P oc.
Na l.
Acad.
Sci.
USA
Vol.
83,
pp.
5904-5908,
Augus
1986
Biochemis y
A ini y
ch oma og aphy
wi h
an
immobilized
RNA
enzyme
(RNase
P/ca aly ic
RNA/ca aly ic
subuni
Ml
RNA/C5
p o ein)
AGUSTIN
VIOQUE
AND
SIDNEY
ALTMAN
Depa men
o
Biology,
Yale
Uni e si y,
New
Ha en,
CT
06520
Communica ed
by
Clemen
L.
Ma ke ,
May
5,
1986
ABSTRACT
Ml
RNA,
he
ca aly ic
subuni
o
Esche ichia
coli
RNase
P,
has
been
co alen ly
linked
a
i s
3'
e minus
o
aga ose
beads.
Unlike
Ml
RNA,
which
is
ac i e
in
solu ion
in
he
absence
o
he
p o ein
componen
(C5)
o
RNase
P,
he
RNA
linked
o
he
beads
is
ac i e
only
in
he
p esence
o
C5
p o ein.
A ini y
ch oma og aphy
o
c ude
ex ac s
o
E.
coli
on
a
column
p epa ed
om
he
beads
o
which
he
RNA
has
been
c osslinked
esul s
in
he
pu i ica ion
o
C5
p o ein
in
a
single
s ep.
The
p o ein
has
been
pu i ied
in
his
manne
om
cells
ha
con ain
a
plasmid,
pINIIIR20,
which
includes
he
gene
ha
codes
o
C5
p o ein.
A
6- old
ampli ica ion
o
he
exp ession
o
C5
p o ein
is
ound
in
hese
cells
a e
induc ion
as
compa ed
o
cells
ha
do
no
ha bo
he
plasmid.
A ini y
ch oma og aphy
has
been
used
o
pu i y
a
a ie y
o
biologically
impo an
molecules
such
as
enzymes,
co ac o s,
and
ho mone
ecep o s
(1,
2).
Immobilized
nucleic
acids
ha e
been
used
o
pu i y
o
iden i y
p o eins,
bo h
ca aly ic
and
nonca aly ic,
wi h
which
hey
in e ac .
Fo
example,
ibo-
somal
p o eins
ha
in e ac
wi h
5S and
5.8S
RNA
ha e
been
iden i ied
by
use
o
columns
made
wi h
hese
RNAs
(3-5).
We
now
epo
he
success ul
p epa a ion
o
a
column
made
wi h
an
immobilized
ca aly ic
RNA,
which
has
been
used
success ully
o
pu i y
he
p o ein
ha ,
oge he
wi h
he
RNA,
makes
up
Esche ichia
coli
RNase
P
in
i o.
RNase
P
is
he
enzyme
esponsible
o
ma u a ion
o
he
5'
e mini
o
RNA
p ecu so
molecules
in
i o
(6).
In
all
o ganisms
in es iga ed
hus
a ,
i
has
been
ound
ha
he
enzyme
is
composed
o
wo
kinds
o
subuni s,
one
o
which
is
RNA
and
one
o
which
is
p o ein,
and
bo h
o
which
a e
essen ial
o
ac i i y
in
i o
(7).
The
RNA
componen
(Ml
RNA)
o
E.
coli
RNase
P
and
o
some
o he
bac e ia
(8,
9)
can,
by
i sel ,
ca y
ou
he
ca aly ic
eac ion
unde
ce ain
condi ions
in
i o.
While
ample
quan i ies
o
hese
RNA
molecules
a e
a ailable,
s udies
o
he
holoenzyme
complex
om
E.
coli
ha e
been
hampe ed
by
he
di icul ies
in
he
pu i ica ion
o
su icien
quan i ies
o
he
pu e
p o ein
com-
ponen
(C5
p o ein)
o
biochemical
expe imen a ion.
The
gene
o
his
p o ein
( npA)
in
E.
coli
has
been
cloned
(10)
bu
does
no
o e exp ess
he
gene
p oduc
o
a
signi ican
deg ee.
We
ha e
inc eased
ou
yield
and
acili a ed
p epa a ion
o
C5
p o ein
by
using
a ini y
ch oma og aphy,
wi h
immobilized
Ml
RNA,
o
cell
ex ac s
p epa ed
om
E.
coli
cells
ha
ha bo
a
no el
subclone
o
he
cis on
and
con ain
he
npA
gene
adjacen
o
an
inducible,
s ong
p omo e .
In
addi ion
o
CS
p o ein
(M
13,700),
we
ha e
iden i ied
a
second
p o ein
(M
27,000)
ha
binds
s ongly
o
Ml
RNA.
METHODS
Bac e ial
S ains
and
Plasmids.
E.
coli
s ain
HB101
(F-,
hsdS20,
ecA13,
a a14,
p oAZ,
lacYI,
galk2,
psL20,
xyl-5,
m l-i,
supE44,
X-)
was
used
as
he
hos
cell
in
he
cloning
expe imen s.
E.
coli
s ain
MRE600
was
used
as
a
sou ce o
RNase
P.
Cells
we e
g own
in
he
ollowing
media
acco ding
o
he
equi ed
condi ions
o
each
expe imen :
LB
medium,
sup-
plemen ed
when
necessa y
wi h
ampicillin
(50
ug/ml);
M9
medium
supplemen ed
wi h
glucose
(2
mg/ml),
leucine
(50
pug/ml),
p oline
(100
gg/ml),
h eonine
(60
,.g/ml),
hiamine
(1
/ig/ml),
and,
when
necessa y,
ampicillin
(25
ug/ml).
Plasmids
pINIIIompAl
[gi
o
M.
Inouye
(11);
a
pBR322
de i a i e
ha
con ains
he
ipp
p omo e
and
he
inducible
lac
p omo e -ope a o
egion
ups eam
om
he
ompA
gene]
and
pFHC1008
[dona ed
by
F.
Hansen
(10)]
we e
used
in
he
cons uc ion
o
he
subclones.
Plasmid
pRR1,
ca ying
he
Ml
RNA
gene,
has
been
desc ibed
(12).
DNA
Manipula ions.
Plasmid
isola ion
and
ou ine
DNA
manipula ions
we e
pe o med
as
desc ibed
elsewhe e
(13).
Res ic ion
enzymes,
Klenow
agmen
o
E.
coli
DNA
polyme ase,
and
T4
DNA
ligase
we e
pu chased
om
New
England
Biolabs.
Cal
in es inal
phospha ase
was
pu chased
om
Boeh inge
Mannheim.
Pu i ica ion
o
Ml
RNA
and
RNase
P.
