Volume 5 Number 3 March 1978
N u c l e i c A c i d s Research
The effect of specific structural modification on the biological activity of E.coli arginine tRNA
T.A. Kruse and B.F.C. Clark
Division of Biostructural Chemistry, Institute of Chemistry, Aarhus University, Aarhus, Denmark anc
M. Sprinzl
Max-Planck-lnstitut fur Experimentelle Medizin, Abteilung Chemie, GSttingen, GFR
Received 5 January 1978
ABSTRACT
Escherichia coli arginine tRNAi has been modified at position s 2 C32 with
iodoacetamide and a spin labelled derivative. The small effects on the charging ability of tRNA by the modifications suggest that the synthetase does
not bind to the tRNA in this region of the anticodon loop before the anticodon. A ternary complex of elongation factor Tu, GTP and the modified ArgtRNA, can be formed allowing future studies of enzymatic binding to the ribosome. Using the triplet binding assay the native Arg-tRNAi decodes all 4
codons beginning with CG. The modified Arg-tRNAi has a restricted decoding
but the decoding pattern is still unusual according to the Wobble Hypothesis.
INTRODUCTION
Now that a three-dimensional structure is known for yeast phenylalanine
tRNA1'2 and this is believed to be generally correct for other tRNA species,
there is considerable enthusiasm for attempting to relate the chemical structure with the biological activity of this group of macromolecules. Furthermore the information gained from a study of how tRNA molecules interact with
and recognize other nucleic acids and proteins will probably have wider applicability in the general field of gene expression where larger RNAs are involved.
Within the general context of attempting to relate structure and function
of tRNA we have chosen to study the biological effect of chemically modifying
specific locations in particular tRNA species. The present work described
herein, using monofunctional reagents although complete in itself is preliminary to.work involving bifunctional reagents using the same tRNA site as in
this work for crosslinking to complexed proteins. We have also used a derivative of our standard reagent iodoacetamide (Fig. IB) containing a spin label3'1*
for electron spin resonance studies which might give information on whether
the tRNA changes shape during interaction with a protein. These reagents have
Abbreviations used: acm = carbamoylmethylSL = spin label
t> Information Retrieval Limited 1 Falconberg Court London W1V5FG England
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Nucleic Acids Research
also been used i n a companion study * on yeast tRNA
in which s2C has been
enzynrically introduced.
Our general approach is to test the modified tRNA f o r several biological
functions. The £ . c o l i tRNA used in these experiments is tRNA Arg
s 6 7
' '
(Fig.
2
1A) because i t contains one 2-thiocytidine (s C32) residue two bases before
the anticodon so t h i s provides our specific modifiable s i t e . The biological
a c t i v i t i e s tested are charging with an ami no acid by E. c o l i arginyl-tRNA
synthetase, formation of a ternary complex o f modified Arg-tRNA with elongat i o n factor EF-Tu, and GTP, and binding to ribosomes induced by oligonucleotides of defined sequence.
MATERIALS AND METHODS
Unfractionated tRNA from E. coli K12 strain CA 265 wo.s obtained from Microbiological Research Establishment, Porton Down. A four-step procedure was
used to purify tRNA Arg : 1) BD--:ellulose column-chromatogra;jhy
e
at 4°C using
a linear NaCl-gradient from 0.45 M to 0.75 M in a buffer containing 10 mM
Tris HC1 pH 7.6, 10 mM MgCl2, 2 mM NaN3 and 1 mM Na2S2O3; tRNAArg was eluted
at approx. 0.6 M NaCl, 2) column-chromatography on Sepharose 4B
9
applying
a decreasing (NH^SC,- gradient from 1.3 M to 0.7 M in a buffer containing
20 mM NaOAc pH 4.5, 10 mM MgCl2, and 1 mM Na2S203; tRNAArg was eluted at
approx. 1.0 M (NH1))2SOi(, 3) reverse phase chromatography (on RPC-5)
10
ap-
plying a linear NaCl gradient from 0.5-0.6 M i n a buffer containing 10 mM
Tris HC1 pH 7.5, 10 mM MgCl2 and 2 mM Na2S203; tRNAArg was eluted at approx.
0.54 M NaCl, 4) rechromatography on RPC-5. Fractions were assayed for arginine
acceptor a c t i v i t y according to Holladay et a l . 1 1 . The major tRNAi1"9 was obtained at a purity of 1500-1600 pmoles/A 26 o-unit, and it
reacted with approx.
