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Controlled degradation of yeast tRNAPhe by spleen phosphodiesterase in the presence of ethidium bromide
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 4 7 7 - 4 8 3
Vol. 152, No. 1, 1988 April 15, 1988
CONTROLLED DEORADATIONOF YEAST tRNA Pbe BY SPLEEN PHOSPH£~IESTERASE
IN THE PRESENCE OF ETHIDIUH BROHID£
Z b i g n i e w d. LeSniKowsKi
Polish
Academy o f S c i e n c e s , C e n t r e o f M o l e c u l a r and M a c r o m o l e c u l a r Studies, Department of Bioorganic Chemistry 9 0 - 3 6 £ LOdZ, Boczna 5, P o l a n d
Received March 8, 1988
SUMMARY: D e g r a d a t i o n o f y e a s t tRNA Phe W i t h spleen phOSphodiesterase in t h e p r e s e n c e o f e t h i d i u m b r o m i d e has been s t u d i e d . I t was f o u n d t h a t in t h e P r e sence of the intercalating dye, the digestion i s h a l t e d a f t e r a l i m i t e d number of nucleotides i s r e m o v e d , P o s s i b l e e x p l a n a t i o n s o f t h e o b s e r v e d Phenomenon in c o n n e c t i o n w i t h t R N A - e t h i d i u m brOmide c o m p l e x f o r m a t i o n a r e d i s c u s s e d . © 1988 A c a d e m i c
Routine
Press,
application
structurally still
Inc.
a
of
modified matter
the total tRNAs,
of
future.
tRNA's structure-function natural tRNA
digestion.
fragments
can be o b t a i n e d
Both,
chemical
emPloYed.
TI3e a n t i c o d o n
(6,10,11),
3'-terminal
cally
misacylated
tRNAs f r o m t h e i r Relatively shortened rature
It
3"-end,
less
(7-9)
for
preparation
the first
i s one o f t h e r e a s o n s why in t h e s t u d i e s
of
chemically
are
of
has
by e n d o n u c l e o l y t i c as w e l l
as
a
major
product
according
cleavage or exonucleolytic
enzymatic
tools
for
methods
(10-15)
anticodon
limited
(15).
Unfortunately
to the "retardation
this sites"
of chemi-
degradation
of
tRNA S t u d i e s .
been p a i d t o t h e P r e p a r a t i o n
tRNA w i t h
are
lOOp c l e a v a g e
(5) o r s y n t h e s i s
b o t h based on
useful
of
altered
in a w i d e use ( 4 - 6 ) .
spleen
In t h e c a s e o f y e a s t tRNA Pne, t h e tRNA t r u n c a t e d as
and e n z y m a t i c a l l y
of
t h e a m i n o a c y l s t e m . The m e t h o d d e s c r i b e d
i s b a s e d on t h e d i g e s t i o n
fragments
efforts
and is
tRNA Phe ( 1 4 ) ,
attention
natural (1-3)
r e p l a c e m e n t b a s e d on s p e c i f i c
provide
of
successful
a m i n o a c i d s t e m base changes
yeast
5'-terminus
of
relationship,
tRNAs and t h e i r fragments
synthesis
in s p i t e
tRNAS w i t h in the
plnospnodiesterase
by £7 n u c l e o t i d e s
approach usually pattern
a
lite(15).
is
isolated
yields
several
(15,16).
One o f t h e m a i n g o a l s o f o u r s t u d y was t o wor~ o u t t h e m e t h o d o f r e m o v i n g p o s sibly spleen
short
oligoribonucleotides
i~ospiqodiesterase
Abbreviations: diesterase.
f r o m t h e tRNA 5 ' - t e r ~ n i n u s .
towards the three-dimensional
y e a s t tRNAPhesPDE: y e a s t tRNA Phe t r e a t e d
477
Tl~e s e n s i t i v i t y
structure with
of
of the sub-
spleen
I~OSpho-
0006-291X/88 $1.50 Copyr~ht © 1988byAcademic Press, Inc. Allrighuofreproduction in anyform rese~ed.
Vol. 152, No. 1, 1988
strate acids making
(17)
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and t h e Known f a c t
by i n t e r c a l a t i n g use
intercalating
of
agents
(18,19),
dye i n t e r a c t i o n .
is believed
protection
of
particular
For the controlled ethidium
t o Rave m a j o r
sites
in
p r o m p t e d us t o a p p r o a c h t h i s
t h e p r o m o t i o n o f new r e t a r d a t i o n
by spleen phospl~odiesterase, agent
of
site(s)
binding
sites
p r o b l e m bY
as a r e s u l t
digestion
b r o m i d e was c h o s e n .
nucleic
of
of yeast This
in the acceptor
tRNA-
tRNA Phe
intercalating
stem
of
yeast
tRNA Phe ( 2 0 , 2 1 ) .
HATER I ALS AND METHODS Y e a s t tRNA Phe was p u r c h a s e d f r o m B o e h r i n g e r Mannheim (1100 p m o l / A 2 6 0 u n i t ) and Institute of Bioorganic C~emistry, P o l i s h Academy o f S c i e n c e s , Pozna~, P o l a n d [1170 pmol/A260 unit after pretreatment with CTP (ATP): tRNA n u c l e o t i d y l transferase]. AlKaline phospllatase from E.coli (EC ~ . 1 , ~ . 1 ) , Polynucleotide K i n a s e f r o m T4 i n f e c t e d E . c o l i B (EC 2 . 7 . 1 . 7 . 8 ) and n u c l e a s e P1 f r o m P e n i c i l lium citrinum (EC 3 . 1 . ~ 0 . x ) w e r e f r o m P b a r m a c i a , Plsospl~odiesterase II from B o v i n e S p l e e n (EC 3. t . 1 6 . 1 ) and e t b i d i u m b r o m i d e w e r e p u r c h a s e d f r o m Sigma. [y_~2p] ATP (5000 C i / m ~ o l ) was PUrChased f r o m A m e r s h a m . ^ c r y l a m i d e and N,N'-bisacrylamide w e r e f r o m B i o m o l F e i n c h e m i K a l i e n GmDH, u r e a was f r o m BRL, tris(hydroxymethyl)aminomethane was f r o m J . T . B a K e r Clsemicals B . V . , b o r i c a c i d , EDTA, magnesium c h l o r i d e , sodium acetate were Merck products,
