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 language | English |
|---|---|
| Pages (from-to) | 819-829 |
| Number of pages | 11 |
| Journal | Biological Chemistry |
| Volume | 379 |
| Issue number | 7 |
| DOIs | |
| Publication status | Published - 1998 Jan 1 |
| Externally published | Yes |
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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
A novel cell-free system for peptide synthesis driven by pyridine. / Nitta, Itaru; Nambu, Hirohide; Okado, Takaaki; Yoshinari, Shigeo; Ueda, Takuya; Endo, Yaeta; Nierhaus, Knud H.; Watanabe, Kimitsuna.
In: Biological Chemistry, Vol. 379, No. 7, 01.01.1998, p. 819-829.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - A novel cell-free system for peptide synthesis driven by pyridine
AU - Nitta, Itaru
AU - Nambu, Hirohide
AU - Okado, Takaaki
AU - Yoshinari, Shigeo
AU - Ueda, Takuya
AU - Endo, Yaeta
AU - Nierhaus, Knud H.
AU - Watanabe, Kimitsuna
PY - 1998/1/1
Y1 - 1998/1/1
N2 - 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.
AB - 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.
KW - Aminoglycoside
KW - In-vitro translation
KW - Non-enzymatic tRNA binding
KW - Pre-translocational ribosome
KW - Puromycin reaction
KW - Ribosome-inactivating protein
UR - http://www.scopus.com/inward/record.url?scp=0031849448&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0031849448&partnerID=8YFLogxK
U2 - 10.1515/bchm.1998.379.7.819
DO - 10.1515/bchm.1998.379.7.819
M3 - Article
VL - 379
SP - 819
EP - 829
JO - Biological Chemistry
JF - Biological Chemistry
SN - 1431-6730
IS - 7
ER -