AbstractThe effect of a low-Mr factor, partially purified from E. coli B, was investigated in E. coli, reticulocyte, and wheat germ lysate in vitro protein synthesis systems. Equal concentrations of factor were needed to inhibit protein synthesis in the eukaryotic system as compared to the prokaryotic system. Experiments suggested that the factor inhibits the initiation step in the eukaryotic systems

Clark, Virginia L.

Skelly, Susan M.

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A low-Mr factor isolated from Escherichia coli inhibits eukaryotic in vitro protein synthesis 

Volume 162, number 1 FEBS 0831 October 1983 
A low-M, factor isolated from Escherichia coli inhibits 
eukaryotic in vitro protein synthesis 
Susan M. Skelly and Virginia L. Clark* 
Department of Microbiology, Box 672, University of Rochester Medical Center, Rochester, NY 14642, USA 
Received 17 June 1983 
The effect of a low-M, factor, partially purified from E. coli B, was investigated in E. coli, reticulocyte, 
and wheat germ lysate in vitro protein synthesis systems. Equal concentrations of factor were needed to 
inhibit protein synthesis n the eukaryotic system as compared to the prokaryotic system. Experiments 
suggested that the factor inhibits the initiation step in the eukaryotic systems. 
Translation Initiation Escherichia coli Protein synthesis nhibitor 
Protein synthesis nhibition Cell-free system 
1. INTRODUCTION 2. MATERIALS AND METHODS 
The isolation from E. coli K-12 of a low-M, 
substance that inhibits protein synthesis in vitro 
has been reported in [l]. Using an in vitro transla- 
tion system containing ‘S-100’ supernatant frac- 
tion, washed ribosomes from E. coli, R-17 RNA as 
a template and crude initiation factors, it was 
demonstrated that this factor inhibits formation of 
the 70 S initiation complex [2]. Although the 
chemical identity of the factor is not known, we 
have shown that it has MI <500, it is weakly ca- 
tionic, it is highly polar, and it is stable to 
periodate oxidation, alkaline hydrolysis (1 M 
NaOH, lOO”C, 0.5 h), and acid hydrolysis (6 M 
HCl, 1 lO”C, 96 h) [1,2]. 
We describe here the purification procedure for 
the factor and its effect in vitro in wheat germ and 
reticulocyte lysate protein synthesis systems. The 
factor was able to inhibit eukaryotic protein syn- 
thesis completely, most probably at the initiation 
level. 
*To whom correspondence should be addressed 
2.1. Partial purification of factor 
Escherichia coli B cells were grown in 0.4% 
glucose, minimal salts and 1 pg thiamine/ml [l] to 
2 x 10’ cells/ml and harvested by centrifugation. 
The pellet was resuspended in 100th vol. of the 
above media, colicin A at a multiplicity of -10 was 
added, and the culture was incubated at 37°C for 
5 min with vigorous shaking; 0.25 &i 
[‘4C]glucose/ml was then added and incubation 
continued for 35 min. The incubation was stopped 
when trichloroacetic acid was added to 10% final 
cont. After centrifugation the supernatant fluid 
was extracted with ether up to pH >5. The super- 
natant fluid was then concentrated by flash 
evaporation, absolute ethanol was added to 90% 
final cont. and the precipitate was discarded. A se- 
cond ethanol precipitation was done and the super- 
natant fluid was applied to an activated alumina 
column (Matheson, Coleman and Bell type F-20). 
Further details of the purification procedure are 
described under fig.l-4. One unit of factor was 
defined as the amount necessary to inhibit fl- 
galactosidase induction in vivo by 50% in 20 min 
with 1 mg cellular protein as in [l]. 
Published by Elsevier Science Publishers B. V. 
16 00145793/83/%3.00 0 1983 Federation of European Biochemical Societies 
