome subunit has not been proposed to interact with mRNA or closed-loop factors eIF4E, eIF4G, and PAB1. Using analytical ultracentrifugation with fluorescent detection system, we have identified a 57S translation complex that contains the 60S ribosome, mRNA, and the closed-loop factors. Previously published data by others also indicate the presence of a 50S-60S translation complex containing these same components. We have found that the abundance of this complex increased upon translational cessation, implying formation after ribosomal dissociation. Stoichiometric analyses of the abundances of the closed-loop components in the 57S complex indicate this complex is most similar to polysomal and monosomal translation complexes at the end of translation rather than at the beginning or middle of translation. In contrast, a 39S complex containing the 40S ribosome bound to mRNA and closed-loop factors was also identified with stoichiometries most similar to polysomal complexes engaged in translation, suggesting that the 39S complex is the previously studied 48S translation initiation complex. These results indicate that the 60S ribosome can associate with the closed-loop mRNA structure and plays a previously undetected role in the translation process.
Topics: Eukaryotic Initiation Factor-4E; Eukaryotic Initiation Factor-4G; Polyribosomes; Protein Biosynthesis; RNA, Messenger; Ribosome Subunits, Large, Eukaryotic; Ribosomes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 30065356
DOI: 10.1038/s41598-018-29832-6 -
Annual Review of Biophysics 2009The ribosome is a complex macromolecular machine responsible for protein synthesis in the cell. It consists of two subunits, each of which contains both RNA and protein... (Review)
Review
The ribosome is a complex macromolecular machine responsible for protein synthesis in the cell. It consists of two subunits, each of which contains both RNA and protein components. Ribosome assembly is subject to intricate regulatory control and is aided by a multitude of assembly factors in vivo, but can also be carried out in vitro. The details of the assembly process remain unknown even in the face of atomic structures of the entire ribosome and after more than three decades of research. Some of the earliest research on ribosome assembly produced the Nomura assembly map of the small subunit, revealing a hierarchy of protein binding dependencies for the 20 proteins involved and suggesting the possibility of a single intermediate. Recent work using a combination of RNA footprinting and pulse-chase quantitative mass spectrometry paints a picture of small subunit assembly as a dynamic and varied landscape, with sequential and hierarchical RNA folding and protein binding events finally converging on complete subunits. Proteins generally lock tightly into place in a 5' to 3' direction along the ribosomal RNA, stabilizing transient RNA conformations, while RNA folding and the early stages of protein binding are initiated from multiple locations along the length of the RNA.
Topics: Binding Sites; Computer Simulation; Models, Chemical; Models, Molecular; Protein Binding; Protein Conformation; Ribosomal Proteins; Ribosome Subunits, Small
PubMed: 19416066
DOI: 10.1146/annurev.biophys.050708.133615 -
Biophysical Journal Dec 2017A major challenge in the study of biomolecular assemblies is to identify reaction coordinates that precisely monitor conformational rearrangements. This is central to...
A major challenge in the study of biomolecular assemblies is to identify reaction coordinates that precisely monitor conformational rearrangements. This is central to the interpretation of single-molecule fluorescence resonance energy transfer measurements, where the observed dynamics depends on the labeling strategy. As an example, different probes of subunit rotation in the ribosome have provided qualitatively distinct descriptions. In one study, changes in fluorescence suggested that the 30S body undergoes a single rotation/back-rotation cycle during the process of mRNA-tRNA translocation. In contrast, an alternate assay implicated the presence of reversible rotation events before completing translocation. For future single-molecule experiments to unambiguously measure the relationship between subunit rotation and translocation, it is necessary to rationalize these conflicting descriptions. To this end, we have simulated hundreds of spontaneous subunit rotation events (≈8°) using a residue-level coarse-grained model of the ribosome. We analyzed nine different reaction coordinates and found that the apparently inconsistent measurements are likely a consequence of ribosomal flexibility. Further, we propose a metric for quantifying the degree of energetic coupling between experimentally measured degrees of freedom and subunit rotation. This analysis provides a physically grounded framework that can guide the development of more precise single-molecule techniques.
Topics: Fluorescence Resonance Energy Transfer; Molecular Conformation; Molecular Dynamics Simulation; Ribosome Subunits; Rotation
PubMed: 29262370
DOI: 10.1016/j.bpj.2017.10.021 -
Current Opinion in Structural Biology Dec 2012The ribosome undergoes numerous large-scale conformational changes during protein synthesis, but the molecular basis for these changes have been unclear. Recent... (Review)
Review
The ribosome undergoes numerous large-scale conformational changes during protein synthesis, but the molecular basis for these changes have been unclear. Recent cryo-electron microscopic and X-ray crystallographic structures of both the bacterial and eukaryotic ribosome now provide snapshots of the wide range of motions that occur within the ribosome. X-ray crystallographic structures of the ribosome have also pinpointed local deformations in ribosomal RNA that occur when the two ribosomal subunits rotate with respect to each other. These structural results establish the foundation for unraveling the mechanics of the ribosome that are universal, and those that differ in bacteria and eukaryotes.
