Vol.
175,
No.
20
E ec s
o
Ribosome-Inac i a ing
P o eins
on
Esche ichia
coli
and
Ag obac e ium
ume aciens
T ansla ion
Sys ems
TOMAS
GIRBES,'
LUIGI
BARBIERI,2
MIGUEL
FERRERAS,'
F.
JAVIER
ARIAS,'
M.
ANGELES
ROJO,'
ROSARIO
IGLESIAS,'
CARLOS
ALEGRE,'
CRISTINA
ESCARMIS,3
AND
FIORENZO
STIRPE2*
Depa amen o
de
Bioquimica,
Biologia
Molecula
y
Fisiologia,
Facul ad
de
Ciencias,
Uni e sidad
de
Valladolid,
E-47005
Valladolid,'
and
Cen o
de
Biologia
Molecula ,
Mad id,3
Spain,
and
Dipa imen o
di
Pa ologia
Spe imen ale,
Uni e si ai
di
Bologna,
I-40126
Bologna,
I aly2
Recei ed
13
May
1993/Accep ed
9
Augus
1993
The
e ec s
o
30
ype
1
and
o
2
( icin
and
olkensin)
ype
2
ibosome-inac i a ing
p o eins
(RIPs)
on
Esche ichia
coli
and
Ag obac e ium
ume aciens
cell- ee
ansla ion
sys ems
we e
compa ed
wi h
he
e ec s
on
a
abbi
e iculocy e
ansla ion
sys em.
The
depu ina ing
ac i i y
o
RIPs
on
E.
coli
ibosomes
was
also
e alua ed.
Only
six
ype
1
RIPs
inhibi ed
endogenous
mRNA-di ec ed
ansla ional
ac i i y
o
E.
coli
lysa es,
wi h
submic omola
50%
inhibi o y
concen a ions.
Fou
RIPs
had
simila
ac i i ies
on
poly(U)-di ec ed
phenylalanine
polyme iza ion
by
E.
coli
ibosomes,
and
h ee
RIPs
inhibi ed
poly(U)-di ec ed
polyphenylala-
nine
syn hesis
by
A.
ume aciens
ibosomes,
wi h
submic omola
50%1
inhibi o y
concen a ions.
Ribosome-inac i a ing
p o eins
(RIPs)
om
plan s
( e-
iewed
in
e e ence
34),
ei he
single-chain
( ype
1
RIPs)
o
wo-chain
( ype
2
RIPs,
i.e.,
icin
and
ela ed
oxins),
a e
N-glycosidases
which
hyd olyze
he
N-glycosidic
bond
o
ade-
nine
in
a
highly
conse ed
egion
o
RNA
(7).
As
a
esul
o
his
al e a ion,
euka yo ic
ibosomes
become
unable
o
bind
elonga ion
ac o s,
wi h
he
consequen
a es
o
p o ein
syn hesis.
On
he
o he
hand,
p o ein
syn hesis
by
p oka yo ic
ibosomes
is
no
signi ican ly
a ec ed
by
icin
o
o he
RIPs
(4,
5,
12, 18,
25,
29),
and
ully
unc ional
ansgenic
RIPs
could
be
exp essed
in
Esche ichia
coli
(10,
24,
31).
Ne e heless,
a
leas
icin
(8)
and
Mi abilis
jalapa
an i i al
p o ein
(MAP)
(14)
depu ina e
naked
23S
and
16S
E.
coli
RNAs
a
A-2660
and
a
A-1014,
espec i ely,
and
MAP
could
no
be
exp essed
a
high
le els
in
E.
coli
because
o
he
oxici y
o
he
p o ein
o
his
o ganism
(15).
I
was
hen
ound
ha
MAP
inhibi ed
p o ein
syn hesis
by
E.
coli
ibosomes,
al hough
a
concen a ions
some
100- old
highe
han
hose
a ec ing
euka yo ic
ibosomes
(13).
Mo e
ecen ly
i
was
epo ed
ha
o he
ype
1
RIPs,
al hough
a
high
concen a ions,
depu ina e
23S
RNA
o
E.
coli
ibo-
somes
(16,
17,
26).
These
esul s
p omp ed
us
o
s udy
in
a
quan i a i e
manne
he
ac i i ies
o
se e al
RIPs
on
h ee
p oka yo e
ansla ion
sys ems.
Ma e ials.
E.
coli
MRE
600
was
supplied
by
J.
P.
Balles a,
Mad id,
Spain.
Ag obac e ium
ume aciens
was
ob ained
om
he
Depa amen o
de
Mic obiologia,
Uni e si y
o
Valencia,
Valencia,
Spain.