Volume 162, number I FEBS LETTERS October 1983 
2.2. In vitro protein synthesis 3. RESULTS AND DISCUSSION 
The rabbit reticulocyte and wheat germ lysates 
were purchased from Bethesda Research 
Laboratories and 10 pg globin message/ml was us- 
ed in a 0.01 ml reaction volume to direct transla- 
tion. The E. coli cell-free extract was prepared as 
in [3] and 0.5 mg QB RNA/ml was used in a 
0.01 ml reaction volume to direct translation [2]. 
After incubation at 37°C for the indicated times, 
a sample was removed, spotted on Whatman 3MM 
paper and boiled in 10% trichloroacetic acid for 
10 min. The filters were washed once in H20, twice 
in ethanol, once in acetone, dried and 3 ml scin- 
tillation fluid was added to determine the amount 
of ~3sS]methionine incorporated into acid- 
insoluble material. 
The purification procedure for factor described 
here yields material that is homogeneous on 
cation-exchange chromatography (fig. l-4). It is 
not pure, however, as determined by mass spec- 
trometry. This lack of purity probably does not 
contribute artifactual inhibition of protein syn- 
thesis, since the effect of this factor preparation is 
a very specific inhibition of formation and 
destabilization of the E. coli 70 S initiation com- 
plex [2]. 
Since the E. coli factor inhibits prokaryotic in 
vitro protein synthesis, it was of interest to deter- 
mine if it could also inhibit eukaryotic in vitro pro- 
tein synthesis. The ability of various concentra- 
8 
t 
FACTOR 
l ACTIVITY 
ML ELUATE 
Fig, 1. Alumina column chromatography: The supernatant fluid, prepared as in section 2, was applied to a dry cohxmn 
of alumina (2.5 cm x 41 cm). The column was eluted with a non-linear ethanol-Hz0 gradient as indicated. 
17 
Volume 162, number 1 FEBSLETTERS October 1983 
5 
4 
I 
z 3 
“0 
2 
1 
I 
FACTOR 
ACTLVITY 
f 1 
0 
10 20 30 40 
FRACTION NUMBER 
Fig.2 Paper chromatography: The fractions from the 
alumina column containing factor activity were pooled 
and applied to Whatman 3MM paper (1.5 cm x 60 cm). 
After descending chromatography overnight with N- 
butanol : ethanol : water (40: 11: 19, by vol.), the strip 
was dried, cut into i cm fractions, and water was used 
to elute the factor. 
tions of factor to inhibit two different eukaryotic 
systems was compared with its inhibition of the E, 
coli in vitro system (fig.5). As can be seen, essen- 
tially complete inhibition by factor is achieved in 
all three systems. A Tetrahymena thermophiia in
vitro synthesis system using poly(U) as template 
was also completely inhibited (not shown). 
Since the factor inhibits the initiation of transIa- 
tion in prokaryotes 121, attempts were made to 
determine if initiation is also the site of inhibition 
Fig.4. Dowex 50 (H’) column chromatography: The 
pooled factor peak from the silicic acid column was 
dried, dissolved in I&O, and applied to a Dowex 50 (H+) 
column (1.2 cm x 20 cm). The column was eluted with 
a linear gradient of O-4 N NHbOH, the N&OH was 
removed by evaporation, and the residue, dissolved in 
HzO, was used in the protein synthesis inhibition 
studies. 
ETHANOL METHANOL 
FACTOR 
ACTl VITY 
FRACTION NUMBER 
Fig.3. Silicic acid column chromatography: The pooled 
factor peak from the paper chromatography was dried, 
dissolved in absolute ethanol, and applied to a silicic 
acid column (1.2 cm x 20 cm). The column was eluted 
successively by absohtte ethanol and methanol. 
FACTOR 
ACTIVITY 
!--I 
10 20 30 40 30 
FRACTION NUMBER 
18 
Volume 162, number 1 FEBS LETTERS October 1983 
1 J 
1 2 3 4 5 6 
UNITS OF FACTOR 
Fig.5. Inhibition of translation by the factor using the 
S-30 (o), rabbit reticulocyte (0) and wheat germ (A) in 
