Ribosomes in a Stacked Array: ELUCIDATION OF THE STEP IN TRANSLATION ELONGATION AT WHICH THEY ARE STALLED DURING S-ADENOSYL-l-METHIONINE-INDUCED TRANSLATION ARREST OF CGS1 mRNA

Yui Yamashita,Yoshitomo Kadokura,Naoyuki Sotta,Toru Fujiwara,Ichigaku Takigawa,Akiko Satake,Hitoshi Onouchi,Satoshi Naito

doi:10.1074/jbc.m113.526616

Yui Yamashita, Yoshitomo Kadokura + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m113.526616

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Abstract

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.

Highlights

S-Adenosyl-L-methionine arrests ribosomes on CGS1 mRNA at the pre-translocation step, and ribosomes stack behind it
Identification of the Peptidyl-tRNA Band That Was Formed on the AdoMet-arrested Ribosome—In the in vitro translation condition used in this study (50 fmol RNA ␮lϪ1 translation mixture), three major translation arrest products were detectable (Fig. 1), and we previously determined that at least two additional ribosomes were stacked behind the arrested ribosome [12]
X-ray crystallographic studies of prokaryotic ribosomes showed that the mRNA cavity on the small subunit covered ϳ30 nt of mRNA [28, 29], corroborating the report that the length of mRNA protected from RNase digestion by a eukaryotic translating ribosome was about 30 nt [3, 6]

Summary

Background

S-Adenosyl-L-methionine arrests ribosomes on CGS1 mRNA at the pre-translocation step, and ribosomes stack behind it. In vitro studies revealed that AdoMet induces translation arrest at Ser, 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. A recent report revealed that CHX blocks movement of the deacylated tRNA toward the E-site within the large subunit at an early step of translocation [13] This enables reinterpretation of our previous data as demonstrating that AdoMet induces translation arrest at the pretranslocation step in CGS1. Primer extension inhibition (toeprint) experiments are used to determine the location of an arrested ribosome on a mRNA, based on the established distance between the P-site of the small subunit and toeprint signal [15, 16], but the location of the stacked ribosomes must be identified by other methods.

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION