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Ribosomes in a stacked array

Yamashita, Yui, Kadokura, Yoshitomo, Sotta, Naoyuki, Fujiwara, Toru, Takigawa, Ichigaku, Satake, Akiko, Onouchi, Hitoshi and Naito, Satoshi 2014. Ribosomes in a stacked array. Journal of Biological Chemistry 289 (18) , pp. 12693-12704. 10.1074/jbc.m113.526616

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Expression of CGS1, which codes for an enzyme of methionine biosynthesis, is feedback-regulated by mRNA degradation in response to S-adenosyl-l-methionine (AdoMet). In vitro studies revealed that AdoMet induces translation arrest at Ser-94, upon which several ribosomes stack behind the arrested one, and mRNA degradation occurs at multiple sites that presumably correspond to individual ribosomes in a stacked array. Despite the significant contribution of stacked ribosomes to inducing mRNA degradation, little is known about the ribosomes in the stacked array. Here, we assigned the peptidyl-tRNA species of the stacked second and third ribosomes to their respective codons and showed that they are arranged at nine-codon intervals behind the Ser-94 codon, indicating tight stacking. Puromycin reacts with peptidyl-tRNA in the P-site, releasing the nascent peptide as peptidyl-puromycin. This reaction is used to monitor the activity of the peptidyltransferase center (PTC) in arrested ribosomes. Puromycin reaction of peptidyl-tRNA on the AdoMet-arrested ribosome, which is stalled at the pre-translocation step, was slow. This limited reactivity can be attributed to the peptidyl-tRNA occupying the A-site at this step rather than to suppression of PTC activity. In contrast, puromycin reactions of peptidyl-tRNA with the stacked second and third ribosomes were slow but were not as slow as pre-translocation step ribosomes. We propose that the anticodon end of peptidyl-tRNA resides in the A-site of the stacked ribosomes and that the stacked ribosomes are stalled at an early step of translocation, possibly at the P/E hybrid state.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Biosciences
Publisher: American Society for Biochemistry and Molecular Biology
ISSN: 0021-9258
Date of Acceptance: 7 March 2014
Last Modified: 10 Nov 2021 02:23

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