Ml
RNA
was
pu i ied
om
p epa a i e
polyac ylamide
gels
o
o al
RNA
om
E.
coli
HB101
ha
con ained
he
plasmid
pRR1
as
desc ibed
ea lie
(12).
Pa ially
pu i ied
RNase
P
was
ob ained
om
cellula
ex ac s
o
s ain
MRE600
a e
ch oma og aphy
on
DEAE-
Sephadex
and
p ecipi a ion
wi h
(NH4)2SO4
as
desc ibed
(14).
P epa a ion
o
P o ein
Labeled
wi h
35S.
Cells
we e
g own
o e nigh
in
M9
medium
a
370C,
washed
wice
wi h
M9
medium
om
which
sul a e
ions
we e
omi ed,
and
incuba ed
o
1
h
a
370C
in
he
sul a e- ee
medium.
[35S]Me hionine
(10
,uCi/ml;
1
Ci
=
37
GBq)
was
hen
added,
and
incuba ion
was
con inued
o
6
mo e
h .
Fo
s udies
o
he
induc ion
o
cells
ha
ca y
he
cons uc ed
pINIIIR20
exp ession
plas-
mid,
2
mM
isop opyl
3-D- hiogalac oside
(iP SGal)
was
added
a
he
same
ime
as
he
label.
Immobiliza ion
o
Ml
RNA.
P io
o
immobiliza ion,
an
aliquo
o
Ml
RNA
was
dephospho yla ed
and
labeled
a
he
5'
end
wi h
[y-32P]ATP
and
polynucleo ide
kinase
(P omega
Bio ec,
Madison,
WI)
as
desc ibed
(15).
The
labeled
RNA
(0.8
mg)
was
hen
oxidized
wi h
10
mM
sodium
pe ioda e
(16).
To
examine
he
e iciency
o
oxida ion,
he
abili y
o
he
pe ioda e- ea ed
Ml
RNA
o
ac
as
a
subs a e
in
an
RNA
ligase
condensa ion
wi h
cy idine
3',5'-[5'-32P]bisphospha e
was
es ed
(17).
We
concluded
ha
>95%
o
he
Ml
RNA
was
oxidized.
The
5'-end-labeled
and
oxidized
Ml
RNA
(0.8
mg;
4
x
106
cpm, Ce enko
adia ion)
was
coupled
o
0.5
ml
o
aga ose-
adipic
acid
dihyd azide
(P-L
Biochemicals)
as
desc ibed
(4),
washed
wi h
2
M
NaCl
un il
no
u he
adioac i i y
was
eleased,
and
packed
in
a
Pas eu
pipe e.
Abou
90%
o
he
RNA
was
ac ually
coupled
o
he
gel
as
es ima ed
om
he
adioac i i y
eco e ed
in
he
washes.
Abb e ia ions:
iP SGal,
isop opyl
/-D- hiogalac oside;
bp,
base
pai (s).
5904
The
publica ion
cos s
o
his
a icle
we e
de ayed
in
pa
by
page
cha ge
paymen .
This
a icle
mus
he e o e
be
he eby
ma ked
"ad e isemen "
in
acco dance
wi h
18
U.S.C.
§1734
solely
o
indica e
his
ac .
P oc.
Na l.
Acad.
Sci.
USA
83
(1986)
5905
Subcloning
o
he
npA
Gene
ha
Codes
o
C5
P o ein.
The
exp ession
plasmid
pINIIIR20
was
cons uc ed
o
inc ease
he
p oduc ion
o
C5
p o ein.
The
inducible
exp ession
ec o
pINIIIompA1
(11)
was
cu
a
he
unique
BamHI
si e,
and
he
s agge ed
ends
we e
illed
in
by
polyme iza ion
wi h
he
Klenow
agmen
o
E.
coli
polyme ase
I
and
dephospho yl-
a ed
wi h
cal
in es inal
phospha ase.
Plasmid
pFHC1008
(10),
which
con ains
pa
o
he
pmH
ope on
and
includes
he
comple e
sequence
o
pmH
( he
gene
o
ibosomal
p o ein
L34)
and
npA
( he
gene
o
C5
p o ein),
was
cu
a
he
unique
BssHII
and
A a
I
si es,
and
a
776-base-pai
(bp)
agmen
ha
con ained
he
pmH
and
npA
genes
was
pu i ied.
This
agmen
was
cu
wi h
Rsa
I
o
gi e
ou
agmen s
(538,
142,
67,
and
33
bp).
The
538-bp
agmen
con ained
mos
o
he
pmH
gene
and
he
comple e
npA
gene.
The
s agge ed
ends
we e
illed
wi h
he
Klenow
agmen ,
and
he
mix u e
o
agmen s
was
liga ed
o
he
linea ized
ec o
wi h
T4
DNA
ligase.
T ans o man s
we e
selec ed
by
ampicillin
esis ance,
and
hose
ca ying
he
538-bp
agmen
in
he
igh
o ien a ion
we e
iden i ied
by
es ic ion
analysis.
In
his
cons uc
he
pmH
gene
is
in
phase
wi h
he
coding
sequence
o
he
signal
pep ide
o
he
OmpA
p o ein
(11).
When
selec ion
o
ans o man s
was
ca ied
ou
in
LB
medium,
all
o
he
clones
ha
con ained
he
538-bp
agmen
had
he
agmen
inse ed
in
he
w ong
o ien a ion.
Howe e ,
when
selec ion
was
ca ied
ou
in
M9
medium,
he
igh
cons uc ion
was
ob ained.
This
esul
sugges s
ha
he
exp ession
o
he
ca boxyl- e minal-modi ied
L34
p o ein
does
no
pe mi
iabili y.
Leaky
induc ion
o
he
lac
gene
p omo e
by
chemical
con aminan s
in
LB
medium
(18)
is
a oided
in
minimal
medium.
No e
also
ha
E.
coli
s ain
HB101
ca ying
pINIIIR20
canno
g ow
in
he
p esence
o
2
mM
iP SGal.
Pu i ica ion
o
C5
P o ein.
Ex ac ion
o
RNAs.
The
RNAs
p esen
in
S30
ex ac s
o
in
RNase
P,
pa ially
pu i ied
a
leas
h ough
he
DEAE-Sephadex
s ep
(14),
we e
ex ac ed
wi h
LiCl/EDTA
by
he
me hod
o
Dijk
and
Li lechild
(19)
as
used
o
p epa a ion
o
ac i e
yeas
ibosomal
p o eins
(20).
(We
also
ha e
used
LiCl/EDTA
o
elu e
basic
p o eins
om
immobilized
RNA
as
is
desc ibed
below.)
One
olume
o
ex ac
was
mixed
wi h
2
olumes
o
10.5
M
LiCl,
and
EDTA
(pH
8.0,
adjus ed
wi h
NaOH)
was
added
o
a
concen a ion
o
10
mM.