RIPs
we e
pu i ied
om
membe s
o
a ious
plan
amilies
as
ollows:
Ca yophyllaceae,
Dian hus
ca yophyllus
lea es
(di-
an hins),
Lychnis
chalcedonica
seeds
(lychnin),
and
Sapona ia
o icinalis
lea es
(sapo in-L
p o eins),
oo s
(sapo in-R),
and
seeds
(sapo in-S
p o eins);
Cucu bi aceae,
B yonia
dioica
oo s
(b yodin-R),
Ci ullus
colocyn his
seeds
(colocins),
Momo dica
cochinchinensis
seeds
(momo cochin-S),
Momo dica
cha an ia
seeds
(momo din
1),
and
T ichosan hes
ki ilowii
seeds
( i-
choki in);
Eupho biaceae,
Gelonium
mul i lo um
seeds
(gelo-
nin),
Hu a
c epi ans
la ex
(H. c epi ans
RIP
5),
C o on
iglium
seeds
(c o ins),
Ja opha
cu cas
seeds
(cu cins),
Maniho
u ilis-
*
Co esponding
au ho .
sima
seeds
(manu in
1),
and
Ricinus
communis
seeds
( icin
60);
Poaceae,
Ho deum
ulga e
seeds
(ba ley
RIP
1);
Aspa agaceae,
Aspa agus
o icinalis
seeds
(aspa ins);
Passi lo aceae,
Adenia
olkensii
( olkensin);
Phy olaccaceae,
Phy olacca
ame icana
in
i o
cell
cul u es
(PAP-C),
oo s
(PAP-R),
and
seeds
(PAP-S).
Ricin
was
pu i ied
as
desc ibed
by
Nicolson
e
al.
(23),
olkensin
was
pu i ied
as
desc ibed
by
Ba bie i
e
al.
(1),
and
o he
RIPs
we e
pu i ied
essen ially
as
desc ibed
by
Ba bie i
e
al.
(3).
L-["4C]Leucine
(speci ic
ac i i y,
11.4
GBq/mmol)
was
om
Ame sham
In e na ional,
Ame sham,
Uni ed
Kingdom,
and
L-[3H] aline
(speci ic
ac i i y,
1.22
TBq/mmol)
was
om
New
England
Nuclea ,
Chicago,
Ill.
Chlo oace aldehyde
was
p epa ed
as
desc ibed
by
McCann
e
al.
(19).
All
o he
eagen s
we e
o
analy ical
o
molecula
biology
g ade
and,
when
possible,
RNase
ee.
T ansla ion
sys ems.
P o ein
syn hesis
was
de e mined
wi h
a
e iculocy e
lysa e
as
desc ibed
p e iously
(35)
and
wi h
a
cell- ee
sys em
om
E.
coli.
F om
E.
coli
cul u es
in
loga i h-
mic
g ow h
polysomes
we e
isola ed
as
desc ibed
p e iously
(11),
and
high-speed
(100,000
x
g)
supe na an
(S-100)
was
p epa ed
(22)
and
s o ed
in
small
po ions
in
liquid
ni ogen
un il
used.
Reac ion
mix u es
con ained
he
ollowing
in
a
inal
olume
o
50
,ul:
50
mM
T is-HCl
bu e
(pH
7.8),
80
mM
NH4Cl,
10
mM
magnesium
ace a e,
0.05
mM
amino
acid
mix u e
(minus
aline),
1
mM
ATP,
0.02
mM
GTP,
0.02
mM
CTP,
5
mM
phosphoenolpy u a e,
1.5
,ug
o
py u a e
kinase,
1
mM
di hio h ei ol,
81
nM
L-[3H] aline,
5
,ug
o
RNA
mix u e
om
E.
coli,
60
o
S-100,
40.5
,ug
o
E.
coli
ibosomes.
A e
incuba ion
o
15
min
a
37°C,
100
jig
o
bo ine
se um
albumin
and
1
ml
o
10%
(w / ol)
cold
ichlo oace ic
acid
we e
added
and
he
acid-insoluble
adioac i i y
was
e alua ed
as
desc ibed
p e iously
(11).
The
concen a ion
gi ing
50%
inhibi ion
(IC50)
was
calcu-
la ed
by
linea - eg ession
analysis.
De e mina ion
o
he
N-glycosidase
ac i i y.
Adenine
e-
leased
om
RIP- ea ed
E.
coli
ibosomes
was
de e mined
by
high-p essu e
liquid
ch oma og aphy
(20,
37)
as
desc ibed
by
Ba bie i
e
al.
(2).
The
posi ion
o
he
adenine
emo ed
was
de e mined
by
acid
aniline
clea age
o
depu ina ed
RNA.
To
his
pu pose,
E.
coli
o A.
ume aciens
ibosomes
(200
,ug)
we e
incuba ed
wi h
0.6
,ug
o
c o in
2
o
15
min
a
37°C
in
50
,ul
o
6721
JOURNAL
OF
BAC ERIOLOGY,
OC .
1993,
p.
6721-6724
0021-9193/93/206721-04$02.00/0
Copy igh
©
1993,
Ame ican
Socie y
o
Mic obiology
on June 5, 2020 by gues h p://jb.asm.o g/Downloaded om
6722
NOTES
TABLE
1.