Gel e l e c t r o p h o r e s i 5 Denaturating polyacrylamide g e l s ( 2 0 0 x 2 0 0 x O . 5 mm) c o n t a i n e d lOX ( w / v ) a c r y l amide/N,N'-methylenebisacrylamide ( 1 9 : 1 . ) , 7M u r e a , 90 mR T r i s - b o r a t e , pH 8 . 3 , and 2 mR EDTA ( 2 2 ) . E l e c t r o p l ~ o r e s i s was u s u a l l y c a r r i e d o u t a t 3 0 0 - 5 0 0 V, The r u n s w e r e t e r m i n a t e d when t h e f r o n t o f b r o m o p r ~ n o l b l u e m a r k e r was a t t h e D o t tom o f t h e g e l . Analytical gels were stained wit~ methylene blue or autoradiograpl~ed by using Agfa-Gevaert 0 5 r a y M~ film. In the case o f preparative gels, RNA bands were located by U V shadowing or autoradiograplsy, cut out with a razor blade and recovered from tl~e gel by extraction (23). L)epho~phorylation The r e a c t i o n m i x t u r e c o n t a i n i n g 2 0 , 0 A260 u n i t s o f y e a s t tRNA Plqe and t 4 u n i t s o f E . c o l i a l k a l i n e p h o s p h a t a s e i n 400 pl o f 25 rnM T r i s - H C I , pH 8 . 0 , was inc u b a t e d f o r 30 m i n a t +50Oc, t h e n EDTA and d i t h i o t h r e i t o l w e r e added t o f i n a l c o n c e n t r a t i o n o f 4 rnH and 3 mR, r e s p e c t i v e l y . The i n c u b a t i o n was c o n t i n u e d f o r 15 m i n a t r o o m t e m p e r a t u r e and rise r e a c t i o n m i x t u r e was e x t r a c t e d w i t h 200 pl o f p h e n o l s a t u r a t e d w i t h b u f f e r c o n t a i n i n g 25 mR T r I S - H C I , pH 8 , 0 , 4 mR EDTA and 3 m R d i t h i o t h r e i t o l . The p h e n o l f r a c t i o n was r e e x t r a c t e d w i t h 200 pl of buffer as above saturated with Phenol. W a t e r fractions were combined, extracted six times with 200 Ul portions of ethyl ether tl~en I M NaOAC, pH 6.2, was added tO final concentration 0.2 M and the product was precipitated with four volumes of ethanol. Tl~e resulting solution was chilled with liquid nitrogen and left for 30min at -20Oc. The Pellet of dephosPnorylated yeast tRNA Phe was centrifuged o f f , washed w i t h e t h a n o l and e t h y l e t h e r and d r i e d u n d e r vacuum. P r e p a r a t i v e d e g r a d a t i o n o f 5 " - d e p D o s p D o r y l a t e d yeas~ tRl~ PDe b y ~ p l e e n p h o $ p h o d i e s t e r a s e i n tne p r e s e n c e o f e t ~ i d i u m O r o m i d e D e p h o s p h o r y l a t e d y e a s t tRNA Phe, 5 . 0 A260 u n i t s in 20 pl 10 n~ NaOAC, pH 6 . 2 , was h e a t e d f o r t 0 min a t +60°C t h e n i t was a l l o w e d t o c o o l down s l o w l y t o r o o m t e m p e r a t u r e i n rise c o u r s e o f a b o u t 2 h. To t h i s s o l u t i o n 1 4 . 5 Pl lOOrnM NaOAc, pH 6 . 2 , and 66 Pl 5 n~l e t N i d i u r n b r o m i d e w e r e a d d e d . The r e s u l t i n g m i x t u r e was i n c u b a t e d f o r 30 min a t +4°C. The r e a c t i o n was t h e n i n i t i a t e d by t h e a d d i t i o n o f 66 p l ( 6 , 6 u n i t s ) o f s p l e e n p h o s p h o d i e s t e r a s e . Final volume of reaction m i x t u r e was 165 p l , concentration o f NaOAc, e t h i d i u m b r o m i d e and s p l e e n pl~osp h o d i e s t e r a s e w e r e 10 mM, 2 mM and 40 u n i t s / m l , respectively. After 15-20 min
478
Vol. 152, No. 1, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
o f t h e i n c u b a t i o n a t +20°C 1 M NaOAC pH 6 . 2 was added t o a f i n a l concentration of 0.2 M and t h e p r o d u c t was p r e c i p i t a t e d with four volumes of ethanol. The resulting s o l u t i o n was c o o l e d down w i t h l i q u i d n i t r o g e n and l e f t f o r 30 m i n a t -20°C, The P e l l e t o f 5 " - t r u n c a t e d y e a s t tRNA Phe was c e n t r i f u g e d down, washed w i t h e t h a n o l and e t h y l e t h e r , and d r i e d u n d e r vacuum. Labeling of 5'-ends w i t h T4 p o l y n u c l e o t i d e K i n a s e / [ y - ~ 2 P ] A T P was p e r formed according to the literature method (24). Two d i m e n s i o n a l a n a l y s i s o f t h e 5 " - t e r m i n a l n u c l e o t i d e was p e r f o r m e d as d e s c r i b e d (25) e x c e p t t h a t f o r t h e c h r o m a t o g r a p h y in t h e s e c o n d d i m e n s i o n , n-butyl alcohol/acetic acid/H20 (5:£:~, v / v / v ) as s o l v e n t s y s t e m was used i n s t e a d of isopropyl alcohol/concentrated HCI/H20 ( 7 0 : 1 5 : 1 5 , v / v / v ) , RESULTS AND DISCU6SION Spleen Pl3osphodiesterase hydrolyses ~'-rnononucleotides ral
fragw~nts
gestion mide,
of tlqe
cleotides
(17).
nucleic
The t r e a t m e n t
shortened at their
degradation is removed,
process yielding
o f tRNAs w i t h
5'-end
Y e a S t tRNA Phe i s p e r f o n l l e d
a c i d s f r o m t h e 5 " - e n d by r e l e a s i n g
(15.16).
t h e enzyme l e a d s t o s e v e -
We h a v e f o u n d t h a t
in the presence of
is halted
after
merely slightly
l-2mM
only a limited
if
the di-
ethidium
bro-
number o f n u -
s h o r t e n e d tRNA (FIGURE
1
and
FIGURE 2 ) . The
5'-terminal
was d e t e r m i n e d lysis ged
of
nucleoside
as A by means o f
a n u c l e a s e Pt d i g e s t
autoradiograPhy
three
tRNA Phe n a m e l y AS,
intact
traces of
In an e f f o r t
in tRNA,
A was e s t a b l i s h e d . the
thin-layer
5"-[3£P]-labeled
in t h e 5 " - p a r t
site
Y e a s t tRNA PIle ( y e a s t tRN&Pnespl) E)
two dimensional
also
A9 and A14.
promoted retardation terminal
of
revealed
adenosine residues
and o f
of 5"-truncated
yeast
There are
the
of
aminoacyl
to determine
By c o m P a r i n g t h e c h a i n the
A
Prolon-
o f G and U (FIGURE ~ ) ,
the exact position
y e a s t tRNA Phe w i t h
chromatograPhy anatRNAPhesPDE,
stem
the ethidium
YeaSt bromide
o f t h e y e a s t tlRNAPhe 5 ' -
lengths of
ladder obtained
on
the 5"-truncated a
8X
polyacryl-
3'
£
c 5'. A
G.C C.G G.C G'U A.U U.A U " AG U
DD O
GGA
ACA
C C Urn1A G
mG CU u CG U G A ~ ~
m7G
m2G A G C-G C'G A.U G -mSC A. CF.
tOA
u
Y
C~A
F I G U R E I. S e c o n d a r y s t r u c t u r e of y e a s t tRNA Pl3e siqowing presumable e t h i d i u m broml Cle inducecl s p l e e n p h o s P h o d ie s t e r a s e r e t a r d a t ion s ire.