The
mix
was
gen ly
agi a ed
o e nigh
a
4°C
and
cen i uged
a
100,000
x
g
o
10
h .
The
supe na an ,
con aining
he
p o eins,
was
dialyzed
agains
bu e
A
(50
mM
T is
HCl,
pH
7.4/10
mM
MgCl2/100
mM
NH4Cl/10
mM
2-me cap oe hanol).
A ini y
ch oma og aphy
on
immobilized
MI
RNA.
The
dialyzed
supe na an
om
he
p e ious
s ep
was
loaded
on
he
a ini y
column,
and
he
sample
was
eci cula ed
h ough
he
column
o e nigh
by
a
pe is al ic
pump.
The
column
was
hen
washed
and
elu ed
wi h
bu e
A
ha
con ained
di e en
concen a ions
o
NH4Cl
as
indica ed
in
he
legends
o
Figs.
2
and
3.
C5
p o ein
was
elu ed
in
he
inal
s ep
wi h
7
M
LiCl/10
mM
EDTA,
pH
8.0.
Elec opho esis
o
P o eins.
P o eins
we e
analyzed
on
0.1%
NaDodSO4/4
M
u ea/12.5%
ac ylamide
gels
by
he
me hod
o
Laemmli
(21).
To al
cellula
p o eins
we e
ana-
lyzed
om
a
cell
pelle
( om
0.5
ml
o
cul u e)
ha
was
boiled
o
3
min
in
loading
bu e
and
hen
loaded
di ec ly
on
he
gel.
O he
p o ein
ac ions
we e
p ecipi a ed
wi h
ichlo oace ic
acid
in
he
p esence
o
15
,ug
o
bo ine
se um
albumin,
when
necessa y,
be o e
boiling
in
loading
bu e
and
loading.
P o ein
concen a ions
we e
de e mined
by
he
me hod
o
Low y
e
al.
(22)
wi h
bo ine
se um
albumin
as
a
s anda d.
Assays
o
RNase
P
Ac i i y.
Recons i u ion
assays
we e
pe o med
by
he
di ec
mixing
me hod
(8)
in
50
mM
T is
HCl,
pH
7.4/10
mM
MgCl2/100
mM
NH4Cl.
PEG
(5%
w / ol)
was
added
whe e
indica ed.
Ml
RNA
ac i i y
was
assayed
in
50
mM
T is
HCl,
pH
7.4/100
mM
MgCl2/100
mM
NH4Cl/5%
PEG.
The
p ecu so
o
E.
coli
RNATY
was
used
as
subs a e
(23)
in
all
o
he
expe imen s
epo ed
he e.
All
incuba ions
we e
ca ied
ou
a
37°C
o
he
imes
indica ed.
The
eac ion
p oduc s
we e
analyzed
on
10%
(w / ol)
poly-
ac ylamide
gels
as
desc ibed
elsewhe e
(8),
and
he
amoun
o
p oduc
o med
was
es ima ed
by
scanning
densi ome y
o
au o adiog aphs.
When
immobilized
Ml
RNA
was
assayed,
i s
concen a ion
was
es ima ed
om
he
adioac i i y
o
i s
5'-end
label,
de e mined
by
scin illa ion
coun ing.
RESULTS
P ope ies
o
he
Ml
RNA
Column.
The
3'
e minus
o
Ml
RNA
can
be
al e ed
in
a ious
ways
wi h
li le
o
no
e ec
on
he
ca aly ic
ac i i y
o
he
RNA
(9,
24).
We
e i ied
(da a
no
shown)
ha
Ml
RNA
om
E.
coli,
oxidized
wi h
pe ioda e
o
c ea e
2'
and
3'
ke o
g oups,
has
simila
ac i i y
o
ha
o
in ac
Ml
RNA,
jus
as
Ma sh
and
Pace
(17)
ound
o
he
RNA
componen
o
Bacillus
sub ilis
RNase
P.
The
oxidized
Ml
RNA
was
coupled
h ough
a
dihyd azide
adipic
acid
space
o
aga ose
beads
as
desc ibed,
and
a
slu y
o
he
esul ing
ma e ial
was
es ed
o
RNase
P
ac i i y.
We
chose
he
pa icula
beads
and
space
o
educe
p oblems
o
s e ic
hind ance.
When
he
Ml
RNA-bead
complex
was
assayed
o
ac i i y
(Fig.
1),
he
ca aly ic
ac i i y
was
almos
ully
eco e ed
in
he
econs i u ion
assay
wi h
C5
p o ein
(lanes
5
and
6)
bu
was
almos
comple ely
los
when
es ed
unde
condi ions
whe e
he
ee
RNA
exhibi ed
ac i i y
by
i sel
(lanes
2
and
3).
No
ac i i y
was
obse ed
when
C5
p o ein
o
Ml
RNA
was
assayed
alone
in
bu e
con aining
10
mM
MgCl2.
Fu he mo e,
es ima es
o
ela i e
a es
o
clea age
we e
made
om
mo e
ex ensi e
kine ic
s udies
(da a
no
shown).
When
ee
and
immobilized
Ml
RNA
we e
assayed
in
he
p esence
o
C5
p o ein,
hey
had
abou
he
same
ac i i y.
Bu
abou
5%
o
he
expec ed
ac i i y
o
ee
Ml
RNA
was
eco e ed
when
he
slu y
o
immobilized
RNA
was
assayed
in
he
absence
o
C5
p o ein.
Howe e ,
when
he
slu y
was
analyzed
by
gel
elec opho esis,
abou
5%
o
he
RNA
an
as
ee
Ml
RNA
in
he
gel,
while
he
es
did
no
en e
he
gel.
F om
his
esul ,
we
in e
ha
a
small
amoun
o
Ml
RNA
was
leached
o
he
beads
and
could
accoun
o
he
ca aly ic
ac i i y
de ec ed
in
he
absence
o
p o ein.
Howe e ,
we
canno
ule
ou
he
possibili y
ha
his
small
amoun
o
1
2
3
5
-*
4
5
6
_
-p RNA-
IeRNAS
FIG.
1.
Compa ison
o
he
ac i i y
o
immobilized
Ml
RNA
wi h
ha
o
ee
oxidized
Ml
RNA.
Assays
we e
cam ed
ou
as
desc ibed
in
he
p esence
o
5%
PEG.
p RNA,
RNA,
and
5'
indica e
he
posi ions
o
he
subs a e
RNA
p ecu so
molecule
and
he
wo
clea age
p oduc s,
espec i ely.