Inhibi ion
o
cell- ee
ansla ion'
IC50
(nM)
in
ansla ion
sys em
RIP
e iculocy e
E.
cli
A.
ume aciens,
mRNA
mRNA
Poly(U)
poly(U)
di ec ed
di ec ed di ec ed
di ec ed
C o in
2
0.48
63
80
192
C o in
3
0.20
13
40
NDb
Cu cin
1
0.19
>1,000
550
ND
Dian hin
32
0.12
>1,000
>1,000
991
Hu a
c epi ans
RIP
5
0.17
450
>1,000
>1,000
Momo din
I
0.06
857
190
190
Sapo in-Rl
0.86
927
>
1,000
>
1,000
Sapo in-R2
0.47
423
>
1,000
>
1,000
a
The
ollowing
RIPs
had
IC50S
o
>
1,000
nM
in
all
h ee
sys ems
es ed:
ype
1
RIPs,
aspa in
1,
aspa in
2,
ba ley
RIP
1,
b yodin-R,
colocin
1,
colocin
3,
cu cin
2,
cu cin
3,
dian hin
30,
lychnin,
manu in
1,
momo chochin-S,
momo din
I,
PAP-C,
PAP-R,
PAP-S,
sapo in-Ll,
sapo in-L2,
sapo in-R3,
sapo in-S5,
sa-
po in-S6,
and
ichoki in;
ype
2
RIPs
( educed
wi h
50
mM
di hio h ei ol
o
30
min
a
37°C),
icin
60
and
olkensin.
b
ND,
no
de e mined.
40
mM
T is-HCl
bu e
(pH
7.6)
con aining
60
mM
NH4Cl
and
10
mM
magnesium
ace a e.
The
eac ion
was
a es ed
by
adding
2
,u1
o
0.5
M
EDTA
(pH
8.0)
and
500
,ul
o
50
mM
T is-HCl
bu e
(pH
7.6)
con aining
0.5%
sodium
dodecyl
sul a e.
The
RNA
was
ex ac ed
wi h
phenol
and
p ecipi a ed
wi h
e hanol
(28),
and
6
jig
was
dissol ed
in
10
.l1
o
wa e
and
incuba ed
in
da kness
o
10
min
a
0°C
wi h
1
olume
o
2
M
aniline
(pH
4.5).
The
eac ion
was
a es ed
by
dilu ion
wi h
200
,ul
o
wa e ,
aniline
was
emo ed
by
wo
ex ac ions
wi h
e he ,
and
RNA
was
eco e ed
by
p ecipi a ion
wi h
e hanol.
Elec-
opho esis
o
RNA
was
ca ied
ou
in
5%
ac ylamide
gels
a
21
mA
o
40
min
as
desc ibed
elsewhe e
(27).
Sequencing
o he
23S
RNA
agmen .
The
RNA
ag-
men s
gene a ed
om
he
ac ion
o
c o in
2
on
E.
coli
and
A.
ume aciens
ibosomes
we e
isola ed
by
elec opho esis
in
a
5%
polyac ylamide
gel
as
desc ibed
abo e.
The
RNA
ag-
men s
(app oxima ely
1
,ug
o
RNA)
we e
ex ac ed
by
imme -
sion
o
he
c ushed
gel
pieces
in
a
mix u e
o
1
olume
o
50
mM
T is-HCl
bu e
(pH
7.5)
con aining
100
mM
NaCl
and
5
mM
EDTA
and
1
olume
o
phenol
o
15
h
a
4°C,
and
hen
he
agmen s
we e
p ecipi a ed
wi h
2
olumes
o
e hanol
a
-
90°C
o
2
h.
The
RNA
was
5'
dephospho yla ed
wi h
alkaline
phospha ase
acco ding
o
he
supplie 's
ins uc ions.
A e
inac i a ion
o
he
enzyme
a
65°C
in
he
p esence
o
20
mM
e hylene
glycol-bis(P3-aminoe hyl
e he )-N,N,N',N'- e -
aace ic
acid
(EGTA),
he
RNA
was
dep o einized
wi h
wo
ex ac ions
wi h
phenol
and
5'
phospho yla ed
wi h
polynucle-
o ide
phospho ylase
and
[y-32P]ATP.
The
labelled
RNA
was
ex ac ed
wi h
phenol,
he
uninco po a ed
adioac i i y
was
sepa a ed
by
gel
il a ion
h ough
a
Sephadex
G-50
column,
and
he
5'-labelled
RNA
agmen
was
pu i ied
by
elec o-
pho esis
in
an
8
M
u ea-4%
polyac ylamide
gel.
The
la ges
RNA
agmen
was
elu ed
as
desc ibed
abo e
and
sequenced
by
pa ial
diges ion
wi h
RNases
as
desc ibed
by
Esca mis
e
al.
(9).
E ec s
on
euka yo ic
ansla ion
sys ems.
