Browse
Submit Data
Databases
API
Help

Dataset Information

27 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Structures of the ribosome in intermediate states of ratcheting.

ABSTRACT: Protein biosynthesis on the ribosome requires repeated cycles of ratcheting, which couples rotation of the two ribosomal subunits with respect to each other, and swiveling of the head domain of the small subunit. However, the molecular basis for how the two ribosomal subunits rearrange contacts with each other during ratcheting while remaining stably associated is not known. Here, we describe x-ray crystal structures of the intact Escherichia coli ribosome, either in the apo-form (3.5 angstrom resolution) or with one (4.0 angstrom resolution) or two (4.0 angstrom resolution) anticodon stem-loop tRNA mimics bound, that reveal intermediate states of intersubunit rotation. In the structures, the interface between the small and large ribosomal subunits rearranges in discrete steps along the ratcheting pathway. Positioning of the head domain of the small subunit is controlled by interactions with the large subunit and with the tRNA bound in the peptidyl-tRNA site. The intermediates observed here provide insight into how tRNAs move into the hybrid state of binding that precedes the final steps of mRNA and tRNA translocation.

SUBMITTER: Zhang W

PROVIDER: S-EPMC2919209 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Cryoelectron microscopy structures of the ribosome complex in intermediate states during tRNA translocation.

Project description:mRNA-tRNA translocation is a central and highly regulated process during translational elongation. Along with the mRNA, tRNA moves through the ribosome in a stepwise fashion. Using cryoelectron microscopy on ribosomes with a P-loop mutation, we have identified novel structural intermediates likely to exist transiently during translocation. Our observations suggest a mechanism by which the rate of translocation can be regulated.

| S-EPMC3064370 | biostudies-literature

Crystal structures of EF-G-ribosome complexes trapped in intermediate states of translocation.

Project description:Translocation of messenger and transfer RNA (mRNA and tRNA) through the ribosome is a crucial step in protein synthesis, whose mechanism is not yet understood. The crystal structures of three Thermus ribosome-tRNA-mRNA-EF-G complexes trapped with β,γ-imidoguanosine 5'-triphosphate (GDPNP) or fusidic acid reveal conformational changes occurring during intermediate states of translocation, including large-scale rotation of the 30S subunit head and body. In all complexes, the tRNA acceptor ends occupy the 50S subunit E site, while their anticodon stem loops move with the head of the 30S subunit to positions between the P and E sites, forming chimeric intermediate states. Two universally conserved bases of 16S ribosomal RNA that intercalate between bases of the mRNA may act as "pawls" of a translocational ratchet. These findings provide new insights into the molecular mechanism of ribosomal translocation.

| S-EPMC3979973 | biostudies-literature

Structures of the eukaryotic ribosome and its translational states in situ.

Project description:Ribosomes translate genetic information into primary structure. During translation, various cofactors transiently bind to the ribosome that undergoes prominent conformational and structural changes. Different translational states of ribosomes have been well characterized in vitro. However, to which extent the known translational states are representative of the native situation inside cells has thus far only been addressed in prokaryotes. Here, we apply cryo-electron tomography to cryo-FIB milled Dictyostelium discoideum cells combined with subtomogram averaging and classification. We obtain an in situ structure that is locally resolved up to 3 Angstrom, the distribution of eukaryotic ribosome translational states, and unique arrangement of rRNA expansion segments. Our work demonstrates the use of in situ structural biology techniques for identifying distinct ribosome states within the cellular environment.

| S-EPMC9718845 | biostudies-literature

Structures of the human mitochondrial ribosome in native states of assembly.

Project description:Mammalian mitochondrial ribosomes (mitoribosomes) have less rRNA content and 36 additional proteins compared with the evolutionarily related bacterial ribosome. These differences make the assembly of mitoribosomes more complex than the assembly of bacterial ribosomes, but the molecular details of mitoribosomal biogenesis remain elusive. Here, we report the structures of two late-stage assembly intermediates of the human mitoribosomal large subunit (mt-LSU) isolated from a native pool within a human cell line and solved by cryo-EM to ∼3-Å resolution. Comparison of the structures reveals insights into the timing of rRNA folding and protein incorporation during the final steps of ribosomal maturation and the evolutionary adaptations that are required to preserve biogenesis after the structural diversification of mitoribosomes. Furthermore, the structures redefine the ribosome silencing factor (RsfS) family as multifunctional biogenesis factors and identify two new assembly factors (L0R8F8 and mt-ACP) not previously implicated in mitoribosomal biogenesis.

| S-EPMC5633077 | biostudies-literature

Structures of dNTP intermediate states during DNA polymerase active site assembly.

Project description:DNA polymerase and substrate conformational changes are essential for high-fidelity DNA synthesis. Structures of DNA polymerase (pol) β in complex with DNA show the enzyme in an "open" conformation. Subsequent to binding the nucleotide, the polymerase "closes" around the nascent base pair with two metals positioned for chemistry. However, structures of substrate/active site intermediates prior to closure are lacking. By destabilizing the closed complex, we determined unique ternary complex structures of pol β with correct and incorrect incoming nucleotides bound to the open conformation. These structures reveal that Watson-Crick hydrogen bonding is assessed upon initial complex formation. Importantly, nucleotide-bound states representing intermediate metal coordination states occur with active site assembly. The correct, but not incorrect, nucleotide maintains Watson-Crick hydrogen bonds during interconversion of these states. These structures indicate that the triphosphate of the incoming nucleotide undergoes rearrangement prior to closure, providing an opportunity to deter misinsertion and increase fidelity.

| S-EPMC3496073 | biostudies-literature

Structures of intermediate transport states of ZneA, a Zn(II)/proton antiporter.