479
VOI. 152, No. 1, 1988
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
AB
C XC
pA
Q
®"
I
FIGURE 2. lOX P o l y a c r y l a m i d e - T M urea g e l , Lane A: 5 " - d e p ~ o s p l q o r y l a t e d y e a s t tRNAP~e (0.15 A260). Lane B: 5'-depiqospl~orylated y e a s t tRNA Phe (0,3 A260), s p l e e n pnosplqodiesterase (0.4 u n i t ) , lOmM sodium a c e t a t e , pH 6 , 2 (10 p l ) , 30 min, 20°C, Lane C: 5 " - d e p ~ o s p l 3 0 r y l a t e d y e a s t tRNAPhe (0.3 A260), s p l e e n phospl~odiesterase (0.4 u n i t ) , lOmH sodium a c e t a t e , pH 6,2, 1 rnH e t h i d i u m bromide (10 p l ) , 30 min, 200C. FI6URE 3. A n a l y s i s o f tl'~e 5 ' - t e r m i n a l n u c l e o t i d e o f 5 " - [ 3 2 P ] - I a o e l e d 5 " t r u n c a t e d y e a s t tRNA Pl~e by two dimensional c e l l u l o s e t ~ i n - l a y e r chromatography (Nuclease PI d i g e s t ) . F i r s t dimension: i s o l ~ J t y r i c a c i d / O . 5 H armnonia (5:3, v / v ) , second dimension: n - b u t y l a l c o l l o l / a c e t i c acid/H£O ( 5 : 2 : 3 , v/v/v),
arnide
sequencing
gel
tRNA PI3e i s s h o r t e r adenosine
residue
The effect
of
phorylated
yeast
(data
than the in yeast
the
not
was
against
concentration
digestion
the
of
have 0.25-5
more y e a s t
Concentrations
b r o m i d e was a p p l i e d ,
spermine
and
not
of
ethidium
The 5"-terminal
b r o m i d e on t h e c l e a v a g e
8Permidine)
observed
a
mH c o n c e n t r a t i o n
The e f f e c t
decrease
of
ceased,
Na + i o n a l o n e
has little
concentration
of
ethidium
most probably effect
sodium acetate
higher for
binding
excess of excess)
sites
buffer
reasons
frag-
of
impro1-2
the degradation
sPecificity
,
rnH
tRNA in well
as
in tRNA ( 2 6 ) ,
SPecificity
(60 mH) a n d i n t h e
480
short
we
in t h e r a n g e o f
Mg2+ o v e r e t h i d i u m ) .
due t o enzyme i n h i b i t i o n
on d i g e s t i o n
the sub-
t h a n 2 m M do n o t
practical
In
concentration
Even tl3ough Mg2+ ( a s
the digestion
(five-fold
o f Mg2+ ( f i f t y - f o l d
of
o f Mg 2+ on t h e d i g e s t i o n
i n FIGURE 5.
competes for
resistance
as t h e e t h i d i u m
tRNAPhesPDE 7 6 - 1 3 and f e w e r
of ethidium
of dephos-
i 8 Shown in FIGURE 4,
the specific
and t h e r e f o r e
i s shown
n~l Mg£+ c o n c e n t r a t i o n
has almost
ethidium
agent,
of degradation
specificity
presence
the 5'-truncated
b y t h e enzyme i n c r e a s e d
As a r e s u l t
ments were observed.
ethidium
was f o u n d t h a t
b y 13 n u c l e o t i d e s .
tRNA Plqe b y S p l e e n P l 3 O S p h o d i e s t e r a s e
increased.
ve the
it
molecule
tRNAPhesPDE sI3oUId be t h u s A14.
t h e r a n g e o f 0 - 1 mM i n t e r c a l a t i n g strate
Shown)
intact
of
yeast
(17),
At
50
tRNA PI3e
Monovalent
however at a
high
absence of ethidium,
Vol. 152, No. 1, 1988
A B
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A B C D E
C D E w
t
XE
XC
®
®
10X Pal y a c r y I ami de-TM urea ge I, FIGURE 4. D i g e s t i o n o f 5"-depttospt3orylated y e a s t tRNAPhe by spleen p h o s p n o d i e s t e r a s e in the presence o f e t h i d i u m bromide - e f f e c t o f e t h i d i u m c o n c e n t r a t i o n . Lane A: 5'-depRosPt~Orylated y e a s t tRNA Pbe (0.~ A260), spleen p n o s p h o d i e s t e r a s e (0,4 u n i t ) , 10 mM sodium a c e t a t e , iol4 6.2, 1 mH e t h i d i u m bromide (10 p l ) , 30 rain, 2K)°C. Lane B: as above, 0 . 5 mM etMidium. Lane c: as above, 0.25 mM e t h i d i u m . Lane D: as above b u t w i t h o u t e t h i d i u m . Lane E: 5 ' - d e p l 3 o s p ~ o r y l a t e d y e a s t tRNAPl~e (0.15 ^260)" FIGURE 5. IOX P o l y a c r y l a m i d e - 7M urea g e l , D i g e s t i o n o f 5"-depl3ospi3orylated y e a s t tRNAP13e by spleen pl3ospl3odiesterase in the presence o f e t n i a i um 13romicle - e f f e c t o f Mg2+ concentrat ion.
Lane A: 5'-depl~osprK)rylated y e a s t tRNAPtm (0.15 ^E60)' Lane B: 5'-depl~osPl30rylated y e a s t tRNAPr~ (0.~ A260), spleen pr~spt~odiesterase (0.4 u n i t ) , 10 rnH so(liurn a c e t a t e , pH 6.2, 1 mM etl~idium bromide, 15 rain, ¢~:)0C. Lane C: as above, 0 . 5 mM MgCIp. Lane D: as above, 5 mM MgCI2. Lane E: as above, 5O mM MgCI 2.
traces
of
addition possible spleen (2T)
yeast
t o Y e a S t tRNA Pl~e 7 6 - 2 7 explanations
for
phosphodiesterase
a mode t h a t
part
of
£x-ystallograpl3ic the
groups with spleen
P-IO
(28)
after
the molecule
fragment
the observed
anotl3er
of
tRNA c o n f o r m a t i o n
removing a defined
is not
accessible
d a t a shows t h a t
loop of yeast
possible
acts
one of
phenomena w o u l d r e q u i r e show
the
more detailed
possibility
of
product,
digestion
of
One
yeast
b r o m i d e may
by the
of
in tl3e
tRNA PT~ b y
be
changing
intercalating
number of nucleotides
the ethidium
binding
hydrogen bonded with
of
(29].
limited
This
agent
in
the remaining
The e x a c t studies, sPecific
481
sites its
finding
degradation
in t h e p r e s e n c e o f e t h i d i u m :
as " a s p r a g " .