P e ious
epo s
on
he
e ec s
o
ype
1
RIPs
on
E.
coli
ibosomes
led
o
he
hypo hesis
ha
possibly
mos
ype
1
RIPs
can
modi y
E.
coli
and
p esumably
o he
eubac e ial
ibosomes
(16).
Ou
esul s
indica e
ha
only
some
RIPs
a e
ac i e
on
bac e ial
ibosomes.
The
ac i i ies
o
RIPs
on
E.
coli
ansla ion
sys ems
a ied
g ea ly
om
one
p o ein
o
ano he ,
he
mos
ac i e
being
c o in
2
and
c o in
3
om
C o on
iglium
seeds
(Table
1).
I--
100
a
0.
W
z
50
U)
*_
o
25
P-
'0
_
D
_
o
q
o
L
RIP
added
(nM)
FIG.
1.
E ec s
o
dian hin
32
and
c o in
3
on
E.
coli
ibosomes:
adenine
elease
and
p o ein
syn hesis
inhibi ion.
Adenine
eleased
(solid
lines)
and
p o ein
syn hesis
(b oken
lines)
we e
measu ed
in
he
p esence
o
c o in
3
(X)
and
dian hin
32
(0).
The e
was
no
co ela ion
be ween
he
ac i i ies
o
RIPs
on
E.
coli
ansla ion
sys ems
and
hose
on
a
mammalian
( abbi
e iculocy e)
endogenous
mRNA-di ec ed
ansla ion
sys em,
and
he
e ec s
we e
no
modi ied
by
he
addi ion
o
abbi
e iculocy e
lysa e
supe na an
( esul s
no
shown)
which
could
sensi ize
A emia
salina
ibosomes
o
se e al
RIPs
(32).
I
is
no ewo hy
ha
RIPs
which
inhibi ed
E.
coli
ibosomes
had
IC50s
lowe
han
hose
o
well-known
inhibi o s
o
p o ein
syn hesis
un
as
posi i e
con ols.
Thus,
c o in
3
was
2,000- old
mo e
ac i e,
on
a
mola
basis,
han
usidic
acid;
600- old
mo e
han
e acycline;
and
15,000- old
mo e
han
s ep omycin
in
inhibi ing
poly(U)-di ec ed
phenylalanine
polyme iza ion
by
E.
coli
ibosomes.
The
RNA
N-glycosidase
ac i i ies
o
RIPs
on
E.
coli
ibo-
somes
we e
consis en
wi h
he
inhibi o y
e ec
on
p o ein
syn hesis,
as
indica ed
by
he
a e
o
adenine
elease.
Rep e-
sen a i e
esul s
ob ained
wi h
RIPs
wi h
di e en
ac i i ies
a e
gi en
in
Fig.
1.
In
bo h
cases,
50%
inhibi ion
o
p o ein
syn hesis
was
ob ained
wi h
he
same
concen a ion
o
RIP
which
induced
elease
o
0.5
mol
o
adenine
pe
mol
o
ibosomes.
Sapo in-S6
a
a
1:1
mola
a io
wi h
ibosomes
eleased
3.5
mol
o
adenine
pe
mol
o
ibosomes,
in
his
di e ing
om
dian hin
32
and
c o in
2,
which
eleased
1
mol/mol
o
less.
The
same
phenomenon
was
obse ed
wi h
a
li e
and
Musca
domes ica
ibosomes
(2).
Poly(U)-di ec ed
polyphenylalanine
syn hesis
by
A.
ume aciens
ibosomes
was
inhibi ed
only
by
momo din
1,
c o in
2,
and,
less
e icien ly,
by
dian hin
32,
wi h
submic omola
IC50s
(Table
1).
These
esul s
a e
consis en
wi h
hose
epo ed
by
Ha ley
e
al.
(16);
howe e ,
he
quan i a i e
measu emen s
o
p o ein
syn hesis
and
o
adenine
eleased
showed
ha
RIPs
ac ed
on
E.
coli
ibosomes
a
concen a ions
much
highe
han
hose
e ec i e
on
euka yo ic
ibosomes
(Table
1).
This
di e ence
does
no
seem
o
be
due
o
RNA
i sel
since
(i)
he
a ec ed
egion
is
highly
conse ed
and
(ii)
i
has
been
shown
ha
icin
depu ina es
a
he
same
a e
pu i ied
RNA
om
a
li e
o
om
E.
coli
and
an
a i icial
35-me
(6,
8).
In
all
cases,
he
concen a ions
o
icin
equi ed
o
depu ina e
pu i ied
RNA
a e
much
highe
han
hose
e ec i e
on
a
li e
ibosomes.
J.
BAc- ERIOL.
on June 5, 2020 by gues h p://jb.asm.o g/Downloaded om
NOTES
6723
1
2 3
4
5
1
1
1
1
1
Kb
1.0-.
Aniline
4- agmen s
FIG.
2.
RNA
N-glycosidase
ac i i y
o
c o in
2
on
ibosomes
om
E.
coli
and
A.
ume aciens.