1500 pmoles '"C-iodoacetamide per one A 2 6 o-unit tRNA, indicating one mole of
S2C per mole tRNA. The sequence of IE. c o l i tRNAi rg is shown in Fig. 1A and is
consistent with the major tRNA/ g p u r i f i e d by us although no f u l l sequence
analysis was carried out.
Cellextracts
Ribosomes were prepared
12
from E. c o l i MRE 600 c e l l paste obtained from
Microbiological Research Establishment, Porton Down. The S 100 from ribosome
preparation was either used as crude £ . c o l i enzyme mixture after 1% streptomycin c u t , 70% ammonium sulphate precipitation and batchwise removal of nuc l e i c acids on Sephadex - A 50, or i t was used for the preparation of p u r i 880
Nucleic Acids Research
A
C
C
A
pG > C
C • G
A > U
U > A
C • G
C • G
G . C
A
D
,
C U C G
. . . .
G A G U
G
G
A
D
U
C
L i
c e
NH2
A
C> G A G G
C
I
U
ft
T
W
X
7
C mG
C • G G
u
c<
A
G
G •
G <
c
A
A
C
A
A
c. .u . c. c
s*U
G
o <V~
0
OH
1
R, = — C H 2 — C O — N H 2
R 2 = — CH 2 —CO—NH—(
,N—0
c
A
Fig. 1A:
Nucleotide sequences of E. coli
r 5 6 7
9 » ' . The modification site
tRNA"
2
s C32 is marked by an arrow.
Fig. IB: Structure of the s2C-residue after alkylation with iodoacetamide or spin label.
fied arginyl-tRNA synthetase according to F. von der Haar 13. The purified
synthetase was used for kinetic measurements, the crude enzyme mixture for
other assays.
'"C-iodoacetamide with a specific activity of 57 mCi/mmol was obtained
from the Radiochemical Center, Amersham. The spin label 4-(2-iodoacetamido)2,2,6, 6-tetramethyl piperidino-oxyl (see Fig. IB) was obtained from SYVA
(Palo Alto, Ca., USA).
Alkylations were carried out at 37°C in 10 mM K P C pH 7.2 with 1 mM reagent except for the preparative alkylations with the spin label where the reagent concentration was 2 mM. The tRNA concentration was 0.5 mg/ml in analytical experiments and up to 4 mg/ml (^ 0.12 mM) in preparative incubations.
For preparative purposes the incubation time was approx. 20 hr.
The modification was removed by incubating the alkylated tRNA in 50 mM
HEPES pH 7.0, 10 mM MgCl 2 , 50 mM KC1, 50 mM NH..C1 and 28 mM g-mercaptoethanol
or 1,2-ethanedithiol as indicated at 37°C for 4 hr. The tRNA was desalted by
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Nucleic Acids Research
gel f i l t r a t i o n and precipitated by addition of 2 vol. 96% ethanol before use.
Carbamoylmethylation of tRNAArg was followed by use of '"C-labelled reagent. The extent of alkylation was determined as the amount of TCA-precipitable radioactivity, that was collected on 3 MM-filter paper discs and counted by liquid s c i n t i l l a t i o n .
The specificity of the reaction was analysed by three different methods:
1) Nucleoside analysis by cation exchange column chromatography as described
earlier 1", 2) the extent of alkylation of the s"U-residue in tRNAArg was determined by following the ratio between UV-absorption at 335 nm (characterist i c for s^U) and absorption at 260 nm during reaction with iodoacetamide, and
3) native and alkylated tRNAArg was digested with RNase Ti and analysed by
chromatography on an RPC-5-column (see Fig. 2). 2.25 A26o tRNA/ 9 , or 3 A260
tRNAArg ('"C-acm s2C32) were incubated at 37°C for 3 hr. with 250 units RNase
Ti in 100 ul 100 mM Tris HC1 pH 7.6. The digests were applied to an RPC-5-column (0.5 x 80 cm), and eluted with f i r s t a gradient of 250 ml total volume
ammonium carbonate pH 9.2 from 0.05 M - 2.00 M, and in order to elute the
larger fragments a second gradient 0.1 M - 0.6 M NaCl in 50 mM NH^C03, 100
ml total volume. Chromatography was performed at room temperature at 16<-18
atm pressure, giving a flowrate of approx. 90 ml/hr. Fractions of 2.8 ml were
collected and the UV-absorption of the eluant was monitored on an ISC0-UA5
monitor with a Type 6-optical unit attached. In the case of tRNAirg (llfC-acm
s2C32) 0.5 ml samples were taken from each fraction and counted in Aquasol.