Project description:Efflux pumps belonging to the ubiquitous resistance-nodulation-cell division (RND) superfamily transport substrates out of cells by coupling proton conduction across the membrane to a conformationally driven pumping cycle. The heavy metal-resistant bacteria Cupriavidus metallidurans CH34 relies notably on as many as 12 heavy metal efflux pumps of the RND superfamily. Here we show that C. metallidurans CH34 ZneA is a proton driven efflux pump specific for Zn(II), and that transport of substrates through the transmembrane domain may be electrogenic. We report two X-ray crystal structures of ZneA in intermediate transport conformations, at 3.0 and 3.7 Å resolution. The trimeric ZneA structures capture protomer conformations that differ in the spatial arrangement and Zn(II) occupancies at a proximal and a distal substrate binding site. Structural comparison shows that transport of substrates through a tunnel that links the two binding sites, toward an exit portal, is mediated by the conformation of a short 14-aa loop. Taken together, the ZneA structures presented here provide mechanistic insights into the conformational changes required for substrate efflux by RND superfamily transporters.

| S-EPMC3832018 | biostudies-literature

Structures of the bacterial ribosome in classical and hybrid states of tRNA binding.

Project description:During protein synthesis, the ribosome controls the movement of tRNA and mRNA by means of large-scale structural rearrangements. We describe structures of the intact bacterial ribosome from Escherichia coli that reveal how the ribosome binds tRNA in two functionally distinct states, determined to a resolution of ~3.2 angstroms by means of x-ray crystallography. One state positions tRNA in the peptidyl-tRNA binding site. The second, a fully rotated state, is stabilized by ribosome recycling factor and binds tRNA in a highly bent conformation in a hybrid peptidyl/exit site. The structures help to explain how the ratchet-like motion of the two ribosomal subunits contributes to the mechanisms of translocation, termination, and ribosome recycling.

| S-EPMC3176341 | biostudies-literature

Structures of the scanning and engaged states of the mammalian SRP-ribosome complex.

Project description:The universally conserved signal recognition particle (SRP) is essential for the biogenesis of most integral membrane proteins. SRP scans the nascent chains of translating ribosomes, preferentially engaging those with hydrophobic targeting signals, and delivers these ribosome-nascent chain complexes to the membrane. Here, we present structures of native mammalian SRP-ribosome complexes in the scanning and engaged states. These structures reveal the near-identical SRP architecture of these two states, show many of the SRP-ribosome interactions at atomic resolution, and suggest how the polypeptide-binding M domain selectively engages hydrophobic signals. The scanning M domain, pre-positioned at the ribosomal exit tunnel, is auto-inhibited by a C-terminal amphipathic helix occluding its hydrophobic binding groove. Upon engagement, the hydrophobic targeting signal displaces this amphipathic helix, which then acts as a protective lid over the signal. Biochemical experiments suggest how scanning and engagement are coordinated with translation elongation to minimize exposure of hydrophobic signals during membrane targeting.

| S-EPMC4497383 | biostudies-literature

Visualization of the hybrid state of tRNA binding promoted by spontaneous ratcheting of the ribosome.

Project description:A crucial step in translation is the translocation of tRNAs through the ribosome. In the transition from one canonical site to the other, the tRNAs acquire intermediate configurations, so-called hybrid states. At this stage, the small subunit is rotated with respect to the large subunit, and the anticodon stem loops reside in the A and P sites of the small subunit, while the acceptor ends interact with the P and E sites of the large subunit. In this work, by means of cryo-EM and particle classification procedures, we visualize the hybrid state of both A/P and P/E tRNAs in an authentic factor-free ribosome complex during translocation. In addition, we show how the repositioning of the tRNAs goes hand in hand with the change in the interplay between S13, L1 stalk, L5, H68, H69, and H38 that is caused by the ratcheting of the small subunit.

| S-EPMC2614368 | biostudies-literature

Cryo-EM structures reveal two distinct conformational states in a picornavirus cell entry intermediate.

Project description:The virions of enteroviruses such as poliovirus undergo a global conformational change after binding to the cellular receptor, characterized by a 4% expansion, and by the opening of holes at the two and quasi-three-fold symmetry axes of the capsid. The resultant particle is called a 135S particle or A-particle and is thought to be on the pathway to a productive infection. Previously published studies have concluded that the membrane-interactive peptides, namely VP4 and the N-terminus of VP1, are irreversibly externalized in the 135S particle. However, using established protocols to produce the 135S particle, and single particle cryo-electron microscopy methods, we have identified at least two unique states that we call the early and late 135S particle. Surprisingly, only in the "late" 135S particles have detectable levels of the VP1 N-terminus been trapped outside the capsid. Moreover, we observe a distinct density inside the capsid that can be accounted for by VP4 that remains associated with the genome. Taken together our results conclusively demonstrate that the 135S particle is not a unique conformation, but rather a family of conformations that could exist simultaneously.

| S-EPMC7549760 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data