EEF3 promotes late stages of tRNA translocation on the ribosome
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA672335 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12907686.fastq.gz | Fastqsanger.gz | |||
| SRR12907687.fastq.gz | Fastqsanger.gz | |||
| SRR12907688.fastq.gz | Fastqsanger.gz | |||
| SRR12907689.fastq.gz | Fastqsanger.gz | |||
| SRR12907690.fastq.gz | Fastqsanger.gz |
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Project description:In addition to the conserved translation elongation factors eEF1A and eEF2, fungi require a third essential elongation factor, eEF3. While eEF3 has been implicated in tRNA binding and release at the A and E sites, its exact mechanism of action is unclear. Here we show that eEF3 acts at the mRNA–tRNA translocation step by promoting the dissociation of the tRNA from the E site, but independent of aminoacyl-tRNA recruitment to the A site. Depletion of eEF3 in vivo leads to a general slow-down in translation elongation due to accumulation of ribosomes with an occupied A site. Cryo-EM analysis of native eEF3-ribosome complexes shows that eEF3 facilitates late steps of translocation by favoring non-rotated ribosomal states as well as by opening the L1 stalk to release the E-site tRNA. Additionally, our analysis provides structural insights into novel translation elongation states, enabling presentation of a revised yeast translation elongation cycle.
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Project description:In higher eukaryotes, the large numbers of nuclear-encoded tRNA genes partially ensure the robustness of cytoplasmic protein translation. Here we discover that a loss-of-function in n-Tr20, a member of the nuclear-encoded tRNA Arg UCU family that is expressed specifically in the central nervous systems leads to low but detectable levels of ribosome stalling. In the absence of GTPBP2, a novel binding partner of the ribosome recycling protein Pelota, ribosome stalling increases, leading to widespread neurodegeneration. Our results not only define GTPBP2 as a ribosome rescue factor, but also unmask the disease potential of mutations in nuclear-encoded tRNA genes. In this submission we provide ribosome footprinting data from the cerebella of four strains derived from the C57BL/6J strain with combinations of n-Tr20 and GTPBP2 mutations. Examination of ribosome stalling in cerebella from 4 mouse strains derived from the: C57BL/6J (B6J) strain. The nmf205-/- strain has a homozygous mutation in the gene GTPBP2 while the B6J strain has normal GTPBP2. The n-Tr20 J/J strain has a defect in the n-Tr20 tRNA while the n-Tr20 N/N strain has a functional n-Tr20 tRNA. The 4 strains are the 2x2 combinations of these defects and correctly functioning sequences. 2 replicates for each strain. Please note that only BAM files are included in the records since they form the basis of the study's conclusions. The raw data ribosomal RNA have been filtered and then unique reads mapping to mm10 were computed using tophat and igenome annotations.
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Project description:Protein translation depends on mRNA-specific initiation, elongation, and termination rates. While the regulation of ribosome elongation is well studied in bacteria and yeast, less is known in higher eukaryotes. Here, we combined ribosome and tRNA profiling to investigate the relations between ribosome elongation rates, (aminoacyl-) tRNA levels and codon usage in mammals. We modeled codon-specific ribosome dwell times and translation fluxes from ribosome profiling, considering pair-interactions between ribosome sites. In mouse liver, the model revealed site and codon specific dwell times, as well as codon pair-interactions clustering by amino acids. While translation fluxes varied significantly across diurnal time and feeding regimen, codon dwell times were highly stable, and conserved in human. Fasting had no effect on codon dwell times in mouse liver. Profiling of total and aminoacylated tRNAs revealed highly heterogeneous levels with specific isoacceptor patterns and a correlation with codon usage. tRNAs for isoleucine, asparagine, aspartate and arginine were lowly loaded and conserved in fasted mice. Finally, codons with low levels of charged tRNAs and high codon usage relative to tRNA abundance exhibited long dwell times. Together, these analyses pave the way towards understanding the complex relation between tRNA loading, codon usage and ribosome dwell times in mammals.
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Project description:In addition to the conserved translation elongation factors eEF1A and eEF2, fungi require a third essential elongation factor, eEF3. While eEF3 has been implicated in tRNA binding and release at the ribosomal A and E sites, its exact mechanism of action is unclear. Here, we show that eEF3 acts at the mRNA-tRNA translocation step by promoting the dissociation of the tRNA from the E site, but independent of aminoacyl-tRNA recruitment to the A site. Depletion of eEF3 in vivo leads to a general slowdown in translation elongation due to accumulation of ribosomes with an occupied A site. Cryo-EM analysis of native eEF3-ribosome complexes shows that eEF3 facilitates late steps of translocation by favoring non-rotated ribosomal states, as well as by opening the L1 stalk to release the E-site tRNA. Additionally, our analysis provides structural insights into novel translation elongation states, enabling presentation of a revised yeast translation elongation cycle.
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