RNA
samples
we e
p epa ed
and
analyzed
by
elec opho esis
as
desc ibed
in
Ma e ials
and
Me hods.
Lanes:
1,
RNA
ma ke s;
2
and
3,
ibosomes
om
A.
ume aciens
incuba ed
wi h
c o in
2;
4
and
5,
ibosomes
om
E.
coli
incuba ed
wi h
c o in
2.
Samples
in
lanes
3
and
5
we e
ea ed
wi h
aniline
as
desc ibed
in
Ma e ials
and
Me hods.
A ows
indica e
he
agmen
spli
by
aniline.
Thus
he
high
ac i i y
on
whole
ibosomes
seems
o
be
due
o
ibosomal
p o eins,
which
ei he
in e ac
wi h
RIPs
in
some
way
o
keep
RNA
in
a
con o ma ion
which
allows
he
RIPs
o
ac .
Consis en
wi h
p e ious
obse a ions
wi h
icin
and
ab in
(25),
he
ype 2
RIPs
icin
and
olkensin
a
he
highes
concen a ion
es ed
(3.3
[IM)
did
no
signi ican ly
a ec
ei he
endogenous
RNA-di ec ed
o
poly(U)-di ec ed
eac ions
by
bo h
E.
coli
and
A.
ume aciens
ibosomes.
The
RNA
o
E.
coli
and
A.
ume aciens
ibosomes
was
ea ed
wi h
c o in
3
o
dian hin
32
and
acid
aniline
o
p omo e
he
clea age
o
he
depu ina ed
RNA
(7).
T ea men
wi h
c o in
2
( aken
as
an
example
o
an
ac i e
RIP)
and
acid
aniline
led
o
he
elease
o
a
agmen
o
app oxima ely
235
nucle-
o ides
(Fig.
2).
The
i s
16
nucleo ides
a
he
5'
end
o
he
agmen s
o
A.
ume aciens
and
E.
coli
we e
de e mined
and
compa ed
wi h
hose
ob ained
om
o he
RIP- ea ed
ibo-
somes
(Table
2).
The
i s
9
nucleo ides
in
he
agmen s
eleased
by
c o in
2
om
bo h
A.
ume aciens
and
E.
coli
RNA
coincided
wi h
a
nucleo ide
sequence
o
he
helix
90
o
domain
VI
o
he
23S
RNA
o
E.
coli.
This
sugges s
ha
he
depu ina ion
si e
was
he
A-2660
o
he
23S
RNA
o
E.
coli
and
ha
he
a ge
o
c o in
2
on
A.
ume aciens
RNA
was
he
equi alen
o
he
a ge
in
E.
coli.
The
5'-GAGGACC
sequence
belongs
o
he
highly
conse ed
loop
ha
in e ac s
wi h
elonga ion
ac o s
G
and
Tu
in
E.
coli
(21).
Mo eo e ,
i
was
shown
ecen ly
ha
he
binding
o
elonga ion
ac o
G
o
E.
coli
ibosomes
as
a
s able
TABLE
2.
Compa ison
o
he
nucleo ide
sequence
a
he
si e
o
depu ina ion
o
RNA
om
di e en
species
by
RIPs
RNA
Nucleo ide
sequencea
Re e ence
A.
ume aciens
agmen
GAGGACC
This
s udy
E.
coli
agmen
GAGGACC
This
s udy
E.
coli
23S
AGUACGAGAGGACC
16
Saccha omyces
ce e isiae
26S
AGUACGAGAGGAAC
33
Ra
li e
28S
AGUACGAGAGGAAC
7
Xenopus
oocy e
28S
AGUACGAAAGGACC
30
a
The
as e isk
indica es
he
depu ina ion
si e.
complex
wi h
usidic
acid
p o ec ed
ibosomes
agains
he
ac ion
o
c o in
2
(17).
Al hough
RIPs
om
he
same
sou ce
can
be
conside ed
o
be
isoenzymes
in
ha
hey
ca alyze
he
same
eac ion,
he
ma ked
di e ences
in
he
ac i i ies
o
a ious
RIPs
on
he
same
E.
coli
sys em
con i m
ha
hese
p o eins
a e
di e en
om
each
o he
(36).
These
di e ences
exis
among
RIPs
om
axonomically
ela ed
plan s
(e.g.,
c o ins
and
gelonin
om
membe s
o
he
amily
Eupho biaceae),
om
he
same
plan
(e.g.,
sapo ins
om
seeds
and
oo s),
and
e en
om
he
same
issue
(e.g.,
dian hins).
The
obse ed
a ia ions
in
hei
e ec s
on
E.
coli
ibosomes
can
be
due
ei he
o
a
so
o
speci ici y
o
he
a ious
RIPs
o
di e en
ibosomes
o
o
he
equi emen
by
ce ain
RIPs
o
some
co ac o (s)
which
may
no
be
p esen
in
he
E.
coli
sys em
used
in
he
expe imen s
and
which
mus
be
di e en
om
hose
p esen
in
abbi
e iculocy e
lysa e
(32).
