title: 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.
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.
authors.affiliation: From the Division of Life Science, Graduate School of Life Science, Hokkaido University, Sapporo 060-8589, Japan.
authors.forename: Yui
authors.initials: Y
authors.lastname: Yamashita
authors.forename: Yoshitomo
authors.initials: Y
authors.lastname: Kadokura
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Sotta
authors.forename: Toru
authors.initials: T
authors.lastname: Fujiwara
authors.forename: Ichigaku
authors.initials: I
authors.lastname: Takigawa
authors.forename: Akiko
authors.initials: A
authors.lastname: Satake
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Onouchi
authors.forename: Satoshi
authors.initials: S
authors.lastname: Naito
mesh_terms: D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D019757:Carbon-Oxygen Lyases / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010441:Peptide Chain Elongation, Translational; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011691:Puromycin / Q000031:analogs & derivatives / Q000378:metabolism; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D029681:Arabidopsis Proteins; D010455:Peptides; D012333:RNA, Messenger; D018749:RNA, Plant; D012346:RNA, Transfer, Amino Acyl; C031736:peptidyl-puromycin; C019037:tRNA, peptidyl-; D011691:Puromycin; D012436:S-Adenosylmethionine; C022696:O-succinylhomoserine (thiol)-lyase; D019757:Carbon-Oxygen Lyases
keywords: MRNA decay; Ribosome function; Ribosomes; Translation; Translation regulation; peptidyl-tRNA; puromycin reaction; ribosome stacking; translation arrest