Name: Identification of nascent peptides of peptidyl-tRNA dissociated from ribosome in the early elongation stage in Escherichia coli pthts cells
Keywords: Translation

OmicsDI

Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Identification of nascent peptides of peptidyl-tRNA dissociated from ribosome in the early elongation stage in Escherichia coli pthts cells

ABSTRACT: Accurate translation of mRNAs into functional proteins is a fundamental process in all living organisms. In bacteria, in the early stage of translation elongation, peptidyl-tRNAs (pep-tRNAs) with short nascent chains frequently dissociate from the ribosome (pep-tRNA drop-off). The dissociated pep-tRNAs are deacylated and recycled by peptidyl-tRNA hydrolase (PTH), which is an essential enzyme in bacteria. Here, we establish a highly sensitive method for direct profiling of pep-tRNAs using RNA isolation method and mass spectrometry. We isolated each tRNA species with peptide from Escherichia coli pthts cells using reciprocal circulating chromatography and precisely analyzed their nascent peptides. As a result, we successfully detected 703 peptides consisted of 402 cognate peptides and 301 non-cognate peptides with single amino-acid substitution. Detailed analysis of individual pep-tRNAs revealed that most of the substitutions in the miscoded peptides take place at the C-terminal drop-off site. We further examined this observation using a reporter construct and found that the non-cognate pep-tRNAs produced by mistranslation rarely participate in the next round of elongation but dissociate from the ribosome, suggesting that pep-tRNA drop-off is an active mechanism by which the ribosome rejects miscoded pep-tRNAs in the early elongation, thereby contributing to quality control of protein synthesis after peptide bond formation.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Escherichia Coli

SUBMITTER: Asuteka Nagao

LAB HEAD: Tsutomu Suzuki

PROVIDER: PXD030805 | Pride | 2023-05-18

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Similar Datasets

Identification of nascent peptides of peptidyl-tRNA dissociated from ribosome in the early elongation stage in Escherichia coli pthts cells

Project description:Accurate translation of mRNAs into functional proteins is a fundamental process in all living organisms. In bacteria, in the early stage of translation elongation, peptidyl-tRNAs (pep-tRNAs) with short nascent chains frequently dissociate from the ribosome (pep-tRNA drop-off). The dissociated pep-tRNAs are deacylated and recycled by peptidyl-tRNA hydrolase (PTH), which is an essential enzyme in bacteria. Here, we establish a highly sensitive method for direct profiling of pep-tRNAs using RNA isolation method and mass spectrometry. We isolated each tRNA species with peptide from Escherichia coli pthts cells using reciprocal circulating chromatography and precisely analyzed their nascent peptides. As a result, we successfully detected 703 peptides consisted of 402 cognate peptides and 301 non-cognate peptides with single amino-acid substitution. Detailed analysis of individual pep-tRNAs revealed that most of the substitutions in the miscoded peptides take place at the C-terminal drop-off site. We further examined this observation using a reporter construct and found that the non-cognate pep-tRNAs produced by mistranslation rarely participate in the next round of elongation but dissociate from the ribosome, suggesting that pep-tRNA drop-off is an active mechanism by which the ribosome rejects miscoded pep-tRNAs in the early elongation, thereby contributing to quality control of protein synthesis after peptide bond formation.

2023-05-18 | PXD030808 | Pride

Identification of nascent peptides of peptidyl-tRNA dissociated from ribosome in the early elongation stage in Escherichia coli pthts cells

2023-05-18 | PXD030807 | Pride

Identification of nascent peptides of peptidyl-tRNA dissociated from ribosome in the early elongation stage in Escherichia coli pthts cells

2023-05-18 | PXD030806 | Pride

Selective control of metabolism by the GCN2 eIF2 kinase pathway in Saccharomyces cerevisiae

Project description:When eukaryotic cells are deprived of amino acids, uncharged tRNAs accumulate and activate the conserved GCN2 protein kinase. We examine how yeast growth and tRNA charging or aminoacylation is affected during amino acid depletion in the presence and absence of GCN2. tRNA charging is measured using a microarray technique which allows for simultaneous measurement of all cytosolic tRNAs. A fully prototrophic and its isogenic GCN2 deletion strain were used. We measured relative tRNA charging levels in yeast strains with an intact and deleted GCN2.

2010-08-01 | E-GEOD-22452 | biostudies-arrayexpress

tRNA Over-Expression in Breast Cancer

Project description:Increased proliferation and elevated levels of protein synthesis are characteristic of transformed and tumor cells. Though components of the translation machinery are often misregulated in cancers, how tRNA plays a role in cancer cells has not been explored. We compare genome-wide tRNA expression in tumorigenic versus non-tumorigenic breast cell lines, as well as tRNA expression in breast tumors versus normal breast tissues. In tumorigenic versus non-tumorigenic cell lines, nuclear-encoded tRNAs increase by up to 3-fold and mitochondrial-encoded tRNAs increase by up to 5-fold. In tumors versus normal breast tissues, both nuclear and mitochondrial-encoded tRNAs increase by up to 10-fold. This tRNA over-expression is selective and coordinates with the properties of cognate amino acids. Nuclear- and mitochondrial-encoded tRNAs exhibit distinct expression patterns, indicating that tRNAs can be used as biomarkers for breast cancer. We analyzed tRNA expression levels in 2 non-tumorigenic breast cell lines, 6 tumorigenic breast cancer cell lines, 3 normal breast tissue samples, and 9 breast tumor samples. We used a non-tumorigenic breast cell line (MCF10A) as a reference sample in all hybridizations. All data is dye-swapped.