Lanes:
1
and
4,
no
addi ion;
2,
10
ng
o
oxidized
Ml
RNA
assayed
o
15
min
in
bu e
ha
con ained
100
mM
MgCl2;
3,
10
ng
o
immobilized
Ml
RNA
assayed
as
in
lane
2;
5,
10
ng
o
oxidized
Ml
RNA
assayed
o
5
min
in
bu e
ha
con ained
10
mM
MgCl2
and
an
excess
o
pu i ied
C5
p o ein;
6,
10
ng
o
immobilized
Ml
RNA
assayed
as
in
lane
5.
Biochemis y:
Vioque
and
Al man
5906
Biochemis y:
Vioque
and
Al man
ac i i y
was,
in
ac ,
due
o
he
immobilized
RNA.
Analysis
o
he
slu y
a e
he
econs i u ion
assay
indica ed
ha
he
p esence
o
he
C5
p o ein
did
no
induce
he
elease
o
RNA
om
he
beads
(da a
no
shown).
The e o e,
he
ca aly ic
ac i i y
de ec ed
in
he
assay
in
he
p esence
o
C5
p o ein
was
due,
in
ac ,
o
he
coupled
RNA-bead
complex.
We
conclude
ha
immobiliza ion
o
he
3'
e minus
o
Ml
RNA
in e e es
wi h
a
c i ical
s ep
in
he
ac i a ion
o
he
enzyme,
in
he
absence
o
he
p o ein
co ac o .
Howe e ,
he
RNA
coupled
o
he
beads
appea s
o
e ain
he
desi ed
speci ici y
o
he
binding
o
C5
p o ein
so
ha
he
column
can
be
used
o
he
success ul
pu i ica ion
o
C5
p o ein.
To
analyze
he
condi ions
ha
allow
he
pu i ica ion
o
C5
p o ein
on
he
a ini y
column,
we
used
a
p o ein
mix u e
p epa ed
om
RNase
P
ha
had been
pa ially
pu i ied
h ough
he
DEAE-Sephadex
s ep
and
(NH4)2SO4
p ecipi a-
ion
as
desc ibed
by
S a k
e
al.
(14).
The
RNAs
p esen
in
his
p epa a ion
we e
ex ac ed
by
ea men
wi h
7
M
LiCl/10
mM
EDTA.
The
esul ing
p o ein
mix u e
was
essen ially
ee
o
RNA
(<1%,
as
de e mined
by
do ing
a
sample
on
aga ose
gel
ha
con ains
e hidium
b omide
and
isualizing
he
ma e ial
wi h
a
UV
ansillumina o ).
No
RNase
P
ac i i y
was
mani es ed
by
his
ma e ial
unless
exogenous
Ml
RNA
was
added.
The
C5
p o ein
in
he
p epa a ion
did
no
bind
o
DEAE-Sephadex
(da a
no
shown)
in
con as
o
he
holoenzyme,
which
does
bind
as
a
consequence
o
i s
RNA
con en .
These
esul s
con i med
ha
he
LiCI/EDTA
ex ac
was
ee
o
Ml
RNA,
and
he
C5
p o ein
con ained
in
i
was
s ill
ac i e
in
he
RNase
P
econs i u ion
assay.
Al hough
p o ein
ex ac s
o
B.
sub ilis
RNase
P
ha
ga e
RNase
P
ac i i y
in
he
econs i u ion
assay
ha e
been
made
using
ace ic
acid
o
emo e
RNA
(25),
his
me hod
has
no
yielded
p epa a ions
o
C5
p o ein
om
E.
coli
RNase
P
ha
a e
ac i e
in
econs i u ion
assays.
A
dialyzed
ex ac
o
RNase
P
om
which
he
RNA
had
been
emo ed
by
ea men
wi h
LiCl/EDTA
was
loaded
on
he
a ini y
column,
and
he
p o eins
ha
we e
elu ed
om
he
immobilized
RNA
a e
shown
in
Fig.
2.
The
posi ion
o
C5
p o ein
in
he
PAGE
analysis
o
he
elua es
is
shown
by
he
a ow
adjacen
o
lane
16.
(No e
ha
C5
p o ein
has
an
anomolous
elec opho e ic
mobili y.
I
mig a es
mo e
slowly
han
expec ed
om
i s
molecula
weigh .)
Op imized
condi-
ions
o
he
binding
o
his
p o ein
o
he
column
include
ecycling
o
he
load
sample
and
he
use
o
PEG
in
he
loading
bu e .
PEG
inc eases
binding
e iciency
by
enhancing
he
e ec i e
local
concen a ion
o
he
p o ein.
The
p o ein
composi ion
o
he
load
sample
and
he
low- h ough
ac ion
a e
shown
in
Fig.
2,
lanes
2
and
3.
The e
is
some
Msmea ing
o
hese
samples
because
o
he
p esence
o
PEG
in
he
bu e .
P o eins
in
subsequen
elua es
om
he
column
a e
shown
in
lanes
5-15.
Many
p o eins
we e
bound
o
he
column,
bu
mos
we e
elu ed
wi h
bu e
ha
con ained
0.37
M
NH4Cl
(lanes
5-8).
A
p o ein
o
appa en
M
27,000
bound
s ongly
o
he
column
bu
could
be
sepa a ed
om
CS
p o ein
by
elu ion
wi h
bu e
ha
con ained
0.7
M
NH4Cl
(lanes
11-13).
A e
his
elu ion,
only
C5
p o ein
emained
bound
o
he
column,
bu
i
could
be
elu ed
along
wi h
almos
all
ma e ial
ha
is
ac i e
in
he
RNase
P
econs i u ion
assay
by
7
M
LiCl/10
mM
EDTA
(lanes
14
and
15).
This
inal
elu ion
s ep
has
been
adap ed
success ully
om
he
me hod
used
o
sepa a e
ibosomal
p o eins
om
RNA
(19).
Each
ac ion
was
assayed
o
RNase
P
ac i i y
in
he
absence
and
p esence
o
Ml
RNA
in
bu e
ha
con ained
10
mM
MgCl2.
The
only
column
ac ion
exhibi ing
signi ican
ac i i y
in
he
p esence
o
added
Ml
RNA
was
he
one
co esponding
o
ha
seen
in
lane
14
o
Fig.
2
(LiCI/EDTA
wash).
The
p o ein
species
elu ed
om
he
column
ha
has
been
labeled
as
"C5
p o ein"
comig a es
wi h
ac i e
C5
p o ein
pu i ied
by
a
di e en
(and
less
e icien )
echnique
(8).
This
1
2 3
4
5
6
7
8 9
10
11
12
13
14
,5
16
i.
I
i
'a
..
.,
Al_
Fi2a
',.................
4.
4
9
FIG.
2.
Elec opho e ic
analysis
o
he
ac ions
elu ed
om
he
a ini y
column.