( d a t a n o t shown)
t o enzyme a c t i o n .
tRNA Phe,
explanation
Pl~osphodiesterase dye
as a m a j o r
limited
t o baCKbone Pl~osphate P - 8 and P-15
tercalating report
(?)
were detected
in t h e p r e s e n c e o f e t h i d i u m
and s t a b i l i z a t i o n
such
in
tRNAPhesF1) E 7 6 - 1 3 f r a g m e n t
that
of
and e f f e c t i v e
is
located
yeast
of
amino
consistent tRNA Pl3e by
a molecule
interpretation The results
is
exocy¢lic
of
in-
the observed
presented shortening
in
this
of y e a s t
Vol. 152, No. 1, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
tRNA Phe a t t h e 5 " - e n d y i e l d i n g structure
-
function
fication.
Our r e s u l t s
etbidium
bromide binding
Studies
on
well
presence of different
tRNA Phe
76-13
relationship
studies
also support
the assumption
sites
the reconstruction
as on t h e d i g e s t i o n
yeast
fra£vnent
and a l s o f o r
o f y e a s t tRNA Pne e x i s t s
that
further one
intercalating
tRNA8 w i t h
for
5'-endmodiof
the
major
a t t h e P - I O lOOp.
of the 5"-end of 5'-truncated
of other
suitable
y e a s t tRNA Pl~e as
SPleen p n o s p n o d i e s t e r a s e
a g e n t s a r e in p r o g r e s s
in
the
in o u r L a b o r a t o r y .
ACKNC~LEDGEMENTS This WOrl< was done in p a r t d u r i n g t h e a u t h o r ' s v i s i t in t ~ e Max-PlancK I n Stitute for Experimental Medicine, Prof. F r i t z Crarner L a b o r a t o r y , Gottingen, 1987. F i n a n c i a l Support o f Max-PlancK G e s e l l s c h a f t i s acKnowledged.
REFERENCES 1.
2.
OhtsuNa, E . , Tanal~a, S . , TanaKa, T . , HiyaKe, T . , Mar~13arn, A . F . , NaKagawa, E . , w a K a b a y a s h i , T . , Naniyama, Y . , N l S h i k a v a , S . , FuKumoto, R., Uemura, H . , D o i , T . , ToKunaga, T. and II
2_!, 3,
4. 5. 6. 7, 8, 9. t0. 11. 12. 13. 14. 15. 16, t7. 18. 19. £0. 21. 22.
ale-a19.
k o w a r y , P . , Sampson, d . , M i l i g a n , d , , Groebe, D. and OnlenbecK, O,C. (1986) in s t r u c t u r e and Dynamics o f RNA (van K n i p P e n b e r g , P.H. and Hi I h e r s , C.W. e d s . ) , NATO ASI S e r i e s , V o l . t10, pp. 6 9 - 7 6 , Plenum P r e s s , New Y o r ~ . T h i e b e , R., H a r b e r s , K. and z a c n a u , H.G. (197~_) E u r . J . B i o c n e m . 266, 144-152. D o i , T . , M o r i o K a , H . , H a t s u g i , d , , OntsuKa, E, and I K e h a r a , H. (1985) FEBS L e t t . 190, 125-128, a) B r u c e , A.G, and U n l e n b e c k , O.C, (1982) B i o c h e m i s t r y 2_J, 855-861. b) B r u c e , A.G, and UnlenbecK, O,C. (1982_) B i o c l l e m i s t r y 21, 3921-3936, T n i e b e , R. and z a c n a u , H.G, (1971) Methods Enzymol, 20, t 7 9 - 1 8 2 . N i s h i K a w a , K. and N e c h t , S.M. (1982) d , B i o l . O l 3 e m . 257 10836-10539. W i n t e r m e y e r , W. and Zachau, H.G. (1975) FEBS L e t t . 58, 306-309. D o i , T . , Yamane, A , , M a t s u g i , J . , OhtsuKa, E. and INehara, M. (1985) NUcl , A c i d s Res, t_33, 3685-3697, OlqtsuKa, E , , D o i , T , , FuKumoto, R . , M a t s u g i , J, and II~ehara, N. (1983) N u c I . A c i d s Res. 11, 3863-3872. Uemura, H . , I m a i , M., Olqtsuka, E . , I K e h a r a , H. arid $611, D, (1982) NU¢l . A c i d s Re8. 1__0, 6531-6539. S t u l b e r g , M.P. and Isnam, K,R. (1974) Methods EnzYmol. 2 9 , 477-482. H e c K l e r , T , G . , Chang, L i - H o , Zama, Y . , NaKa, T , , Cllorghade, H.S. and H e c n t , S.M. (1984) B i o c t 3 e m i s t r y 2_~, 1468-1473. P h i l i p p s e n , P, and Zacnau, H.G. (1974) Methods EnzYmol. 2 9 , 475-477. P h i l i p p s e n , P, and Zacnau, H,G, (1972) B i o c n i m . B i o p n y s , A c t a £77, 523-5~8. B e r n a r d i , A. and B e r ~ a r d i , G. ( t 9 7 t ) Tl~e Enzymes 4, 329-336. H a r b e r s , K . , T h i e b e , R. and Zachau, H.G, (1972) E u r , d . B i o c l q e m . 26, 13,~-143. T a n n e r , N.K. and Cech, T.R. (1985) N U C I . A c i d 8 Res. 1~3, 7759-7779. J o n e s , C . R . , B o l t o n , P.H. and Kear~ns, D.R. (1978) B i o c l ~ e m i s t r y 1~7, 601-607 Liel3man, M. R u b i n , O. and S u n d a r a l i n g a m , M. (1977) P r o C . N a t I . A c a d . Sci.USA 744, 4821-4825. Donis-Keller, H . , Maxam, A.M. and G i l b e r t , W. (1979) N u c I . A c i d S Res. 4 , £5£7-£538.
482
Vol. 152, No. 1, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
23. Haxam, A.M. and G i l b e r t , W. (1980) Methods Enzymol. 65, 499-560. 24. Chaconas, G., van de Sande, J.H. and Church, R,B. (1975) Bioc~em.Biophys.Res.Cofm~n, 6._.66, 962-969, 25, KuChino, Y,, Kato, M., S u g i s a k i , H, and Nisnimura, S. (1979) NucI.Acid8 Res. 6, 3459-3469. £6. $aKai, T . T . , T o r g e t , R., Freda, C.E. and Cohen, S.S. (1975) NUCI.ACiO9 Res. 2, 1005-10££ 27. N i e l s e n , P.E. (1981) [email protected] 655, 89-95. 28, G o l d f i e l d , E.M., Luxon, B.A,, Bowie, V. and Gorenstein, D.G. (1983) B i o c h e m i s t r y 22, 3336-3344. 29. Q u i g l e y , G.J., T e e t e r , M.M. and Rich, A, (1978) Proc.Natl.Acad.SCi.USA 75, 64-68.
