A novel cell-free system for peptide synthesis driven by pyridine

Itaru Nitta, Hirohide Nambu, Takaaki Okado, Shigeo Yoshinari, Takuya Ueda, Yaeta Endo, Knud H. Nierhaus, Kimitsuna Watanabe

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

Previously we demonstrated that ribosomes can synthesize polypeptides in the presence of high concentrations (40-60%) of pyridine without any protein factors. Here we analyze additional ribosomal parameters in 60% pyridine using Escherichia coli ribosomes. Ribosomal subunits once exposed to pyridine failed to re-associate to 70S ribosomes in aqueous buffer systems even in the presence of 20 mM Mg2+, whereas they formed 70S complexes in the presence of 60% pyridine. Two-dimensional gel electrophoresis of ribosomal proteins revealed that some proteins located at the protuberances of the large subunit, e.g. L7/L12 and L11 forming the elongation factor-binding domain, were released in the pyridine system. The aminoglycoside neomycin, a strong inhibitor of the ribosomal (factor-independent) translocation reaction, completely blocked poly(Phe) synthesis and translocation activities in the pyridine system, whereas these activities were not affected at all by gypsophilin, a ribotoxin that inhibits factor-dependent translocation. Another inhibitor of the ribosomal translocation, thiostrepton, had no effect concerning the two activites, which is consistent with the fact that this antibiotic requires L11 for its binding to the ribosome. These results suggest that the ribosomes can perform a translocation reaction in the pyridine system, but in a factor-independent (spontaneous) manner.

Original languageEnglish
Pages (from-to)819-829
Number of pages11
JournalBiological Chemistry
Volume379
Issue number7
DOIs
Publication statusPublished - 1998 Jan 1
Externally publishedYes

    Fingerprint

Keywords

  • Aminoglycoside
  • In-vitro translation
  • Non-enzymatic tRNA binding
  • Pre-translocational ribosome
  • Puromycin reaction
  • Ribosome-inactivating protein

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Clinical Biochemistry

Cite this

Nitta, I., Nambu, H., Okado, T., Yoshinari, S., Ueda, T., Endo, Y., Nierhaus, K. H., & Watanabe, K. (1998). A novel cell-free system for peptide synthesis driven by pyridine. Biological Chemistry, 379(7), 819-829. https://doi.org/10.1515/bchm.1998.379.7.819