2010-05-22 | E-GEOD-17945 | biostudies-arrayexpress

Engineered transfer RNAs for suppression of premature termination codons

Project description:PTCs cause a multitude of human diseases and there are no established therapeutic options for their therapeutic management. Herein, we report the high-throughput cloning and identification, characterization and functional analysis of anticodon-edited tRNA which display efficacious PTC reversion in eukaryotic cells and mouse skeletal muscle. Notably, our screen identifies ACE-tRNA, in total, with the potential to repair a vast majority of known human disease-causing PTC, but this therapeutic will require overcoming tissue and delivery specific challenges. However, the engineered tRNA, once delivered, faithfully encode their cognate amino acid, thus abrogating spurious effects on downstream protein stability, folding, and trafficking, and consequently negating the need for tandem therapies involving protein folding or trafficking agents. When transfected as cDNA, ACE-tRNAs rescued multiple full-length proteins via PTC suppression; a NLuc luciferase reporter, a model protein HDH, and two disease nonsense mutations in CFTR.

2019-02-11 | PXD012581 | Pride

Pytheas S. cerevisiae tRNA sample analysis

Project description:Mass spectrometry remains an important method for analysis of modified nucleosides ubiquitously present in cellular RNAs, in particular for ribosomal and transfer RNAs that play crucial roles in mRNA translation and decoding. Furthermore, modifications have effect on the lifetimes of nucleic acids in plasma and cells and are consequently incorporated into RNA therapeutics. To provide an analytical tool for sequence characterization of modified RNAs, we developed Pytheas, an open-source software package for automated analysis of tandem MS data for RNA. This dataset contains the analysis of a mixture of S. cerevisiae tRNAs, decorated with a variety of complex RNA modifications.

2022-05-04 | PXD030844 | Pride

Transfer RNA (tRNA) processing and RNA m5C-methylation profiles of mouse skin squamous tumours

Project description:Purpose: High-throughput RNA sequencing has accelerated discovery of the complex regulatory roles of small RNAs, such those derived from tRNAs. Also recent advances in high-throughput RNA sequencing has revealed the complex RNA modification landscape and the complex role these nucleosides modifactions have in cell signalling, stem cell biology, development and cancer. The goal of this study is to establish how m5C-tRNA methylation and tRNA-derived small RNAs can affect stem cell fucntion in cancer. Methods: four replicates of tRNAs and RNA buisulphite sequencing of wild-type (WT) and NSun2 -/- mouse skin squamous tumours were generated by deep sequencing, using Illumina HiSeq platform. Results: Our analyses reveal that inhibition of post-transcriptional cytosine-5 methylation locks stem cells in this distinct translational inhibition programme that results in tumour progression but that also sentizes cancer cells to genotoxic stress. Transfer RNA (tRNA) sequencing and RNA Bisulphite sequencing of wild-type (WT) and NSun2 -/- mouse skin squamous tumours

2016-06-16 | E-GEOD-72065 | biostudies-arrayexpress

Loss of cytosine-5 methylation in tRNA triggers stress responses in a disease model for Intellectual Disability

Project description:Mutations in the cytosine-5 RNA methyltransferase NSun2 can cause Intellectual Disability (ID) and symptoms commonly found in patients with Dubowitz syndrome. By analysing gene expression data with the global cytosine-5 RNA methylome in NSun2-deficient mice, we find that loss of cytosine-5 RNA methylation increases the fragmentation of transfer RNAs (tRNA) leading to an accumulation of 5M-bM-^@M-^Y halves. Cleavage of tRNAs by Angiogenin is a common cellular stress response to silence translational programmes, and we show that Angiogenin binds tRNAs lacking site-specific NSun2-methylation with higher affinity. Furthermore, cells lacking functional NSun2 up-regulate stress markers, and deletion of NSun2 compromises cellular survival in response stress stimuli including UV-light and oxidative stress. The decreased tolerance of NSun2 null cells towards oxidative stress can be rescued through inhibition of Angiogenin. In conclusion, cytosine-5 RNA methylation is an essential post-transcriptional mechanism during cellular stress responses and NSun2-mediated tRNA methylation protects from Angiogenin-dependent stress-induced RNA cleavage. RNA Methylation profiling by high throughput sequencing small non-coding RNA profiling by high throughput sequencing Pol III Chromatin-IP profiling by high throughput sequencing

2014-11-06 | E-GEOD-44746 | biostudies-arrayexpress

tRNA gene identity acts as a determinant of gene positioning

Project description:Here we report that the spatial organization of yeast tRNA genes depends upon both locus position and tRNA identity; supporting the idea that the genomic organization of tRNA loci utilizes tRNA dependent signals within the nucleoprotein-tRNA complexes that form into clusters. We use high-throughput sequencing coupled to Circular Chromosome Conformation Capture to detect interactions with two wild type tRNAs and these same positions replaced with suppressor tRNAs (SUP4-1). Detect DNA-DNA interactions (Circular chromosome conformation capture; 4C) with two wild type tRNAs and these same positions replaced with suppressor tRNAs (SUP4-1) Supplementary files: Alignment files generated by Topography v1.19 software.

2012-06-13 | E-GEOD-30103 | biostudies-arrayexpress

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

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