vitro systems. Concentrations of factor as shown were 
added prior to the lysate and the reaction continued for 
20 min. 
in the eukaryotic systems (table 1). When ihe fac- 
tor was added to any of the 3 systems before the 
initiation of translation, essentially complete in- 
hibition of synthesis was observed. No detectable 
high-Mr proteins were observed by 
SDS-polyacrylamide gel electrophoresis when the 
factor was added before protein synthesis was in- 
itiated (not shown). To differentiate between ef- 
fects on initiation and elongation, the factor was 
added 12 min after initiation. The reaction was 
allowed to proceed for 15 min, and the inhibition 
of protein synthesis during the latter 15 min was 
determined (table 1). In contrast to addition of fac- 
tor at 0 min, the addition of factor at 12 min did 
not completely inhibit further protein synthesis. 
The control experiments adding inhibitors of 
elongation, chloramphenicol and cycloheximide, 
resulted in complete inhibition of protein synthesis 
during this time period. The control experiment 
adding kasugamycin, a specific inhibitor of initia- 
tion in prokaryotes [4,5] gave results similar to 
those obtained with factor. These results suggest 
Table 1 
Inhibition of protein synthesis using eukaryotic and 
prokaryotic in vitro transIationa1 systems 
In vitro system 
E. coli S-30 lysate 
70 Inhibition 
Inhibitora 0 minb 12 minC 
None 0 0 
CAP - 100 
KSG - 52 
Factor 95 63 
Rabbit reticulocyte 
lysate 
None 0 0 
CH _ 100 
Factor 94 67 
Wheat germ lysate None 0 0 
CH - 96 
Factor 99 74 
a Chloramphenicol (CAP), kasugamycin (KSG), 
cycloheximide (CH) and the E. coli factor were added 
to final cont. 2OOpg/ml, 400 gg/ml, 50pM and 600 
units/ml, respectively 
b Inhibitors were added before the addition of the 
designated lysate; the reaction was allowed to continue 
for 20 min 
’ Inhibitors were added to the in vitro assay 12 min after 
the initiation of translation and the reaction was 
allowed to continue for a further 15 min. 
Incorporation of 13’S]methionine into trichloroacetic 
acid-insoluble material that occurred only in the latter 
I5 min interval was determined as in section 2 
that once peptidyl chains have been initiated, the 
factor does not inhibit their completion, and that, 
therefore, the factor acts at the level of initiation 
of protein synthesis in eukaryotes as well as in 
prokaryotes, 
It is extremely intriguing that a low-i% com- 
pound isolated from a prokaryote is capable of af- 
fecting eukaryotic protein synthesis. The results 
presented here suggest hat there is some step, pro- 
bably in initiation, that is common to both pro- 
karyotes and eukaryotes, and that this step is the 
target of action of the E. coli factor. 
ACKNOWLEDGEMENTS 
This study was supported by American Cancer 
Society institutions research grant IN-18Y 234; 
19 
Volume 162, number 1 FEBS LETTERS October 1983 
S.M.S. is a recipient of a National Institutes of 
He&h predoctoral fellowship grant GM-07102. 
131 Wilhelm, J.M. and Haseikorn, R. (1971) Methods 
Enzymol. 20, 527-536. 
1441 Tanaka, N., Nishimura, T., Yamaguchi, Hi., 
REFERENCES 
Y~amoto, C., Yosbida, Y., Sashikata, K. and 
Umezawa. H. (1965) J. Antibiot. 18. 139-144. 
f1j Clark, V.L. (1979) J. Bacterial. 137, 349-349. 
I21 Clark, V.L. (1980) Proc. Natl. Acad. Sci. USA 
77,118L-1184. 
f5l Okuyama; H.,‘Ma&iyama, N., Kinbshita, T, and 
Tanaka, N. (1971) Biochem. Biophys. Res. Com- 
mun. 43, 196-199. 


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A low-Mr factor isolated from Escherichia coli inhibits eukaryotic in vitro protein synthesis

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