Why
and
how
RIPs
which
ha e
li le
e ec
on
E.
coli
p o ein
syn hesis
a e
oxic
o
his
o ganism
when
exp essed
in
i
emain
o
be
sol ed.
I
is
possible
ha
(i)
he
in acellula
concen a-
ion
in
mic ocompa men s
builds
up
o
a
le el
su icien
o
al e
E.
coli
ibosomes
(and
indeed
all
RIPs
showed
some
ac i i y
a
concen a ions
abo e
5,000
nM;
esul s
no
shown)
o
(ii)
ha
some
componen (s)
o
his
o ganism
may
po en ia e
he
e ec s
o
RIPs.
The
esea ch
was
suppo ed
by
g an s
om
CICYT
(B1092-0231),
Jun a
de
Cas illa
y
Le6n,
and
Ibe d ola
S.A.
o
T.
Gi bes
and
by
a
uni e si y
g an -in-aid
o
J.
M.
Fe e as
and
R.
Iglesias.
The
esea ch
in
Bologna
was
suppo ed
by
a
con ac
om
he
Consiglio
Nazionale
delle
Riche che,
Rome,
wi hin
he
special
p ojec
"Bio ecnologie
e
Bios umen azione,"
and
by
he
Minis e o
della
Pubblica
Is uzione,
he
Associazione
I aliana
pe
la
Rice ca
sul
Canc o,
and
he
Pallo i's
Legacy
o
Cance
Resea ch.
F.J.A.
and
M.A.R.
hold
ellowships
om
Ibe due o
and
J.M.F.
holds
a
ellowship
om
he
Minis e io
de
Educaci6n
y
Ciencia,
Mad id.
REFERENCES
1.
Ba bie i,
L.,
A.
I.
Falasca,
and
F.
S i pe.
1984.
Volkensin,
he
oxin
o
Adenia
olkensii
(kilyambi i
plan ).
FEBS
Le .
171:277-279.
2.
Ba bie i,
L.,
J.
M.
Fe e as,
A.
Ba aco,
P.
Ricci,
and
F.
S i pe.
1992.
Some
ibosome-inac i a ing
p o eins
depu ina e
ibosomal
RNA
a
mul iple
si es.
Biochem.
J.
286:1-4.
3.
Ba bie i,
L.,
C.
S oppa,
and
A.
Bolognesi.
1987.
La ge
scale
ch oma og aphic
pu i ica ion
o
ibosome-inac i a ing
p o eins.
J.
Ch oma og .
408:235-243.
4.
Camma ano,
P.,
A.
Teichne ,
P.
Londei,
M.
Acca,
B.
Nicolaus,
J.
L.
Sanz,
and
R.
Amils.
1985.
Insensi i i y
o
a chaebac e ial
ibosomes
o
p o ein
syn hesis
inhibi o s.
E olu iona y
implica-
ions.
EMBO
J.
4:811-816.
5.
Elha d ,
D.,
and
A.
Bock.
1982.
An
in
i o
polypep ide
syn hesiz-
ing
sys em
om
me hanogenic
bac e ia:
sensi i i y
o
an ibio ics.
Mol.
Gen.
Gene .
188:128-134.
6.
Endo,
Y.,
Y.
L.
Chan,
A.
Lin,
K.
Tsu ugi,
and
I.
G.
Wool.
1988.
The
cy o oxins
cx-sa cin
and
icin
e ain
hei
speci ici y
when
es ed
on
a
syn he ic
oligo ibonucleo ide
(35-me )
ha
mimics
a
egion
o
28S
ibosomal
ibonucleic
acid.
J.
Biol.
Chem.
263:7917-7920.
7.
Endo,
Y.,
K.
Mi sui,
M.
Mo izuki,
and
K.
Tsu ugi.
1987.
The
mechanism
o
ac ion
o
icin
and
ela ed
oxic
lec ins
on
euka y-
o ic
ibosomes.
The
si e
and
cha ac e is ics
o
he
modi ica ion
in
28S
ibosomal
RNA
caused
by
he
oxins.
J.
Biol.
Chem.
262:5908-
5912.
8.
Endo,
Y.,
and
K.
Tsu ugi.
1988.
The
RNA
N-glycosidase
ac i i y
o
icin
A-chain.
The
cha ac e is ics
o
he
enzyma ic
ac i i y
o
icin
A-chain
wi h
ibosomes
and
wi h
RNA.
J.
Biol.
Chem.
263:8735-
8739.
9.
Esca mis,
C.,
M.
Toja,
M.
Medina,
and
E.
Domingo.
1992.
Modi ica ions
o
he
5'-un ansla ed
egion
o
oo
and
mou h
disease
i us
a e
p olonged
pe sis ence
in
cell
cul u e.
Vi us
Res.
26:113-125.
10.
E ensen,
G.,
A.
Ma hiesen,
and
A.
Sundan.
1991.