P o ein
ex ac ed
wi h
LiC1/EDTA
om
pa ially
pu i ied
RNase
P
(see
ex )
was
placed
in
bu e
A
ha
con ained
5%
PEG,
and
12
ml
(6
mg)
was
loaded
on
a
0.5-ml
column
con aining
700
,4g
o
bound
Ml
RNA.
The
sample
(lane
2)
was
eci cula ed
on
he
column
o
15
h
a
a
low
a e
o
6
ml/h .
The
low- h ough
ac ion
was
collec ed
(lane
3),
and
he
column
was
washed
in
succession
wi h
15
ml
o
bu e
A
ha
con ained
5%
PEG
(no
shown),
2
ml
o
bu e
A
(lane
4),
h ee
imes
wi h
0.6
ml
o
0.37
M
NH4Cl
in
bu e
A
(lanes
5-7),
h ee
imes
wi h
0.6
ml
o
0.5
M
NH4Cl
in
bu e
A
(lanes
8-10),
h ee
imes
wi h
0.6
ml
o
0.7
M
NH4Cl
in
bu e
A
(lanes
11-13),
and
wice
wi h
0.4
ml
o
7
M
LiCl/10
mM
EDTA
(lanes
14-15).
Lane
1
con ains
myoglobin
(M
17,200)
and
a
smalle
con aminan .
Lane
16
con ains
he
ollowing
molecula
weigh
ma ke s:
phospho ylase
B
(M
92,500),
bo ine
se um
albumin
(M
66,200),
o albumin
(M
45,000),
ca bonic
anhyd ase
(M
31,000),
soybean
ypsin
inhibi o
(M
21,500),
and
lysozyme
(M
14,400).
The
uppe mos
dense
band
in
lanes
2-15
is
he
bo ine
se um
albumin
used
as
ca ie
in
he
p epa a ion
o
he
sample.
Lanes
2
and
3
con ained
one- i h
as
much
sample
as
lanes
4-15.
The
a ow
nex
o
lane
16
indica es
he
posi ion
o
C5
p o ein.
The
gel
was
s ained
wi h
Coomassie
b illian
blue.
species,
elu ed
in
he
inal
s ep
(Fig.
2,
lane
14),
was
ac i e
in
he
econs i u ion
assay
o
RNase
P
ac i i y,
and
so
we
concluded
ha
i
is,
indeed,
C5
p o ein.
Howe e ,
a
species
wi h
simila
mobili y
in
he
gels,
was
elu ed
a
many
o
he
lowe
concen a ions
o
NH4Cl
bu
did
no
mani es
ac i i y
in
he
econs i u ion
assay
wi h
Ml
RNA.
The e
we e
many
basic
p o eins
in
addi ion
o
CS
p o ein
in
hese
elua es
ha
could
compe e
o
binding
si es
on
Ml
RNA.
I
is
also
possible
ha
he
C5
p o ein
in
hese
ac ions
was
i e e sibly
dena u ed.
We
ha e
no
eco e ed
ac i e
CS
p o ein
om
he
p o ein
elu ed
a
a
lowe
concen a ion
o
NH4Cl,
by
dialysis
agains
bu e
A,
o
by
ea men
o
hese
ac ions
wi h
LiCl/EDTA
p io
o
dialysis.
When
ac ions
ha
we e
elu ed
a
0.37
M
NH4Cl
we e
dialyzed
and
ecycled
h ough
he
column,
some
o
he
M
27,000
p o ein
and
he
appa en
C5
p o ein
we e
elu ed
a
highe
sal
concen a ions,
hough
mos
o
he
ma e ial
s ill
was
elu ed
a
0.37
M.
Vi ually
no
ac i e
CS
p o ein
was
eco e ed.
These
da a
indica e
ha
he
appa en
C5
p o ein
ha
was
elu ed
a
he
lowe
sal
con-
cen a ions
may
indeed
be
dena u ed
o
may
be
inhibi ed
in
he
econs i u ion
issay
by
he
concen a ions
o
o he
basic
p o eins,
including
he
M
27,000
p o ein,
which
a e
ela i ely
high
compa ed
o
hose
ound
in
c ude
ex ac s.
Twen y
g ams
o
E.
coli
yielded
abou
5.4
mg
o
LiCl
ex ac
and,
inally,
abou
5
,g
o
abou
15%
o
he
o al
C5
ac i e
p o ein
in
he
o iginal
7
M
LiCI/10
mM
EDTA
ex ac .
P oc.
Na l.
Acad
Sci.
USA
83
(1986)
P oc.
Na l.
Acad.
Sci.
USA
83
(1986)
5907
The
eco e y
o
ac i e
C5
p o ein
wi h
his
me hod
was
a
leas
h ee
imes
be e
han
ha
ob ained
by
p epa a i e
elec opho esis
o
he
same
amoun
o
s a ing
ma e ial
(14),
and
he
pu i ica ion
was
achie ed
in
less
han
hal
he
ime
needed
o
he
la e
me hod.
I
is
di icul
o
es ima e
accu a ely
he
ex en
o
eco e y
o
C5
p o ein
because
econs i u ed
RNase
P
ac i i y
depended
on
he
concen a-
ion
o
added
C5
p o ein
in
a
nonlinea
ashion
(unpublished
da a),
and
pu e
C5
p o ein
was
inac i a ed
on
dilu ion.
The
la e
p oblem
could
be
o e come,
in
pa ,
by
inclusion
o
PEG
in
he
dilu ion
bu e .
Thus,
ou
es ima es
o
o al
eco e ed
ac i i y
may
be
accu a e
only
wi hin
a
ac o
o
2.
I
one
assumes
ha
a
leas
one
C5
p o ein
molecule
can
bind
o
one
Ml
RNA
molecule,
he
capaci y
o
he
Ml
RNA
column
is
0.11
,ug
o
C5
p o ein
pe
Ag
o
Ml
RNA
a ailable
o
binding.
In
he
case
we
desc ibe,
he
heo e ical
capaci y
is
77
Ag
o
C5
p o ein.
The
only
limi a ion
in
scaling
up
his
p ocedu e
is
he
a ailabili y
o
Ml
RNA,
and
ha
is
no
longe
a
se ious
obs acle.
Ampli ica ion
o
C5
P o ein
in
Vi o.
To
enhance
he
yield
o
C5
p o ein
om
c ude
ex ac s
o
cells,
we
subcloned,
nex
o
a
s ong,
inducible
p omo e
in
pINIIlompAl,
he
gene
ha
codes
o
C5
p o ein
( npA)
and
he
adjacen
gene
ha
codes
o
he
ibosomal
p o ein
L34
( pmH)
om
pFHC1008.
C ude
ex ac s
we e
made
om
cells
ha
ha bo ed
he
new
ec o ,
pINIIIR20,
and om
he
pa en
cells
wi h
no
ec o .