483
Vol. 152, No. 1, 1988 April 15, 1988
CONTROLLED DEORADATIONOF YEAST tRNA Pbe BY SPLEEN PHOSPH£~IESTERASE
IN THE PRESENCE OF ETHIDIUH BROHID£
Z b i g n i e w d. LeSniKowsKi
Polish
Academy o f S c i e n c e s , C e n t r e o f M o l e c u l a r and M a c r o m o l e c u l a r Studies, Department of Bioorganic Chemistry 9 0 - 3 6 £ LOdZ, Boczna 5, P o l a n d
Received March 8, 1988
SUMMARY: D e g r a d a t i o n o f y e a s t tRNA Phe W i t h spleen phOSphodiesterase in t h e p r e s e n c e o f e t h i d i u m b r o m i d e has been s t u d i e d . I t was f o u n d t h a t in t h e P r e sence of the intercalating dye, the digestion i s h a l t e d a f t e r a l i m i t e d number of nucleotides i s r e m o v e d , P o s s i b l e e x p l a n a t i o n s o f t h e o b s e r v e d Phenomenon in c o n n e c t i o n w i t h t R N A - e t h i d i u m brOmide c o m p l e x f o r m a t i o n a r e d i s c u s s e d . © 1988 A c a d e m i c
Routine
Press,
application
structurally still
Inc.
a
of
modified matter
the total tRNAs,
of
future.
tRNA's structure-function natural tRNA
digestion.
fragments
can be o b t a i n e d
Both,
chemical
emPloYed.
TI3e a n t i c o d o n
(6,10,11),
3'-terminal
cally
misacylated
tRNAs f r o m t h e i r Relatively shortened rature
It
3"-end,
less
(7-9)
for
preparation
the first
i s one o f t h e r e a s o n s why in t h e s t u d i e s
of
chemically
are
of
has
by e n d o n u c l e o l y t i c as w e l l
as
a
major
product
according
cleavage or exonucleolytic
enzymatic
tools
for
methods
(10-15)
anticodon
limited
(15).
Unfortunately
to the "retardation
this sites"
of chemi-
degradation
of
tRNA S t u d i e s .
been p a i d t o t h e P r e p a r a t i o n
tRNA w i t h
are
lOOp c l e a v a g e
(5) o r s y n t h e s i s
b o t h based on
useful
of
altered
in a w i d e use ( 4 - 6 ) .
spleen
In t h e c a s e o f y e a s t tRNA Pne, t h e tRNA t r u n c a t e d as
and e n z y m a t i c a l l y
of
t h e a m i n o a c y l s t e m . The m e t h o d d e s c r i b e d
i s b a s e d on t h e d i g e s t i o n
fragments
efforts
and is
tRNA Phe ( 1 4 ) ,
attention
natural (1-3)
r e p l a c e m e n t b a s e d on s p e c i f i c
provide
of
successful
a m i n o a c i d s t e m base changes
yeast
5'-terminus
of
relationship,
tRNAs and t h e i r fragments
synthesis
in s p i t e
tRNAS w i t h in the
plnospnodiesterase
by £7 n u c l e o t i d e s
approach usually pattern
a
lite(15).
is
isolated
yields
several
(15,16).
One o f t h e m a i n g o a l s o f o u r s t u d y was t o wor~ o u t t h e m e t h o d o f r e m o v i n g p o s sibly spleen
short
oligoribonucleotides
i~ospiqodiesterase
Abbreviations: diesterase.
f r o m t h e tRNA 5 ' - t e r ~ n i n u s .
towards the three-dimensional
y e a s t tRNAPhesPDE: y e a s t tRNA Phe t r e a t e d
477
Tl~e s e n s i t i v i t y
structure with
of
of the sub-
spleen
I~OSpho-
0006-291X/88 $1.50 Copyr~ht © 1988byAcademic Press, Inc. Allrighuofreproduction in anyform rese~ed.
Vol. 152, No. 1, 1988
strate acids making
(17)
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and t h e Known f a c t
by i n t e r c a l a t i n g use
intercalating
of
agents
(18,19),
dye i n t e r a c t i o n .
is believed
protection
of
particular
For the controlled ethidium
t o Rave m a j o r
sites
in
p r o m p t e d us t o a p p r o a c h t h i s
t h e p r o m o t i o n o f new r e t a r d a t i o n
by spleen phospl~odiesterase, agent
of
site(s)
binding
sites
p r o b l e m bY
as a r e s u l t
digestion
b r o m i d e was c h o s e n .
nucleic
of
of yeast This
in the acceptor
tRNA-
tRNA Phe
intercalating
stem
of
yeast
tRNA Phe ( 2 0 , 2 1 ) .
HATER I ALS AND METHODS Y e a s t tRNA Phe was p u r c h a s e d f r o m B o e h r i n g e r Mannheim (1100 p m o l / A 2 6 0 u n i t ) and Institute of Bioorganic C~emistry, P o l i s h Academy o f S c i e n c e s , Pozna~, P o l a n d [1170 pmol/A260 unit after pretreatment with CTP (ATP): tRNA n u c l e o t i d y l transferase]. AlKaline phospllatase from E.coli (EC ~ . 1 , ~ . 1 ) , Polynucleotide K i n a s e f r o m T4 i n f e c t e d E . c o l i B (EC 2 . 7 . 1 . 7 . 8 ) and n u c l e a s e P1 f r o m P e n i c i l lium citrinum (EC 3 . 1 . ~ 0 . x ) w e r e f r o m P b a r m a c i a , Plsospl~odiesterase II from B o v i n e S p l e e n (EC 3. t . 1 6 . 1 ) and e t b i d i u m b r o m i d e w e r e p u r c h a s e d f r o m Sigma. [y_~2p] ATP (5000 C i / m ~ o l ) was PUrChased f r o m A m e r s h a m . ^ c r y l a m i d e and N,N'-bisacrylamide w e r e f r o m B i o m o l F e i n c h e m i K a l i e n GmDH, u r e a was f r o m BRL, tris(hydroxymethyl)aminomethane was f r o m J . T . B a K e r Clsemicals B . V . , b o r i c a c i d , EDTA, magnesium c h l o r i d e , sodium acetate were Merck products,
