An unusual mechanism for EF-Tu activation during tmRNA-mediated ribosome rescue

Mickey R. Miller, Allen Buskirk

Research output: Contribution to journalArticle

Abstract

In bacteria, ribosomes stalled on truncated mRNAs are rescued by transfer-messenger RNA (tmRNA) and its protein partner SmpB. Acting like tRNA, the aminoacyl-tmRNA/SmpB complex is delivered to the ribosomal A site by EF-Tu and accepts the transfer of the nascent polypeptide. Although SmpB binding within the decoding center is clearly critical for licensing tmRNA entry into the ribosome, it is not known how activation of EF-Tu occurs in the absence of a codon-anticodon interaction. A recent crystal structure revealed that SmpB residue His136 stacks on 16S rRNA nucleotide G530, a critical player in the canonical decoding mechanism. Here we use pre-steady-state kinetic methods to probe the role of this interaction in ribosome rescue. We find that although mutation of His136 does not reduce SmpB's affinity for the ribosomal A-site, it dramatically reduces the rate of GTP hydrolysis by EF-Tu. Surprisingly, the same mutation has little effect on the apparent rate of peptide-bond formation, suggesting that release of EF-Tu from the tmRNA/SmpB complex on the ribosome may occur prior to GTP hydrolysis. Consistent with this idea, we find that peptidyl transfer to tmRNA is relatively insensitive to the antibiotic kirromycin. Taken together, our studies provide a model for the initial stages of ribosomal rescue by tmRNA.

Original languageEnglish (US)
Pages (from-to)228-235
Number of pages8
JournalRNA
Volume20
Issue number2
DOIs
StatePublished - Feb 2014
Externally publishedYes

Fingerprint

Peptide Elongation Factor Tu
Transfer RNA
Ribosomes
Messenger RNA
Guanosine Triphosphate
Hydrolysis
Anticodon
Peptides
Mutation
Licensure
Codon
Nucleotides
Anti-Bacterial Agents
Bacteria

Keywords

  • Decoding
  • EF-Tu
  • Ribosome
  • SmpB
  • TmRNA

ASJC Scopus subject areas

  • Molecular Biology

Cite this

An unusual mechanism for EF-Tu activation during tmRNA-mediated ribosome rescue. / Miller, Mickey R.; Buskirk, Allen.

In: RNA, Vol. 20, No. 2, 02.2014, p. 228-235.

Research output: Contribution to journalArticle

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