These
ex ac s
we e
bo h
subjec ed
o
elec opho esis
o
analysis
o
he
p o ein
con en
and
also
we e
passed
o e
he
Ml
RNA
column.
The
p oduc ion
o
p o eins
in
cells
wi h
pINIIIR20,
a e
induc ion
wi h
iP SGal,
is
shown
in
Fig.
3,
lane
2.
Lane
1
shows
p o eins
om
an
ex ac
o
uninduced
cells.
No e
he
massi e
inc ease
in
he
p oduc ion
o
a
p o ein
ha
co e-
sponds
in
mobili y
o
L34,
he
gene
ha
is
adjacen
o
and
ups eam
om
npA,
he
gene
ha
codes
o
C5
p o ein.
The
uppe
a ow
in
lane
2
poin s
o
a
ain
mino
band,
unseen
in
1
23
A-I
0-
4
5
6
7
8
91011121314
45
31
-
21.5
-1
i.:
14.4
.-
$
FIG.
3.
Au o adiog am
o
he
elec opho e ic
analysis
o
p o eins
o
[5S]me hionine-labeled
E.
coli
s ain
HB101
ha
ca ies
pINIIIR20.
Lanes:
1,
uninduced
cells;
2,
cells
induced
wi h
2
mM
iP SGal;
3,
S30
ex ac
p epa ed
om
induced
cells;
4-14,
elu ion
pa e ns
om
he
immobilized
MI
RNA
column
o
he
S30
ex ac
shown
in
lane
3
a e
ex ac ion
o
RNAs
wi h
7
M
LiCl/10
mM
EDTA
(elu ion
was
done
as
in
Fig.
2,
bu
he
0.7
M
NH4Cl
washes
we e
omi ed);
4,
low- h ough
ac ion;
5,
bu e
A
con aining
5%
PEG
wash;
6,
bu e
A
wash;
7-9,
0.37
M
NH4Cl
washes;
10-12,
0.5
M
NH4Cl
washes;
13-14,7
M
LiCl/10
mM
EDTA
washes.
Gels
we e
d ied
and
exposed
o
24
h
(lanes
1-2),
1
wk
(lane
3),
o
4
wk
(lanes
4-14).
The
a ows
nex
o
lane
2
indica e
he
posi ions
o
he
L34
usion
p o ein
(lowe
a ow)
and
C5
p o ein
(uppe
a ow).
The
ha ch
ma ks
nex
o
lanes
2,
3,
and
4
show
he
posi ions
o
he
molecula
weigh
(X
10-3)
ma ke s
be ween
lanes
3
and
4.
The
a ow
nex
o
lane
14
indica es
he
posi ion
o
he
C5
p o ein.
No e
ha
ibosomal
p o ein
L34
is
no
p esen
in
he
p epa a ion
o
he
S30
ex ac
(lane
3)
om
he
o al
cell
p o ein
(lane
2).
he
uninduced
cells,
which
has
he
mobili y
o
C5
p o ein.
This
analysis
did
no
allow
us
o
es ima e
he
amoun
o
ampli ica ion
o
p oduc ion
o
C5
p o ein.
The e o e,
we
ea ed
he
c ude
ex ac
o
pINIIIR20
wi h
7
M
LiCl/10
mM
EDTA
o
emo e
RNA
and
used
he
esul ing
p o ein
in
econs i u ion
assays
o
RNase
P
ac i i y.
The
esul an
ac i i y
o
RNase
P
om
he
c ude
ex ac s
o
E.
coli
s ain
HB101
ha bo ing
plasmid
pINIIIR20
induced
wi h
iP SGal
is
abou
6- old
highe
han
he
ac i i y
in
he
ex ac
om
uninduced
cells.
We
p esumed,
he e o e,
ha
he
p oduc-
ion
o
C5
p o ein
in
he
induced
cells
is
a
leas
6- old
g ea e
han
in
uninduced
cells,
and
his
p esump ion
was
e i ied
by
expe imen s
wi h
adioac i ely
labeled
ex ac s.
When
an
S30
cell
ex ac ,
om
which
RNA
had
been
emo ed
wi h
7
M
LiCl/10
mM
EDTA
(Fig.
3,
lane
3),
o
induced
cells
ha bo ing
pINIIIR20
was
passed
o e
he
immobilized
Ml
RNA
column,
>80%
o
he
p o ein
ound
in
he
column
ac ion
elu ed
wi h
7
M
LiCl/10
mM
EDTA
(Fig.
3,
lane
13)
was
C5
p o ein,
as
judged
by
au o adiog aphy
o
ma e ial
labeled
wi h
35S.
The
yield
o
C5
p o ein
in
his
ac ion
is
4-
o
6- old
g ea e
han
ha
ound
in
ex ac s
o
uninduced
cells
as
de e mined
by
compa isons
o
he
eco -
e y
o
35S-labeled
C5
p o ein.
The
M
27,000
p o ein
was
no
appa en
in
he
ma e ial
elu ed
wi h
7
M
LiCl
om
he
column
ha
was
de i ed
om
he
induced
cells.
Ou
esul s
demon-
s a e
ha
i
is
possible
o
pu i y
a
e y
mino
p o ein
om
E.
coli
(abou
250
copies
pe
cell)
in
one
s ep
on
he
immobilized
Ml
RNA
column.
DISCUSSION
A ini y
ch oma og aphy
o
p o eins
on
immobilized
RNAs
has
been
used
as
an
analy ical
ool
o
de e mine
which
p o eins
in e ac
in
a
speci ic
manne
wi h
di e en
RNAs
(3-5).
We
ha e
used
his
me hod,
no
only
o
pu i y
p o eins
ha
bind
s ongly
o
Ml
RNA,
a
ca aly ic
mac omolecule,
bu
also
o
p epa e
hese
p o eins
in
amoun s
use ul
o
biochemical
expe imen a ion.
In
ac ,
we
ha e
been
able
o
pu i y
C5,
he
p o ein
componen
o
RNase
P,
in
a
single
ch oma og aphic
s ep
om
a
c ude
ex ac
o
E.
coli.
We
also
ha e
ound
ano he
p o ein
o
M
27,000
ha
binds
s ongly
o
Ml
RNA.
In
addi ion,
he
me hod
we
ha e
de eloped
allows
p o ein
pu i ica ion
o
be
achie ed
wi hou
he
use
o
dena u ing
agen s
such
as
NaDodSO4,
u ea,
o
ace ic
acid
o
o
lyophiliza ion.
Al hough
he
high
concen a ions
o
sal s
we
used
o
sepa a e
RNA
om
p o ein
in
he
ini ial
c ude
ex ac
and
in
he
column
elu ion
bu e s
in
gene al
will
a ec
he
e ia y
s uc u e
o
p o eins,
he
eco e ed
p o ein
is
in
a
"na i e"
s a e
(see
e .