Topics: Movement; Protein Biosynthesis; RNA, Ribosomal; Ribosome Subunits; Ribosomes
PubMed: 22871550
DOI: 10.1016/j.sbi.2012.07.011 -
Biochimica Et Biophysica Acta 2009Translation of the genomes of several positive-sense RNA viruses follows end-independent initiation on an internal ribosomal entry site (IRES) in the viral mRNA. There... (Review)
Review
Translation of the genomes of several positive-sense RNA viruses follows end-independent initiation on an internal ribosomal entry site (IRES) in the viral mRNA. There are four major IRES groups, and despite major differences in the mechanisms that they use, one unifying characteristic is that each mechanism involves essential non-canonical interactions of the IRES with components of the canonical translational apparatus. Thus the approximately 200nt.-long Type 4 IRESs (epitomized by Cricket paralysis virus) bind directly to the intersubunit space on the ribosomal 40S subunit, followed by joining to a 60S subunit to form active ribosomes by a factor-independent mechanism. The approximately 300nt.-long type 3 IRESs (epitomized by Hepatitis C virus) binds independently to eukaryotic initiation factor (eIF) 3, and to the solvent-accessible surface and E-site of the 40S subunit: addition of eIF2-GTP/initiator tRNA is sufficient to form a 48S complex that can join a 60S subunit in an eIF5/eIF5B-mediated reaction to form an active ribosome. Recent cryo-electron microscopy and biochemical analyses have revealed a second general characteristic of the mechanisms of initiation on Type 3 and Type 4 IRESs. Both classes of IRES induce similar conformational changes in the ribosome that influence entry, positioning and fixation of mRNA in the ribosomal decoding channel. HCV-like IRESs also stabilize binding of initiator tRNA in the peptidyl (P) site of the 40S subunit, whereas Type 4 IRESs induce changes in the ribosome that likely promote subsequent steps in the translation process, including subunit joining and elongation.
Topics: Animals; Base Sequence; Guanosine Triphosphate; Humans; Models, Molecular; Molecular Conformation; Molecular Sequence Data; Nucleic Acid Conformation; Protein Biosynthesis; Protein Structure, Tertiary; RNA, Messenger; RNA, Transfer; Regulatory Elements, Transcriptional; Ribosome Subunits, Large, Eukaryotic; Ribosome Subunits, Small, Eukaryotic; Ribosomes; Solvents
PubMed: 19539793
DOI: 10.1016/j.bbagrm.2009.06.001 -
Nucleic Acids Research Jun 2024Ribosomal RNA modifications are introduced by specific enzymes during ribosome assembly in bacteria. Deletion of individual modification enzymes has a minor effect on...
Ribosomal RNA modifications are introduced by specific enzymes during ribosome assembly in bacteria. Deletion of individual modification enzymes has a minor effect on bacterial growth, ribosome biogenesis, and translation, which has complicated the definition of the function of the enzymes and their products. We have constructed an Escherichia coli strain lacking 10 genes encoding enzymes that modify 23S rRNA around the peptidyl-transferase center. This strain exhibits severely compromised growth and ribosome assembly, especially at lower temperatures. Re-introduction of the individual modification enzymes allows for the definition of their functions. The results demonstrate that in addition to previously known RlmE, also RlmB, RlmKL, RlmN and RluC facilitate large ribosome subunit assembly. RlmB and RlmKL have functions in ribosome assembly independent of their modification activities. While the assembly stage specificity of rRNA modification enzymes is well established, this study demonstrates that there is a mutual interdependence between the rRNA modification process and large ribosome subunit assembly.
Topics: Escherichia coli; Escherichia coli Proteins; Methyltransferases; Ribosome Subunits, Large; Ribosome Subunits, Large, Bacterial; Ribosomes; RNA, Ribosomal; RNA, Ribosomal, 23S
PubMed: 38554109
DOI: 10.1093/nar/gkae222 -
Nucleic Acids Research Sep 2023Ribosome biogenesis is one of the biggest consumers of cellular energy. More than 20 genetic diseases (ribosomopathies) and multiple cancers arise from defects in the...
Ribosome biogenesis is one of the biggest consumers of cellular energy. More than 20 genetic diseases (ribosomopathies) and multiple cancers arise from defects in the production of the 40S (SSU) and 60S (LSU) ribosomal subunits. Defects in the production of either the SSU or LSU result in p53 induction through the accumulation of the 5S RNP, an LSU assembly intermediate. While the mechanism is understood for the LSU, it is still unclear how SSU production defects induce p53 through the 5S RNP since the production of the two subunits is believed to be uncoupled. Here, we examined the response to SSU production defects to understand how this leads to the activation of p53 via the 5S RNP. We found that p53 activation occurs rapidly after SSU production is blocked, prior to changes in mature ribosomal RNA (rRNA) levels but correlated with early, middle and late SSU pre-rRNA processing defects. Furthermore, both nucleolar/nuclear LSU maturation, in particular late stages in 5.8S rRNA processing, and pre-LSU export were affected by SSU production defects. We have therefore uncovered a novel connection between the SSU and LSU production pathways in human cells, which explains how p53 is induced in response to SSU production defects.
Topics: Humans; Ribosomal Proteins; Ribosome Subunits, Large; Ribosome Subunits, Small; Ribosomes; RNA, Ribosomal; Tumor Suppressor Protein p53
PubMed: 37526268
DOI: 10.1093/nar/gkad637