EF-G-independent reactivity of a pre-translocation-state ribosome complex with the aminoacyl tRNA substrate puromycin supports an intermediate (hybrid) state of tRNA binding

Divya Sharma, Daniel R. Southworth, Rachel Green

Research output: Contribution to journalArticlepeer-review

Abstract

Following peptide-bond formation, the mRNA:tRNA complex must be translocated within the ribosomal cavity before the next aminoacyl tRNA can be accommodated in the A site. Previous studies suggested that following peptide-bond formation and prior to EF-G recognition, the tRNAs occupy an intermediate (hybrid) state of binding where the acceptor ends of the tRNAs are shifted to their next sites of occupancy (the E and P sites) on the large ribosomal subunit, but where their anticodon ends (and associated mRNA) remain fixed in their prepeptidyl transferase binding states (the P and A sites) on the small subunit. Here we show that pre-translocation-state ribosomes carrying a dipeptidyl-tRNA substrate efficiently react with the minimal A-site substrate puromycin and that following this reaction, the pre-translocation-state bound deacylated tRNA:mRNA complex remains untranslocated. These data establish that pre-translocation-state ribosomes must sample or reside in an intermediate state of tRNA binding independent of the action of EF-G.

Original languageEnglish (US)
Pages (from-to)102-113
Number of pages12
JournalRNA
Volume10
Issue number1
DOIs
StatePublished - Jan 2004

Keywords

  • Hybrid state
  • Puromycin
  • Ribosome
  • Translocation

ASJC Scopus subject areas

  • Molecular Biology

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