Gel e l e c t r o p h o r e s i 5 Denaturating polyacrylamide g e l s ( 2 0 0 x 2 0 0 x O . 5 mm) c o n t a i n e d lOX ( w / v ) a c r y l amide/N,N'-methylenebisacrylamide ( 1 9 : 1 . ) , 7M u r e a , 90 mR T r i s - b o r a t e , pH 8 . 3 , and 2 mR EDTA ( 2 2 ) . E l e c t r o p l ~ o r e s i s was u s u a l l y c a r r i e d o u t a t 3 0 0 - 5 0 0 V, The r u n s w e r e t e r m i n a t e d when t h e f r o n t o f b r o m o p r ~ n o l b l u e m a r k e r was a t t h e D o t tom o f t h e g e l . Analytical gels were stained wit~ methylene blue or autoradiograpl~ed by using Agfa-Gevaert 0 5 r a y M~ film. In the case o f preparative gels, RNA bands were located by U V shadowing or autoradiograplsy, cut out with a razor blade and recovered from tl~e gel by extraction (23). L)epho~phorylation The r e a c t i o n m i x t u r e c o n t a i n i n g 2 0 , 0 A260 u n i t s o f y e a s t tRNA Plqe and t 4 u n i t s o f E . c o l i a l k a l i n e p h o s p h a t a s e i n 400 pl o f 25 rnM T r i s - H C I , pH 8 . 0 , was inc u b a t e d f o r 30 m i n a t +50Oc, t h e n EDTA and d i t h i o t h r e i t o l w e r e added t o f i n a l c o n c e n t r a t i o n o f 4 rnH and 3 mR, r e s p e c t i v e l y . The i n c u b a t i o n was c o n t i n u e d f o r 15 m i n a t r o o m t e m p e r a t u r e and rise r e a c t i o n m i x t u r e was e x t r a c t e d w i t h 200 pl o f p h e n o l s a t u r a t e d w i t h b u f f e r c o n t a i n i n g 25 mR T r I S - H C I , pH 8 , 0 , 4 mR EDTA and 3 m R d i t h i o t h r e i t o l . The p h e n o l f r a c t i o n was r e e x t r a c t e d w i t h 200 pl of buffer as above saturated with Phenol. W a t e r fractions were combined, extracted six times with 200 Ul portions of ethyl ether tl~en I M NaOAC, pH 6.2, was added tO final concentration 0.2 M and the product was precipitated with four volumes of ethanol. Tl~e resulting solution was chilled with liquid nitrogen and left for 30min at -20Oc. The Pellet of dephosPnorylated yeast tRNA Phe was centrifuged o f f , washed w i t h e t h a n o l and e t h y l e t h e r and d r i e d u n d e r vacuum. P r e p a r a t i v e d e g r a d a t i o n o f 5 " - d e p D o s p D o r y l a t e d yeas~ tRl~ PDe b y ~ p l e e n p h o $ p h o d i e s t e r a s e i n tne p r e s e n c e o f e t ~ i d i u m O r o m i d e D e p h o s p h o r y l a t e d y e a s t tRNA Phe, 5 . 0 A260 u n i t s in 20 pl 10 n~ NaOAC, pH 6 . 2 , was h e a t e d f o r t 0 min a t +60°C t h e n i t was a l l o w e d t o c o o l down s l o w l y t o r o o m t e m p e r a t u r e i n rise c o u r s e o f a b o u t 2 h. To t h i s s o l u t i o n 1 4 . 5 Pl lOOrnM NaOAc, pH 6 . 2 , and 66 Pl 5 n~l e t N i d i u r n b r o m i d e w e r e a d d e d . The r e s u l t i n g m i x t u r e was i n c u b a t e d f o r 30 min a t +4°C. The r e a c t i o n was t h e n i n i t i a t e d by t h e a d d i t i o n o f 66 p l ( 6 , 6 u n i t s ) o f s p l e e n p h o s p h o d i e s t e r a s e . Final volume of reaction m i x t u r e was 165 p l , concentration o f NaOAc, e t h i d i u m b r o m i d e and s p l e e n pl~osp h o d i e s t e r a s e w e r e 10 mM, 2 mM and 40 u n i t s / m l , respectively. After 15-20 min
478
Vol. 152, No. 1, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
o f t h e i n c u b a t i o n a t +20°C 1 M NaOAC pH 6 . 2 was added t o a f i n a l concentration of 0.2 M and t h e p r o d u c t was p r e c i p i t a t e d with four volumes of ethanol. The resulting s o l u t i o n was c o o l e d down w i t h l i q u i d n i t r o g e n and l e f t f o r 30 m i n a t -20°C, The P e l l e t o f 5 " - t r u n c a t e d y e a s t tRNA Phe was c e n t r i f u g e d down, washed w i t h e t h a n o l and e t h y l e t h e r , and d r i e d u n d e r vacuum. Labeling of 5'-ends w i t h T4 p o l y n u c l e o t i d e K i n a s e / [ y - ~ 2 P ] A T P was p e r formed according to the literature method (24). Two d i m e n s i o n a l a n a l y s i s o f t h e 5 " - t e r m i n a l n u c l e o t i d e was p e r f o r m e d as d e s c r i b e d (25) e x c e p t t h a t f o r t h e c h r o m a t o g r a p h y in t h e s e c o n d d i m e n s i o n , n-butyl alcohol/acetic acid/H20 (5:£:~, v / v / v ) as s o l v e n t s y s t e m was used i n s t e a d of isopropyl alcohol/concentrated HCI/H20 ( 7 0 : 1 5 : 1 5 , v / v / v ) , RESULTS AND DISCU6SION Spleen Pl3osphodiesterase hydrolyses ~'-rnononucleotides ral
fragw~nts
gestion mide,
of tlqe
cleotides
(17).
nucleic
The t r e a t m e n t
shortened at their
degradation is removed,
process yielding
o f tRNAs w i t h
5'-end
Y e a S t tRNA Phe i s p e r f o n l l e d
a c i d s f r o m t h e 5 " - e n d by r e l e a s i n g
(15.16).
t h e enzyme l e a d s t o s e v e -
We h a v e f o u n d t h a t
in the presence of
is halted
after
merely slightly
l-2mM
only a limited
if
the di-
ethidium
bro-
number o f n u -
s h o r t e n e d tRNA (FIGURE
1
and
FIGURE 2 ) . The
5'-terminal
was d e t e r m i n e d lysis ged
of
nucleoside
as A by means o f
a n u c l e a s e Pt d i g e s t
autoradiograPhy
three
tRNA Phe n a m e l y AS,
intact
traces of
In an e f f o r t
in tRNA,
A was e s t a b l i s h e d . the
thin-layer
5"-[3£P]-labeled
in t h e 5 " - p a r t
site
Y e a s t tRNA PIle ( y e a s t tRN&Pnespl) E)
two dimensional
also
A9 and A14.
promoted retardation terminal
of
revealed
adenosine residues
and o f
of 5"-truncated
yeast
There are
the
of
aminoacyl
to determine
By c o m P a r i n g t h e c h a i n the
A
Prolon-
o f G and U (FIGURE ~ ) ,
the exact position
y e a s t tRNA Phe w i t h
chromatograPhy anatRNAPhesPDE,
stem
the ethidium
YeaSt bromide
o f t h e y e a s t tlRNAPhe 5 ' -
lengths of
ladder obtained
on
the 5"-truncated a
8X
polyacryl-
3'
£
c 5'. A
G.C C.G G.C G'U A.U U.A U " AG U
DD O
GGA
ACA
C C Urn1A G
mG CU u CG U G A ~ ~
m7G
m2G A G C-G C'G A.U G -mSC A. CF.
tOA
u
Y
C~A
F I G U R E I. S e c o n d a r y s t r u c t u r e of y e a s t tRNA Pl3e siqowing presumable e t h i d i u m broml Cle inducecl s p l e e n p h o s P h o d ie s t e r a s e r e t a r d a t ion s ire.