19).
The
LiCl/EDTA
me hod
has
been
used
o
ex ac
yeas
ibosomal
p o eins
ha
a e
subsequen ly
able
o
econs i u e
unc ional
60S
yeas
ibo-
somal
subuni s
(20).
The
C5
p o ein
we
pu i ied
is
ac i e
in
he
econs i u ion
assay
o
RNase
P
ac i i y
and,
hus,
is
highly
sui able
o
u he
s udies
o
he
mechanism
o
ac ion
o
RNase
P.
A
p esen
we
do
no
know
i
he
M
27,000
p o ein
is
ela ed
o
he
unc ion
o
Ml
RNA
o
he
p ocessing
o
he
p ecu so
o
Ml
RNA
in
i o.
Al hough
he
size
o
he
M
27,000
p o ein
may
be
compa ible
wi h
ha
o
a
dime
o
C5
p o ein,
i
is
no
likely
ha
such
a
dime
su i es
boiling
in
NaDodSO4
o
elec opho esis
h ough
NaDodSO4
and
u ea,
bo h
p ocedu es
being
pa
o
ou
p o ocols.
In
addi ion,
he
M
27,000
p o ein
is
no
seen
in
he
inal
column
ac ions
o
35S-labeled
ex ac s
o
HB101
cells
ha
do
o
do
no
ha bo
pINIIIR20,
al hough
la ge
amoun s
o
C5
p o ein
a e
ob-
se ed.
This
obse a ion
indica es
ha
he
M
27,000
p o ein
lacks
me hionine
esidues
and,
he e o e,
is
un ela ed
o
C5
p o ein.
The
elu ion
om
he
a ini y
column
o
C5
p o ein
o e
a
wide
ange
o
sal
concen a ions
is
no
unexpec ed
because
Biochemis y:
Vioque
and
Al man
5908
Biochemis y:
Vioque
and
Al man
we
know
ha
Ml
RNA
can
exis
in
a
a ie y
o
con o ma-
ions,
some
o
which
may
p o ide
highe
speci ici y
o
binding
o
C5
p o ein
(and
M
27,000
p o ein)
han
o he s.
Howe e ,
he
amoun
o
ac i e
C5
p o ein
eco e ed
in
hese
ac ions
is
no
well
co ela ed
wi h
he
appa en
amoun
o
p o ein,
mos
o
he
ac i i y
being
eco e ed
in
he
inal
wash
wi h
7
M
LiCl/10
mM
EDTA.
A
his
poin
we
canno
say
o
ce ain
whe he
he
species
ha
comig a es
wi h
C5
p o ein
is
inac i e
o
modi ied
C5
p o ein,
which
binds
less
s ongly
o
Ml
RNA
han
does
na i e
C5
p o ein,
no
do
we
know
whe he
i s
ac i i y
is
simply
inhibi ed
by
he
p esence
o
basic
p o eins
ha
copu i y
wi h
i .
The
amoun
o
C5
p o ein
in
cellula
ex ac s
has
been
inc eased
by
subcloning
a
cis on
ha
con ains
he
gene
coding
o
his
p o ein
( npA)
om
he
plasmid
pFHC1008
o
a
si e
adjacen
o
an
inducible,
s ong
p omo e
in
he
plasmid
pINIIIompAl.
No mally,
npA
is
co ansc ibed
wi h
he
gene
o
ibosomal
p o ein
L34
( pmH)
and
wo
o he
p o eins
(10),
bu
no
signi ican
o e p oduc ion
o
he
npA
gene
p oduc
is
obse ed
in
cells
ha bo ing
pFHC1008,
al hough
L34
is
ampli ied
(10).
When
hese
wo
genes
a e
placed
adjacen
o
he
lpp
and
lac
p omo e s
in
pINIIIR20,
an
=6- old
ampli ica ion
o
C5
p o ein
is
obse ed,
as
judged
by
he
amoun
o
C5
eco e ed
a e
a ini y
ch oma og aphy.
Thus,
a
combina ion
o
he
no el
column
echnique
and
gene ic
enginee ing
has
led
o
a
signi ican
imp o emen
in
he
yield
and
e iciency
o
pu i ica ion
o
C5
p o ein.
In
cons uc ing
he
immobilized
RNA
column,
we
ook
ad an age
o
he
ac
ha
he
3'
e minus
o
Ml
RNA
can
be
al e ed
in
a ious
ways
wi hou
signi ican ly
diminishing
he
unc ion
o
he
molecule
(24).
Fu he mo e,
he
leng h
o
he
b idge
o
he
aga ose
beads
was
chosen
o
minimize
s e ic
p oblems
and,
hus,
we
an icipa ed
ha
he
immobilized
RNA
would
s ill
e ain
i s
ca aly ic
unc ion.
Howe e ,
ca aly ic
ac i i y
a
he
same
a e
as
shown
by
he
equi alen
amoun
o
ee
RNA
is
de ec ed
only
in
he
p esence
o
he
p o ein
co ac o .
This
esul
p omp s
he
specula ion
ha ,
o
he
RNA
o
unc ion
by
i sel ,
i
mus
ha e
some
deg ee
o
con o ma ional
lexibili y
a
i s
3'
e minus
ha
is
absen
when
i
is
co alen ly
linked
o
he
beads.
Con o ma ional
change,
such
as
dime iza ion
(26),
as
a
p elude
o
ca aly ic
unc ion
may
be
slowed
o
p e en ed
when
Ml
RNA
is
linked
o
he
beads.
Indeed,
some
immobilized
p o ein
enzymes
ha e
been
epo ed
o
unde go
con o ma ional
changes
10_-105
imes
mo e
slowly
han
do
he
ee
p o eins
(27).
Fo
he
immobilized
Ml
RNA
o
become
ac i e,
h ough
wha -
e e
con o ma ional
change
may
be
needed,
C5
p o ein
mus
be
p esen
as
an
essen ial
co ac o .
The
equi emen
o
a
p o ein
co ac o
in
his
eac ion
bea s
on
specula ion
ega d-
ing
ea ly
biochemical sys ems.
I
he
i s
enzymes
we e
composed
o
RNA
and
had
p ope ies
simila
o
hose
o
Ml
RNA,
immobiliza ion
on
he
in e io
o
p imi i e
cell
mem-
b anes
o
in
simple
o ganelles
would
ha e
c ea ed
immedia e
selec i e
p essu e
o
he
pa icipa ion
o
p o eins
in
enzy-
ma ic
eac ions.
