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Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses
11:19

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

Published on: February 25, 2011

The arginine attenuator peptide interferes with the ribosome peptidyl transferase center

Jiajie Wei1, Cheng Wu, Matthew S Sachs

  • 1Department of Biology, Texas A&M University, College Station, Texas, USA.

Molecular and Cellular Biology
|April 18, 2012
PubMed

Insights

Fungal arginine attenuator peptide (AAP) stalls translation by inhibiting the ribosome's peptidyl transferase center (PTC) in response to arginine. This mechanism ensures proper gene regulation in response to amino acid availability.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Fungal arginine attenuator peptide (AAP) regulates gene expression via upstream open reading frames (uORFs).
  • AAP functions as a nascent peptide within the ribosome, stalling translation in response to arginine (Arg).

Purpose of the Study:

  • To investigate the effect of AAP and Arg on ribosome peptidyl transferase center (PTC) function.
  • To elucidate the mechanism by which AAP inhibits translation.

Main Methods:

  • Analysis of AAP and Arg effects on PTC function in Neurospora crassa and wheat germ extracts.
  • Assay using transfer of nascent AAP to puromycin.
  • Primer extension inhibition (toeprint) assays and reporter synthesis assays.

Main Results:

  • Wild-type AAP inhibited PTC function in the presence of high Arg concentrations, while a non-stalling mutant AAP did not.
  • Optimal stalling occurred with wild-type AAP length, with inhibitory function localized to a four-residue C-terminal window.
  • Arg may interfere with PTC by inhibiting peptidyltransferase activity or restricting A-site accessibility.

Conclusions:

  • Inhibition of PTC function by AAP in response to Arg underlies ribosome stalling at the uORF termination codon.
  • The unusual mode of PTC inhibition by AAP does not require specific amino acids or a specific nascent peptide length.

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