M§?y._for_tetermining_kinetic_garameters °f the_aminoacy.!ation_of_tRNAlj]
Different amounts of tRNAirg were incubated at 37°C in a reaction mixture
containing: 100 mM HEPES pH 6.7, 10 mM MgCl2, 8 mM KC1, 4 mM ATP, 0.24 mM
CTP.6.4 uM 3H-arginine HC1, specific activity 11000 mCi/mmol and 1.46 x 10~*
A280 purified arginine-tRNA-synthetase in a volume of 100 u l . The reaction
was started by the addition of tRNAArg, 20 pi aliquots were taken after T ,
2 ' , 31 and 4", spotted on 2.5 cm Whatman 3 MM f i l t e r discs, that were washed
2 x 10' in 5% TCA and 1 x V in EtOH, dried and counted in toluene-scintillation liquid. The extent of aminoacylation was calculated using a specific act i v i t y of 3080 cpm/pmole (12.7% counting efficiency) and the results plotted
as a function of time. The best straight line was taken as the i n i t i a l rate
of aminoacylation, V o, at the specific tRNA concentration. In all cases less
than 25% of the tRNAArg was aminoacylated within 4 1 . 1^- and Vmax-values were
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Nucleic Acids Research
determined from Eadie-Hofstee-plots, i.e. V o plottet against V 0 /[tRNA].
EF-Tu-GDP was a gift from Dr. D.L. Miller, Roche Institute of Molecular
Biology, New Jersey, USA. EF-Tu-GTP was prepared from EF-Tu-GDP in the following way 1S: 600 pmoles EF-Tu-GDP (1.5 x 10" 5 M) were incubated for 15' at
37°C with 5 mM phosphoenolpyruvate, 5 x 10"5M GTP, 0.25 mg/ml pyruvate kinase, 50 mM HEPES pH 7.0, 10 mM MgCl 2 , 50 mM NH..C1 and 50 mM KC1 in 40 pi total volume. Then approx. 70 pmoles of the tRNA to be tested were added, the
incubation was continued for 5 more min. and the mixture was injected cnto
an Ultrogel AcA44-column .(0.6 x 50 cm) and eluted at 4°C with elution buffer:
10 mM Tris HC1 pH 7.5, 10 mM MgCl 2 and 100 mM NH..C1, at ^ 60 cm hydrostatic
pressure giving a flowrate of 7 ml/hr. 8-drop-fractions were collected and
4 ml of BBS-3-scintillation liquid were added to each fraction before counting. The sample for measuring the ESR-spectra of EF-Tu bound tRNA contained
in 22 yl of the same buffer 0.4 mg (^ 9 nmoles) EF-Tu-GDP, 10 pg pyruvate
kinase, 50 nmoles GTP, 400 nmoles PEP and 2.3 A 2 6 o (^ 3.5 nmoles) 1(*C-arginyl-tRNA rg labelled with the spin label; all components except the tRNA
were incubated at 37°C for 15' before addition of tRNA. In control/experiments GTP and PEP were omitted in order to avoid phosphorylation of the GDP
bound to EF-Tu. After the ESR-measurement the mixture was injected onto an
AcA44-column and chromatographed as described above. From each fraction 50
pi were counted in BBS-3-scintillation liquid, the rest was dried in desiccator at room temperature, taken into 25 pi H 2 0 and used for determination
of the relative amount of spin label from the height of the central peak in
the ESR-spectra. ESR-spectra of charged or uncharged tRNA^rg (SL s 2 C32) were
recorded at the same tRNA-concentration and the same buffer as above.
Ribgsomal_binding_assay
Non-enzymatic triplet dependent binding of native and alkylated arginyltRNA^ rg was tested according to Leder 16. 150 pmoles 3 H-Arg-tRNA Arg were incubated with 2.4 A26o (^ 60 pmoles) ribosomes and 0.15 A 26 o (^ 5000 pmoles)
triplet in 100 mM Tris HC1 pH 7.2, 50 mM NH..C1 and 15 mM MgCl 2 in 50 pi, 2
ml of the ice cold buffer were added, and the mixture was filtered through
a nitrocellulose Millipore filter that was washed three times with 2 ml buffer. The filter was counted in Brays solution. Controls were made without
the triplets. Different Mg2+-concentrations and different incubation conditions were tested. 15 mM MgCl 2 and incubation for 20 min. at 23°C were found
to give the best results.
