Similar Datasets
Project description:Translation depends on messenger RNA (mRNA)-specific initiation, elongation, and termination rates. While translation elongation is well studied in bacteria and yeast, less is known in higher eukaryotes. Here we combined ribosome and transfer RNA (tRNA) profiling to investigate the relations between translation elongation rates, (aminoacyl-) tRNA levels, and codon usage in mammals. We modeled codon-specific ribosome dwell times from ribosome profiling, considering codon pair interactions between ribosome sites. In mouse liver, the model revealed site- and codon-specific dwell times that differed from those in yeast, as well as pairs of adjacent codons in the P and A site that markedly slow down or speed up elongation. While translation efficiencies vary across diurnal time and feeding regimen, codon dwell times were highly stable and conserved in human. Measured tRNA levels correlated with codon usage and several tRNAs showed reduced aminoacylation, which was conserved in fasted mice. Finally, we uncovered that the longest codon dwell times could be explained by aminoacylation levels or high codon usage relative to tRNA abundance.
| S-EPMC7196831 | biostudies-literature
Project description:Robust landscapes of ribosome dwell times and aminoacyl-tRNAs in response to nutrient stress in liver
| PRJNA521739 | ENA
Project description:Aminoacyl-tRNA synthetases (aaRS) are essential enzymes catalyzing the formation of aminoacyl-tRNAs, the immediate precursors for encoded peptides in ribosomal protein synthesis. Previous studies have suggested a link between tRNA aminoacylation and high-molecular-weight cellular complexes such as the cytoskeleton or ribosomes. However, the structural basis of these interactions and potential mechanistic implications are not well understood. To biochemically characterize these interactions we have used a system of two interacting archaeal aaRSs: an atypical methanogenic-type seryl-tRNA synthetase and an archaeal ArgRS. More specifically, we have shown by thermophoresis and surface plasmon resonance that these two aaRSs bind to the large ribosomal subunit with micromolar affinities. We have identified the L7/L12 stalk and the proteins located near the stalk base as the main sites for aaRS binding. Finally, we have performed a bioinformatics analysis of synonymous codons in the Methanothermobacter thermautotrophicus genome that supports a mechanism in which the deacylated tRNAs may be recharged by aaRSs bound to the ribosome and reused at the next occurrence of a codon encoding the same amino acid. These results suggest a mechanism of tRNA recycling in which aaRSs associate with the L7/L12 stalk region to recapture the tRNAs released from the preceding ribosome in polysomes.
| S-EPMC4005694 | biostudies-literature
Project description:Using explicit-solvent simulations of the 70S ribosome, the barrier-crossing attempt frequency was calculated for aminoacyl-tRNA elbow-accommodation. In seven individual trajectories (200-300 ns, each, for an aggregate time of 2.1 ?s), the relaxation time of tRNA structural fluctuations was determined to be ?10 ns, and the barrier-crossing attempt frequency of tRNA accommodation is ?1-10 ?s(-1). These calculations provide a quantitative relationship between the free-energy barrier and experimentally measured rates of accommodation, which demonstrate that the free-energy barrier of elbow-accommodation is less than 15 k(B)T, in vitro and in vivo.
| S-EPMC2945706 | biostudies-literature
Project description:We used a cell-free system with pure Escherichia coli components to study initial codon selection of aminoacyl-tRNAs in ternary complex with elongation factor Tu and GTP on messenger RNA-programmed ribosomes. We took advantage of the universal rate-accuracy trade-off for all enzymatic selections to determine how the efficiency of initial codon readings decreased linearly toward zero as the accuracy of discrimination against near-cognate and wobble codon readings increased toward the maximal asymptote, the d value. We report data on the rate-accuracy variation for 7 cognate, 7 wobble, and 56 near-cognate codon readings comprising about 15% of the genetic code. Their d values varied about 400-fold in the 200-80,000 range depending on type of mismatch, mismatch position in the codon, and tRNA isoacceptor type. We identified error hot spots (d = 200) for U:G misreading in second and U:U or G:A misreading in third codon position by His-tRNA(His) and, as also seen in vivo, Glu-tRNA(Glu). We suggest that the proofreading mechanism has evolved to attenuate error hot spots in initial selection such as those found here.
| S-EPMC4534242 | biostudies-other
Project description:Several l-aminoacyl-tRNA synthetases can transfer a d-amino acid onto their cognate tRNA(s). This harmful reaction is counteracted by the enzyme d-aminoacyl-tRNA deacylase. Two distinct deacylases were already identified in bacteria (DTD1) and in archaea (DTD2), respectively. Evidence was given that DTD1 homologs also exist in nearly all eukaryotes, whereas DTD2 homologs occur in plants. On the other hand, several bacteria, including most cyanobacteria, lack genes encoding a DTD1 homolog. Here we show that Synechocystis sp. PCC6803 produces a third type of deacylase (DTD3). Inactivation of the corresponding gene (dtd3) renders the growth of Synechocystis sp. hypersensitive to the presence of d-tyrosine. Based on the available genomes, DTD3-like proteins are predicted to occur in all cyanobacteria. Moreover, one or several dtd3-like genes can be recognized in all cellular types, arguing in favor of the nearubiquity of an enzymatic function involved in the defense of translational systems against invasion by d-amino acids.