479
VOI. 152, No. 1, 1988
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
AB
C XC
pA
Q
®"
I
FIGURE 2. lOX P o l y a c r y l a m i d e - T M urea g e l , Lane A: 5 " - d e p ~ o s p l q o r y l a t e d y e a s t tRNAP~e (0.15 A260). Lane B: 5'-depiqospl~orylated y e a s t tRNA Phe (0,3 A260), s p l e e n pnosplqodiesterase (0.4 u n i t ) , lOmM sodium a c e t a t e , pH 6 , 2 (10 p l ) , 30 min, 20°C, Lane C: 5 " - d e p ~ o s p l 3 0 r y l a t e d y e a s t tRNAPhe (0.3 A260), s p l e e n phospl~odiesterase (0.4 u n i t ) , lOmH sodium a c e t a t e , pH 6,2, 1 rnH e t h i d i u m bromide (10 p l ) , 30 min, 200C. FI6URE 3. A n a l y s i s o f tl'~e 5 ' - t e r m i n a l n u c l e o t i d e o f 5 " - [ 3 2 P ] - I a o e l e d 5 " t r u n c a t e d y e a s t tRNA Pl~e by two dimensional c e l l u l o s e t ~ i n - l a y e r chromatography (Nuclease PI d i g e s t ) . F i r s t dimension: i s o l ~ J t y r i c a c i d / O . 5 H armnonia (5:3, v / v ) , second dimension: n - b u t y l a l c o l l o l / a c e t i c acid/H£O ( 5 : 2 : 3 , v/v/v),
arnide
sequencing
gel
tRNA PI3e i s s h o r t e r adenosine
residue
The effect
of
phorylated
yeast
(data
than the in yeast
the
not
was
against
concentration
digestion
the
of
have 0.25-5
more y e a s t
Concentrations
b r o m i d e was a p p l i e d ,
spermine
and
not
of
ethidium
The 5"-terminal
b r o m i d e on t h e c l e a v a g e
8Permidine)
observed
a
mH c o n c e n t r a t i o n
The e f f e c t
decrease
of
ceased,
Na + i o n a l o n e
has little
concentration
of
ethidium
most probably effect
sodium acetate
higher for
binding
excess of excess)
sites
buffer
reasons
frag-
of
impro1-2
the degradation
sPecificity
,
rnH
tRNA in well
as
in tRNA ( 2 6 ) ,
SPecificity
(60 mH) a n d i n t h e
480
short
we
in t h e r a n g e o f
Mg2+ o v e r e t h i d i u m ) .
due t o enzyme i n h i b i t i o n
on d i g e s t i o n
the sub-
t h a n 2 m M do n o t
practical
In
concentration
Even tl3ough Mg2+ ( a s
the digestion
(five-fold
o f Mg2+ ( f i f t y - f o l d
of
o f Mg 2+ on t h e d i g e s t i o n
i n FIGURE 5.
competes for
resistance
as t h e e t h i d i u m
tRNAPhesPDE 7 6 - 1 3 and f e w e r
of ethidium
of dephos-
i 8 Shown in FIGURE 4,
the specific
and t h e r e f o r e
i s shown
n~l Mg£+ c o n c e n t r a t i o n
has almost
ethidium
agent,
of degradation
specificity
presence
the 5'-truncated
b y t h e enzyme i n c r e a s e d
As a r e s u l t
ments were observed.
ethidium
was f o u n d t h a t
b y 13 n u c l e o t i d e s .
tRNA Plqe b y S p l e e n P l 3 O S p h o d i e s t e r a s e
increased.
ve the
it
molecule
tRNAPhesPDE sI3oUId be t h u s A14.
t h e r a n g e o f 0 - 1 mM i n t e r c a l a t i n g strate
Shown)
intact
of
yeast
(17),
At
50
tRNA PI3e
Monovalent
however at a
high
absence of ethidium,
Vol. 152, No. 1, 1988
A B
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A B C D E
C D E w
t
XE
XC
®
®
10X Pal y a c r y I ami de-TM urea ge I, FIGURE 4. D i g e s t i o n o f 5"-depttospt3orylated y e a s t tRNAPhe by spleen p h o s p n o d i e s t e r a s e in the presence o f e t h i d i u m bromide - e f f e c t o f e t h i d i u m c o n c e n t r a t i o n . Lane A: 5'-depRosPt~Orylated y e a s t tRNA Pbe (0.~ A260), spleen p n o s p h o d i e s t e r a s e (0,4 u n i t ) , 10 mM sodium a c e t a t e , iol4 6.2, 1 mH e t h i d i u m bromide (10 p l ) , 30 rain, 2K)°C. Lane B: as above, 0 . 5 mM etMidium. Lane c: as above, 0.25 mM e t h i d i u m . Lane D: as above b u t w i t h o u t e t h i d i u m . Lane E: 5 ' - d e p l 3 o s p ~ o r y l a t e d y e a s t tRNAPl~e (0.15 ^260)" FIGURE 5. IOX P o l y a c r y l a m i d e - 7M urea g e l , D i g e s t i o n o f 5"-depl3ospi3orylated y e a s t tRNAP13e by spleen pl3ospl3odiesterase in the presence o f e t n i a i um 13romicle - e f f e c t o f Mg2+ concentrat ion.
Lane A: 5'-depl~osprK)rylated y e a s t tRNAPtm (0.15 ^E60)' Lane B: 5'-depl~osPl30rylated y e a s t tRNAPr~ (0.~ A260), spleen pr~spt~odiesterase (0.4 u n i t ) , 10 rnH so(liurn a c e t a t e , pH 6.2, 1 mM etl~idium bromide, 15 rain, ¢~:)0C. Lane C: as above, 0 . 5 mM MgCIp. Lane D: as above, 5 mM MgCI2. Lane E: as above, 5O mM MgCI 2.
traces
of
addition possible spleen (2T)
yeast
t o Y e a S t tRNA Pl~e 7 6 - 2 7 explanations
for
phosphodiesterase
a mode t h a t
part
of
£x-ystallograpl3ic the
groups with spleen
P-IO
(28)
after
the molecule
fragment
the observed
anotl3er
of
tRNA c o n f o r m a t i o n
removing a defined
is not
accessible
d a t a shows t h a t
loop of yeast
possible
acts
one of
phenomena w o u l d r e q u i r e show
the
more detailed
possibility
of
product,
digestion
of
One
yeast
b r o m i d e may
by the
of
in tl3e
tRNA PT~ b y
be
changing
intercalating
number of nucleotides
the ethidium
binding
hydrogen bonded with
of
(29].
limited
This
agent
in
the remaining
The e x a c t studies, sPecific
481
sites its
finding
degradation
in t h e p r e s e n c e o f e t h i d i u m :
as " a s p r a g " .
