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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: PXD030807 | Pride | 2023-05-18

REPOSITORIES: Pride

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			Action	DRS
	Ala1_1.RAW	Raw
	Ala1_2.RAW	Raw
	Ala2_1.RAW	Raw
	Ala2_2.RAW	Raw
	Arg2_1.RAW	Raw

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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.

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

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