Di ec
molecula
VOL.
l175,
1
993
on June 5, 2020 by gues h p://jb.asm.o g/Downloaded om
6724
NOTES
cloning
and
exp ession
o
wo
dis inc
ab in
A-chains.
J.
Biol.
Chem.
266:6848-6852.
11.
Gi bes,
T.,
B.
Cab e ,
and
J.
Modolell.
1979.
P epa a ion
and
assay
o
pu i ied
Esche ichia
coli
polysomes
de oid
o
ee
iboso-
mal
subuni s
and
endogenous
GTPase
ac i i ies.
Me hods
Enzy-
mol.
59:353-362.
12.
G eco,
M.,
L.
Mon ana o,
F.
No ello,
C.
Saccone,
S.
Spe i,
and
F.
S i pe.
1974.
Inhibi ion
o
p o ein
syn hesis
by
icin:
expe imen s
wi h
a
li e
mi ochond ia
and
nuclei
and
wi h
ibosomes
om
Esche ichia
coli.
Biochem.
J.
142:695-697.
13.
Habuka,
N.,
K.
Akiyama,
H.
Tsuge,
M.
Miyano,
T.
Ma sumo o,
and
M.
Noma.
1990.
Exp ession
and
sec e ion
o
Mi abilis
an i i al
p o ein
in
Esche ichia
coli
and
i s
inhibi ion
o
in
i o
euka yo ic
and
p oka yo ic
p o ein
syn hesis.
J.
Biol.
Chem.
265:10988-
10992.
14.
Habuka,
N.,
M.
Miyano,
J.
Ka aoka,
and
M.
Noma.
1991.
Esche-
ichia
coli
ibosome
is
inac i a ed
by
Mi abilis
an i i al
p o ein
which
clea es
he
N-glycosidic
bond
a
A2660
o
23
S
ibosomal
RNA.
J.
Mol.
Biol.
221:737-743.
15.
Habuka,
N.,
Y.
Mu akami,
M.
Noma,
T.
Kudo,
and
K.
Ho ikoshi.
1989.
Amino
acid
sequence
o
Mi abilis
an i i al
p o ein,
o al
syn hesis
o
i s
gene
and
exp ession
in
Esche ichia
coli.
J.
Biol.
Chem.
264:6629-6637.
16.
Ha ley,
M.
R.,
G.
Legname,
R.
Osbo n,
Z.
Chen,
and
J.
M.
Lo d.
1991.
Single-chain
ibosome-inac i a ing
p o eins
om
plan s
de-
pu ina e
Esche ichia
coli
23S
ibosomal
RNA.
FEBS
Le .
290:65-
68.
17.
Iglesias,
R.,
C.
Esca mis,
C.
Aleg e,
J.
M.
Fe e as,
and
T.
Gi bes.
1993.
Fusidic
acid-dependen
ibosomal
complexes
p o ec
Esch-
e ichia
coli
om
he
ac ion
o
ype-I
ibosome-inac i a ing
p o ein
c o in-2.
FEBS
Le .
318:189-192.
18.
Lugnie ,
A. A.
J.,
H.
Kun zel,
and
G.
Di heime .
1976.
Inhibi ion
o
Neu ospo a
c assa
and
yeas
mi ochond ial
p o ein
syn hesis
by
icin,
a
oxic
p o ein
inac i e
on
E.
coli
p o ein
syn hesis.
FEBS
Le .
66:202-205.
19.
McCann,
W.
P.,
L.
M.
Hall,
and
W.
K.
Nonidez.
1983.
P epa a ion,
i a ion,
and
s o age
o
chlo oace aldehyde
o
luo ome ic
de-
e mina ion
o
adenine
and
i s
de i a i es.
Anal.
Chem.
55:1455-
1456.
20.
McCann,
W.
P.,
L.
M.
Hall,
W.
Sile ,
N.
Ba on,
and
R.
J.
Whi ley.
1985.
High-p essu e
liquid
ch oma og aphic
me hods
o
de e -
mining
a abinosyladenine-5'-monophospha e,
a abinosyladenine,
and
a abinosylhypoxan hine
in
plasma
and
u ine.
An imic ob.
Agen s
Chemo he .
28:265-273.
21.
Moazed,
D.,
J.
M.
Robe son,
and
H.
F.
Nolle .
1988.
In e ac ion
o
elonga ion
EF-G
and
EF-Tu
wi h
a
conse ed
loop
in
23S
RNA.
Na u e
(London)
334:362-364.
22.
Modolell,
J.,
and
B.
D.
Da is.
1968.
Rapid
inhibi ion
o
polypep-
ide
chain
ex ension
by
s ep omycin.
P oc.
Na l.
Acad.
Sci.
USA
61:1279-1286.
23.
Nicolson,
G.
L.,
J.
Blaus ein,
and
M.
E zle .
1974.
Cha ac e iza-
ion
o
wo
plan
lec ins
om
Ricinus
communis
and
hei
quan-
i a i e
in e ac ion
wi h
a
mu ine
lymphoma.