| S-EPMC2682858 | biostudies-literature
Project description:Mammalian cells reorganize their proteomes in response to nutrient stress through translational suppression and degradative mechanisms using the proteasome and autophagy systems1,2. Ribosomes are central targets of this response, as they are responsible for translation and subject to lysosomal turnover during nutrient stress3-5. The abundance of ribosomal (r)-proteins (around 6% of the proteome; 107 copies per cell)6,7 and their high arginine and lysine content has led to the hypothesis that they are selectively used as a source of basic amino acids during nutrient stress through autophagy4,7. However, the relative contributions of translational and degradative mechanisms to the control of r-protein abundance during acute stress responses is poorly understood, as is the extent to which r-proteins are used to generate amino acids when specific building blocks are limited7. Here, we integrate quantitative global translatome and degradome proteomics8 with genetically encoded Ribo-Keima5 and Ribo-Halo reporters to interrogate r-protein homeostasis with and without active autophagy. In conditions of acute nutrient stress, cells strongly suppress the translation of r-proteins, but, notably, r-protein degradation occurs largely through non-autophagic pathways. Simultaneously, the decrease in r-protein abundance is compensated for by a reduced dilution of pre-existing ribosomes and a reduction in cell volume, thereby maintaining the density of ribosomes within single cells. Withdrawal of basic or hydrophobic amino acids induces translational repression without differential induction of ribophagy, indicating that ribophagy is not used to selectively produce basic amino acids during acute nutrient stress. We present a quantitative framework that describes the contributions of biosynthetic and degradative mechanisms to r-protein abundance and proteome remodelling in conditions of nutrient stress.
| S-EPMC7351614 | biostudies-literature
Project description:Structural analysis of ribosomes in complex with aminoacyl- and/or peptidyl-transfer RNA (tRNA) often suffers from rapid hydrolysis of the ester bond of aminoacyl-tRNAs. To avoid this issue, several methods to introduce an unhydrolyzable amide bond instead of the canonical ester bond have been developed to date. However, the existing methodologies require rather complex steps of synthesis and are often inapplicable to a variety of amino acids including those with noncanonical structures. Here, we report a new method to synthesize 3'-aminoacyl-NH-tRNAs by means of flexizymes-ribozymes capable of charging amino acids onto tRNAs. We show that two types of flexizymes, dFx and eFx, are able to charge various amino acids, including nonproteinogenic ones, onto tRNA or microhelix RNA bearing the 3'-deoxy-3'-amino-adenosine. Due to the versatility of the flexizymes toward any pair of nonproteinogenic amino acids and full-length or fragment tRNAs, this method provides researchers an opportunity to use a wide array of hydrolytically stable 3'-aminoacyl-NH-tRNAs and analogs for various studies.
| S-EPMC6511858 | biostudies-other
Project description:Escherichia coli ItaT toxin reportedly acetylates the α-amino group of the aminoacyl-moiety of Ile-tRNAIle specifically, using acetyl-CoA as an acetyl donor, thereby inhibiting protein synthesis. The mechanism of the substrate specificity of ItaT had remained elusive. Here, we present functional and structural analyses of E. coli ItaT, which revealed the mechanism of ItaT recognition of specific aminoacyl-tRNAs for acetylation. In addition to Ile-tRNAIle, aminoacyl-tRNAs charged with hydrophobic residues, such as Val-tRNAVal and Met-tRNAMet, were acetylated by ItaT in vivo. Ile-tRNAIle, Val-tRNAVal and Met-tRNAMet were acetylated by ItaT in vitro, while aminoacyl-tRNAs charged with other hydrophobic residues, such as Ala-tRNAAla, Leu-tRNALeu and Phe-tRNAPhe, were less efficiently acetylated. A comparison of the structures of E. coli ItaT and the protein N-terminal acetyltransferase identified the hydrophobic residues in ItaT that possibly interact with the aminoacyl moiety of aminoacyl-tRNAs. Mutations of the hydrophobic residues of ItaT reduced the acetylation activity of ItaT toward Ile-tRNAIlein vitro, as well as the ItaT toxicity in vivo. Altogether, the size and shape of the hydrophobic pocket of ItaT are suitable for the accommodation of the specific aminoacyl-moieties of aminoacyl-tRNAs, and ItaT has broader specificity toward aminoacyl-tRNAs charged with certain hydrophobic amino acids.
| S-EPMC7367177 | biostudies-literature
Project description:During protein synthesis, the ribosome simultaneously binds up to three different transfer RNA (tRNA) molecules. Among the three tRNA binding sites, the regulatory role of the exit (E) site, where deacylated tRNA spontaneously dissociates from the translational complex, has remained elusive. Here we use two donor-quencher pairs to observe and correlate both the conformation of ribosomes and tRNAs as well as tRNA occupancy. Our results reveal a partially rotated state of the ribosome wherein all three tRNA sites are occupied during translation elongation. The appearance and lifetime of this state depend on the E-site tRNA dissociation kinetics, which may vary among tRNA species and depends on temperature and ionic strength. The 3-tRNA partially rotated state is not a proper substrate for elongation factor G (EF-G), thus inhibiting translocation until the E-site tRNA dissociates. Our result presents two parallel kinetic pathways during translation elongation, underscoring the ability of E-site codons to modulate the dynamics of protein synthesis.
| S-EPMC5748233 | biostudies-literature