( d a t a n o t shown)
t o enzyme a c t i o n .
tRNA Phe,
explanation
Pl~osphodiesterase dye
as a m a j o r
limited
t o baCKbone Pl~osphate P - 8 and P-15
tercalating report
(?)
were detected
in t h e p r e s e n c e o f e t h i d i u m
and s t a b i l i z a t i o n
such
in
tRNAPhesF1) E 7 6 - 1 3 f r a g m e n t
that
of
and e f f e c t i v e
is
located
yeast
of
amino
consistent tRNA Pl3e by
a molecule
interpretation The results
is
exocy¢lic
of
in-
the observed
presented shortening
in
this
of y e a s t
Vol. 152, No. 1, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
tRNA Phe a t t h e 5 " - e n d y i e l d i n g structure
-
function
fication.
Our r e s u l t s
etbidium
bromide binding
Studies
on
well
presence of different
tRNA Phe
76-13
relationship
studies
also support
the assumption
sites
the reconstruction
as on t h e d i g e s t i o n
yeast
fra£vnent
and a l s o f o r
o f y e a s t tRNA Pne e x i s t s
that
further one
intercalating
tRNA8 w i t h
for
5'-endmodiof
the
major
a t t h e P - I O lOOp.
of the 5"-end of 5'-truncated
of other
suitable
y e a s t tRNA Pl~e as
SPleen p n o s p n o d i e s t e r a s e
a g e n t s a r e in p r o g r e s s
in
the
in o u r L a b o r a t o r y .
ACKNC~LEDGEMENTS This WOrl< was done in p a r t d u r i n g t h e a u t h o r ' s v i s i t in t ~ e Max-PlancK I n Stitute for Experimental Medicine, Prof. F r i t z Crarner L a b o r a t o r y , Gottingen, 1987. F i n a n c i a l Support o f Max-PlancK G e s e l l s c h a f t i s acKnowledged.
REFERENCES 1.
2.
OhtsuNa, E . , Tanal~a, S . , TanaKa, T . , HiyaKe, T . , Mar~13arn, A . F . , NaKagawa, E . , w a K a b a y a s h i , T . , Naniyama, Y . , N l S h i k a v a , S . , FuKumoto, R., Uemura, H . , D o i , T . , ToKunaga, T. and II
2_!, 3,
4. 5. 6. 7, 8, 9. t0. 11. 12. 13. 14. 15. 16, t7. 18. 19. £0. 21. 22.
ale-a19.
k o w a r y , P . , Sampson, d . , M i l i g a n , d , , Groebe, D. and OnlenbecK, O,C. (1986) in s t r u c t u r e and Dynamics o f RNA (van K n i p P e n b e r g , P.H. and Hi I h e r s , C.W. e d s . ) , NATO ASI S e r i e s , V o l . t10, pp. 6 9 - 7 6 , Plenum P r e s s , New Y o r ~ . T h i e b e , R., H a r b e r s , K. and z a c n a u , H.G. (197~_) E u r . J . B i o c n e m . 266, 144-152. D o i , T . , M o r i o K a , H . , H a t s u g i , d , , OntsuKa, E, and I K e h a r a , H. (1985) FEBS L e t t . 190, 125-128, a) B r u c e , A.G, and U n l e n b e c k , O.C, (1982) B i o c h e m i s t r y 2_J, 855-861. b) B r u c e , A.G, and UnlenbecK, O,C. (1982_) B i o c l l e m i s t r y 21, 3921-3936, T n i e b e , R. and z a c n a u , H.G, (1971) Methods Enzymol, 20, t 7 9 - 1 8 2 . N i s h i K a w a , K. and N e c h t , S.M. (1982) d , B i o l . O l 3 e m . 257 10836-10539. W i n t e r m e y e r , W. and Zachau, H.G. (1975) FEBS L e t t . 58, 306-309. D o i , T . , Yamane, A , , M a t s u g i , J . , OhtsuKa, E. and INehara, M. (1985) NUcl , A c i d s Res, t_33, 3685-3697, OlqtsuKa, E , , D o i , T , , FuKumoto, R . , M a t s u g i , J, and II~ehara, N. (1983) N u c I . A c i d s Res. 11, 3863-3872. Uemura, H . , I m a i , M., Olqtsuka, E . , I K e h a r a , H. arid $611, D, (1982) NU¢l . A c i d s Re8. 1__0, 6531-6539. S t u l b e r g , M.P. and Isnam, K,R. (1974) Methods EnzYmol. 2 9 , 477-482. H e c K l e r , T , G . , Chang, L i - H o , Zama, Y . , NaKa, T , , Cllorghade, H.S. and H e c n t , S.M. (1984) B i o c t 3 e m i s t r y 2_~, 1468-1473. P h i l i p p s e n , P, and Zacnau, H.G. (1974) Methods EnzYmol. 2 9 , 475-477. P h i l i p p s e n , P, and Zacnau, H,G, (1972) B i o c n i m . B i o p n y s , A c t a £77, 523-5~8. B e r n a r d i , A. and B e r ~ a r d i , G. ( t 9 7 t ) Tl~e Enzymes 4, 329-336. H a r b e r s , K . , T h i e b e , R. and Zachau, H.G, (1972) E u r , d . B i o c l q e m . 26, 13,~-143. T a n n e r , N.K. and Cech, T.R. (1985) N U C I . A c i d 8 Res. 1~3, 7759-7779. J o n e s , C . R . , B o l t o n , P.H. and Kear~ns, D.R. (1978) B i o c l ~ e m i s t r y 1~7, 601-607 Liel3man, M. R u b i n , O. and S u n d a r a l i n g a m , M. (1977) P r o C . N a t I . A c a d . Sci.USA 744, 4821-4825. Donis-Keller, H . , Maxam, A.M. and G i l b e r t , W. (1979) N u c I . A c i d S Res. 4 , £5£7-£538.
482
Vol. 152, No. 1, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
23. Haxam, A.M. and G i l b e r t , W. (1980) Methods Enzymol. 65, 499-560. 24. Chaconas, G., van de Sande, J.H. and Church, R,B. (1975) Bioc~em.Biophys.Res.Cofm~n, 6._.66, 962-969, 25, KuChino, Y,, Kato, M., S u g i s a k i , H, and Nisnimura, S. (1979) NucI.Acid8 Res. 6, 3459-3469. £6. $aKai, T . T . , T o r g e t , R., Freda, C.E. and Cohen, S.S. (1975) NUCI.ACiO9 Res. 2, 1005-10££ 27. N i e l s e n , P.E. (1981) [email protected] 655, 89-95. 28, G o l d f i e l d , E.M., Luxon, B.A,, Bowie, V. and Gorenstein, D.G. (1983) B i o c h e m i s t r y 22, 3336-3344. 29. Q u i g l e y , G.J., T e e t e r , M.M. and Rich, A, (1978) Proc.Natl.Acad.SCi.USA 75, 64-68.
483