Biochemis y
13:196-
204.
24.
O'Ha e,
M.,
L.
M.
Robe s,
P.
E.
Tho pe,
G.
J.
Wa son,
B.
P io ,
and
J.
M.
Lo d.
1987.
Exp ession
o
icin
A
chain
in
Esche ichia
coli.
FEBS
Le .
216:73-78.
25.
Olsnes,
S.,
L.
Heibe g,
and
A.
Phil.
1973.
Inac i a ion
o
euka y-
o ic
ibosomes
by
he
oxic
plan
p o eins
ab in
and
icin.
Mol.
Biol.
Rep.
1:15-20.
26.
P es le,
J.,
E.
Ho ning,
M.
Schon elde ,
and
K.
Mund y.
1992.
Mechanism
and
si e
o
ac ion
o
a
ibosome-inac i a ing
p o ein
ype-1
om
Dian hus
ba ba us
which
inac i a es
Esche ichia
coli
ibosomes.
FEBS
Le .
297:250-252.
27.
Sallus io,
S.,
and
P.
S anley.
1990.
Isola ion
o
Chinese
hams e
o a y
ibosomal
mu an s
di e en ially
esis an
o
icin,
ab in
and
modeccin.
J.
Biol.
Chem.
265:582-588.
28.
Samb ook,
J.,
E.
F.
F i sch,
and
T.
Mania is.
1989.
Molecula
cloning:
a
labo a o y
manual,
2nd
ed.
Cold
Sp ing
Ha bo
Labo-
a o y
P ess,
Cold
Sp ing
Ha bo ,
N.Y.
29.
Sanz,
J.
L.,
S.
Al amu a,
I.
Mazzio i,
R.
Amils,
and
P.
Camma-
ano.
1987.
Unique
an ibio ic
sensi i i y
o
an
in
i o
polypep ide
syn hesis
sys em
om
he
a chaebac e ium
The moplasma
aci-
dophilum.
Phylogene ic
implica ions.
Mol.
Gen.
Gene .
207:385-
394.
30.
Saxena,
S.,
and
E.
Acke man.
1990.
Mic oinjec ed
oligonucleo i-
des
complemen a y
o
he
ox-sa cin
loop
o
28S
RNA
abolish
p o ein
syn hesis
in
Xenopus
oocy es.
J.
Biol.
Chem.
265:3263-
3269.
31.
Shaw,
P.-C.,
M.-H.
Yung,
R.-H.
Zhu,
W.
K.-K.
Ho,
T.-B.
Ng,
and
H.-W.
Yeung.
1991.
Cloning
o
ichosan hin
cDNA
and
i s
exp ession
in
Esche ichia
coli.
Gene
97:267-272.
32.
Spe i,
S.,
M.
B igo i,
M.
Zamboni,
D.
Ca nicelli,
and
L.
Mon-
ana o.
1991.
Requi emen s
o
he
inac i a ion
o
ibosomes
by
gelonin.
Biochem.
J.
277:281-284.
33.
S i pe,
F.,
S.
Bailey,
S.
P.
Mille ,
and
J.
W.
Bodley.
1988.
Modi ica ion
o
ibosomal
RNA
by
ibosome-inac i a ing
p o eins
om
plan s.
Nucleic
Acids
Res.
16:1349-1357.
34.
S i pe,
F.,
L.
Ba bie i,
M.
G.
Ba elli,
M.
So ia,
and
D.
A.
Lappi.
1992.
Ribosome-inac i a ing
p o eins
om
plan s:
p esen
s a us
and
u u e
p ospec s.
BiolTechnology
10:405-412.
35.
S i pe,
F.,
A.
Gaspe i-Campani,
L.
Ba bie i,
A.
Falasca,
A.
Ab-
bondanza,
E.
Lo enzoni,
and
W.
A.
S e ens.
1983.
Ribosome-
inac i a ing
p o eins
om
he
seeds
o
Sapona ia
o icinalis
L.
(soapwo ),
o
Ag os emma
gi hago
L.
(co n
cockle)
and
o
Aspa -
agus
o icinalis
L.
(aspa agus)
and
om
he
la ex
o
Hu a
c epi ans
L.
(sandbox
ee).
Biochem.
J.
216:617-625.
36.
S i pe,
F.,
and
R.
C.
Hughes.
1989.
Speci ici y
o
ibosome-
inac i a ing
p o eins
wi h
RNA
N-glycosidase
ac i i y.
Biochem.
J.
262:1001-1002.
37.
Zamboni,
M.,
M.
B igo i,
F.
Rambelli,
L.
Mon ana o,
and
S.
Spe i.
1989.
High-p essu e-liquid-ch oma og aphic
and
luo i-
me ic
me hods
o
he
de e mina ion
o
adenine
eleased
om
ibosomes
by
icin
and
gelonin.
Biochem.
J.
259:639-643.
J
.
BACT
ERIOL.
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