S-Adenosyl-l-methionine Induces Compaction of Nascent Peptide Chain inside the Ribosomal Exit Tunnel upon Translation Arrest in the Arabidopsis CGS1 Gene

Noriyuki Onoue,Yui Yamashita,Nobuhiro Nagao,Derek B Goto,Hitoshi Onouchi,Satoshi Naito

doi:10.1074/jbc.m110.211656

Noriyuki Onoue, Yui Yamashita + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m110.211656

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Abstract

Expression of the Arabidopsis CGS1 gene, encoding the first committed enzyme of methionine biosynthesis, is feedback-regulated in response to S-adenosyl-l-methionine (AdoMet) at the mRNA level. This regulation is first preceded by temporal arrest of CGS1 translation elongation at the Ser-94 codon. AdoMet is specifically required for this translation arrest, although the mechanism by which AdoMet acts with the CGS1 nascent peptide remained elusive. We report here that the nascent peptide of CGS1 is induced to form a compact conformation within the exit tunnel of the arrested ribosome in an AdoMet-dependent manner. Cysteine residues introduced into CGS1 nascent peptide showed reduced ability to react with polyethyleneglycol maleimide in the presence of AdoMet, consistent with a shift into the ribosomal exit tunnel. Methylation protection and UV cross-link assays of 28 S rRNA revealed that induced compaction of nascent peptide is associated with specific changes in methylation protection and UV cross-link patterns in the exit tunnel wall. A 14-residue stretch of amino acid sequence, termed the MTO1 region, has been shown to act in cis for CGS1 translation arrest and mRNA degradation. This regulation is lost in the presence of mto1 mutations, which cause single amino acid alterations within MTO1. In this study, both the induced peptide compaction and exit tunnel change were found to be disrupted by mto1 mutations. These results suggest that the MTO1 region participates in the AdoMet-induced arrest of CGS1 translation by mediating changes of the nascent peptide and the exit tunnel wall.

Highlights

As the ribosomal exit tunnel holds 30 – 40 amino acids [33, 34], it was expected that a cysteine residue located between 30 and 40 amino acids upstream of the arrest site (Ser-94) would be near the outlet of the exit tunnel and that the pegylation efficiency for this residue would change prominently depending on the conformation of the nascent peptide
A single cysteine was introduced at 40 amino acids upstream of the arrest site by substituting Ala-55 to cysteine (A55C) to generate the M8:ND5(A55C, C80A) construct (Fig. 1A)
We have demonstrated that the nascent peptide of the CGS1 gene is induced to adopt a compact conformation within an arrested eukaryotic ribosome according to AdoMet, the effector molecule for feedback regulation of CGS1 expression

Summary

Introduction

We have applied the pegylation assay to test the hypothesis that AdoMet induces a change of CGS1 nascent peptide conformation inside the ribosomal exit tunnel during translation arrest. We show here that CGS1 nascent peptide arrested at Ser-94, which includes the MTO1 sequence, forms a compact conformation in response to AdoMet. Further analyses using methylation protection and UV cross-link of the 28 S rRNA showed that this compaction is associated with specific changes in methylation protection and UV cross-link patterns in the ribosomal exit tunnel.

Results

Conclusion