Differential inhibitory effects of antibiotics on the biosynthesis of envelope proteins of Escherichia coli

doi:10.1016/0022-2836(73)90012-0

Journal of Molecular Biology

Volume 79, Issue 2, 15 September 1973, Pages 373-389

https://doi.org/10.1016/0022-2836(73)90012-0 Get rights and content

Abstract

Inhibitory effects of six antibiotics (kasugamycin, tetracycline, chloramphenicol, sparsomycin, puromycin and rifampicin) on the biosynthesis of envelope proteins of Escherichia coli were examined and compared with those on the biosynthesis of cytoplasmic proteins. Kasugamycin, puromycin and rifampicin were much more inhibitory to the over-all biosynthesis of cytoplasmic proteins than to that of envelope proteins. On the contrary, tetracycline and sparsomycin showed much stronger inhibitory effects on the biosynthesis of envelope proteins than on that of cytoplasmic proteins. Chloramphenicol showed little difference in its inhibitory effect on the biosynthesis of envelope proteins and cytoplasmic proteins.

The envelope proteins were labeled with [³H]arginine in the presence of the antibiotics and separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The inhibitory effects of the antibiotics on the biosynthesis of individual envelope proteins were then examined. Inhibition patterns were found to be widely different from one envelope protein to the other. For example, the biosynthesis of one major envelope protein of molecular weight 38,000 was more resistant to kasugamycin, chloramphenicol and sparsomycin than that of the other envelope proteins. On the other hand, the biosynthesis of another major envelope protein (lipoprotein) of about 7500 molecular weight was much more resistant to puromycin and rifampicin than that of the other envelope proteins. In the case of tetracycline, little differential inhibitory effect on the biosynthesis of individual envelope proteins was observed.

Stability of messenger RNAs for individual envelope proteins was also determined from the inhibitory effect of rifampicin on their biosynthesis. It was found that the average of half lives of mRNAs for major envelope proteins examined (5.5 minutes) is twice as long as the average of those of mRNAs for cytoplasmic proteins (2 minutes), except for the lipoprotein of about 7500 molecular weight which has extremely stable mRNA with a half life of 11.5 minutes. From these results the envelope proteins of E. coli appear to be biosynthesized in a somewhat different manner from that of the cytoplasmic proteins. Furthermore, at least some envelope proteins may have their own specific biosynthetic systems.

References (44)

D.W. Allen et al.
Biochim. Biophys. Acta
(1962)
A.I. Aronson et al.
Biochim. Biophys. Acta
(1961)
G.W. Both et al.
J. Mol. Biol
(1972)
D.G. Brown et al.
Biochim. Biophys. Acta
(1970)
E. Craig et al.
J. Mol. Biol
(1972)
E. Cundliffe
J. Mol. Biol
(1970)
J. Forchhammer et al.
J. Mol. Biol
(1972)
R. Greene et al.
J. Mol. Biol
(1967)
M. Inouye
J. Biol. Chem
(1971)
M. Inouye et al.
J. Biol. Chem
(1970)

M. Inouye et al.

J. Biol. Chem

(1972)

M. Kozak et al.

J. Mol. Biol

(1972)

C. Lark et al.

J. Mol. Biol

(1964)

A.J. Levine et al.

J. Mol. Biol

(1968)

H.F. Lodish

J. Biol. Chem

(1971)

H.F. Lodish et al.

J. Biol. Chem

(1972)

J. Rouvière et al.

J. Mol. Biol

(1969)

W.C. Summers

J. Mol. Biol

(1970)

M.N. Thang et al.

Biochim. Biophys. Acta

(1963)

V.K. Vambutas et al.

Biochim. Biophys. Acta

(1970)

M.D. Yudkin et al.

J. Mol. Biol

(1965)

H. Berissi et al.

Nature New Biol

(1971)

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^☆: This research was supported by Public Health Service grant GM19043-01 from the National Institute of General Medical Sciences, by American Cancer Society grant BC67, and by the Research Foundation of the State University of New York. One of us (A. H.) was supported by a fellowship (1F03GM53864-01) from the U.S. National Institutes of Health.

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Journal of Molecular Biology

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