The
p epa a ion
o
enzyma ically
ac i e
immobilized
RNA
may
also
be
o
p ac ical
signi icance
since
ca aly ically
ac i e
immobilized
p o eins
ha e
ound
wide-
sp ead
applica ions
(28).
Finally,
we
no e
ha
ac i e
RNase
P
can
be
econs i u ed
om
Ml
RNA
and
he
app op ia e
p o ein
componen s
om
HeLa
cells
(29)
and
B.
sub ilis
(8).
These
obse a ions
aise
he
possibili y
ha
he
a ini y-ch oma og aphy
p ocedu e
we
desc ibe
he e
may
be
use ul
in
pu i ying
he
p o ein
componen s
o
RNase
P
om
a
a ie y
o
o ganisms.
The
p ocedu e
also
may
be
adap ed
o
he
pu i ica ion
o
p o ein
co ac o s
ha
s imula e
o he
eac ions
known
o
be
ca alyzed
by
RNA
(30)
o
in
which
ibonucleop o eins
a e
in ima ely
in ol ed
(31).
We
hank
Donna
Wesolowski
o
pu i ying
Ml
RNA
and
ou
colleagues
o
aluable
ad ice
and
gi s
o
ma e ials.
A.V.
is
he
ecipien
o
a
long- e m
ellowship
om
he
Eu opean
Molecula
Biology
O ganiza ion.
This
wo k
was
suppo ed
by
g an s
om
he
Uni ed
S a es
Public
Heal h
Se ice
and
he
Na ional
Science
Founda ion
o
S.A.
1.
Le man,
L.
S.
(1953)
P oc.
Na i.
Acad.
Sci.
USA
39,
232-236.
2.
Wilchek,
M.,
Mi on,
T.
&
Kohn,
J.
(1984)
Me hods
Enzymol.
104,
3-55.
3.
Bu ell,
H.
R.
&
Ho owi z,
J.
(1977)
Eu .
J.
Biochem.
75,
533-544.
4.
Ulb ich,
N.
&
Wool,
I.
G.
(1978)
J.
Biol.
Chem.
253,
9049-9052.
5.
Ulb ich,
N.,
Lin,
A.
&
Wool,
I.
G.
(1979)
J.
Biol.
Chem.
254,
8641-8645.
6.
Al man,
S.,
Gue ie -Takada,
C.,
F ank o ,
H.
&
Robe son,
H.
D.
(1982)
in
Nucleases,
eds.
Linn,
S.
&
Robe s,
R.
(Cold
Sp ing
Ha bo
Labo a o y,
Cold
Sp ing
Ha bo ,
NY),
pp.
243-274.
7.
Kole,
R.,
Bae ,
M.,
S a k,
B. C.
&
Al man,
S.
(1980)
Cell
19,
881-887.
8.
Gue ie -Takada,
C.,
Ga dine ,
K.,
Ma sh,
T.,
Pace,
N.
&
Al man,
S.
(1983)
Cell
35,
849-857.
9.
Gue ie -Takada,
C.
&
Al man,
S.
(1984)
Science
223,
285-286.
10.
Hansen,
F.
G.,
Hansen,
E.
B.
&
A lung,
T.
(1985)
Gene
38,
85-93.
11.
Gh ayeb,
J.,
Kimu a,
H.,
Takaha a,
M.,
Hsiung,
H.,
Masui,
Y.
&
Inouye,
M.
(1984)
EMBO
J.
3,
2437-2442.
12.
Reed,
R.
E.,
Bae ,
M.
F.,
Gue ie -Takada,
C.,
Donis-Kelle ,
H.
&
Al man,
S.
(1982)
Cell
30,
627-636.
13.
Mania is,
T.,
F i sch,
E.
F.
&
Samb ook,
J.
(1982)
Molecula
Cloning:
A
Labo a o y
Manual
(Cold
Sp ing
Ha bo
Labo a-
o y,
Cold
Sp ing
Ha bo ,
NY).
14.
S a k,
B.
C.,
Kole,
R.,
Bowman,
E.
J.
&
Al man,
S.
(1978)
P oc.
Na l.
Acad.
Sci.
USA
75,
3717-3721.
15.
Gue ie -Takada,
C.
&
Al man,
S.
(1984)
Biochemis y
23,
6327-6334.
16.
Fahnes ock,
S.
R.
&
Nomu a,
M.
(1972)
P oc.
Na l.
Acad.
Sci.
USA
69,
363-365.
17.
Ma sh,
T.
L.
&
Pace,
N.
R.
(1985)
Science
229,
79-81.
18.
Takaha a,
M.,
Hible ,
D.
W.,
Ba ,
P.
J.,
Ge l ,
J.
A.
&
Inouye,
M.
(1985)
J.
Biol.
Chem.
260,
2670-2674.
19.
Dijk,
J.
&
Li lechild,
J.
(1979)
Me hods
Enzymol.
59,
481-502.
20.
Vioque,
A.
&
Palacian,
E.
(1985)
Mol.
Cell.
Biochem.
66,
55-60.
21.
Laemmli,
U.
K.
(1970)
Na u e
(London)
227,
680-685.
22.
Low y,
0.
H.,
Roseb ough,
N.
J.,
Fa ,
A.
L.
&
Randall,
R.
J.
(1951)
J.
Biol.
Chem.
193,
265-275.
23.
Robe son,
H.
D.,
Al man,
S.
&
Smi h,
J.
D.
(1972)
J.
Biol.
Chem.
247,
5243-5251.
24.
Gue ie -Takada,
C.
&
Al man,
S.
(1986)
Cell
45,
177-183.
25.
Ga dine ,
K.
J.,
Ma sh,
T.
L.
&
Pace,
N.
(1985)
J.
Biol.
Chem.
260,
5415-5419.
26.
Gue ie -Takada,
C.,
Haydock,
K.,
Allen,
L.
&
Al man,
S.
(1986)
Biochemis y
25,
1509-1515.
27.
Da ydo ,
R.
M.,
Ves e man,
B.
G.,
Genkin,
M.
V.,
Blumen eld,
L.
A.
&
K ylo ,
0.
V.
(1981)
Dokl.
Akad.
Nauk.
SSSR
256,
491-494.
28.
Mosbach,
K.,
ed.
(1976)
Me hods
Enzymol.
44,
491-936.
29.
Gold,
H.
A.
&
Al man,
S.
(1986)
Cell
44,
243-249.
30.
K uge ,
K.,
G abowski,
P.
J.,
Zaug,
A.
J.,
Sands,
J.,
Go sch-
ling,
D.
E.
&
Cech,
T.
R.
(1982)
Cell
31,
147-157.
31.
Cech,
T.
R.
&
Bass,
B.
L.
(1986)
Annu.
Re .
Biochem.
55,
599-629.
P oc.
Na l.
Acad.
Sci.
USA
83
(1986)