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Nucleic Acids Research
Trinucieoside diphosphates C-G-C, C-G-G and C-G-U were prepared according
to Leder " , C-G-A-A was a generous g i f t from Dr. J.H. van Boom, Leiden.
ESR-sgectra
ESR-spectra were recorded on a Varian E3-spectrometer using a microwave
power of 5 mW or less to avoid saturation. The samples having a tRNA concentrat i o n of approx. 1.5 x 10'1* M were introduced i n glass c a p i l l a r i e s of about 1
mm inner diameter. The rotational correlation time, T , of the spin label can
be calculated from an expression involving either the linear or the quadrat i c term of the nitrogen quantum number m
18
»19.
The following tensor elements are used A =87 MH , A =A =14 MH , g =
Z
2.0089, g =2.0027
zo
Z
X
j
Z
X
, which gives the following expression for x (involving
the quadratic term i n m):
T = 6.5 xlO-'° x ( | / ^ j
+
-j/hjo|4 2) xAt(o) sec
where AB(o) is the peak-to-peak separation of the central hyperfine component of the derivative spectrum and h(m) is the heights of the peaks.
A l l spectra were recorded four times at 24-26°C with scan times of 8-16
m i n . , and the mean values from the four runs were used in the calculations.
I n no case could any systematic change in the spectra be detected during the
four runs.
RESULTS
As siiown e a r l i e r 2-thiocytidine can be alkylated by a l k y l h a l i d e s , e.g.
iodomethane or iodoacetamide
21
, i n neutral or s l i g h t l y acidic aqueous solu-
tions at low temperatures, ( F i g . I B ) . Z. c o l i tRNAi
9
, was alkylated with i o -
doacetamide in a pseudo - 1st order reaction having a second order rate constant k=2.2 min" 1 M" 1 . For comparison yeast tRNA
, in which the p e n u l t i -
mate nucleoside at the 3'-end normally a c y t i d i n e is enzymatically replaced
by 2 - t h i o c y t i d i n e
stant k=3.1 min"
1
22
is modified under the same conditions with a rate con-
M" 1 .
Nucleoside analysis showed the disappearance of s2C and appearance of
a new nucleoside with the same chromtographic behaviour as acm s2C a f t e r preparative a l k y l a c i o n . I t was also shown that i n agreement with the l i t e r a t u r e
the modification can be removed by certain nucleophiles, e.g. ethanedithiol,
giving r i s e to s2C again, or g-mercaptoethanol, giving rise to C.
The s p e c i f i c i t y of the reaction was analysed by RNase ^ - d i g e s t i o n and
column chromatography on RPC-5, as shown in F i g . 2. Except f o r a small amount
of unreacted ^C-iodoacetamide eluted at the break-through volume, the only
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Nucleic Acids Research
0.07r
0.050.03
1200
800
400
E
a.
Q050.03-
10
20
30
40
110
120
130
fraction
Fig. 2: Analysis of RNase T 1 -digests of A) tRNA^rg modified by
'"C-iodoacetamide, and B) native tRNAA>"g by RPC-5 column chromatography.
Experimental conditions are described under Materials and Methods.
radioactive peak coincides with a new peak in the U.V. absorption-profile.
From the nucleoside analysis of this peak shown in Fig. 3, it is evident
that it contains I, U and acm s2C in nearly equimolar amounts as expected
for acm s2C-U-Ip Ti-fragment. In addition it contains G and smaller amounts
of A and C, the sum of the latter equalling the amount of G, as expected
when some of the dinucleotide peak (containing ApGp and CpGp) is pooled together with the alkylated trinucleotide.
From following the decrease in U.V.-absorption at 338 nm during alkylation and from determination of the amount of radioactivity bound to tRNAi 9
that is not removable by B-mercaptoethanol treatment, it can be concluded
that the extent of alkylation of s'*U under standard conditions is about <W.
Carbamoylmethylation of s2C or conversion
effect on the extent of aminoacylation (Table
has no effect on the kinetics of the reaction
s2C causes a 2-fold decrease in K M and a 2 to
of s2C to C in tRNAi g has no
1). The s2C to C-conversion
either, whereas alkylation of
3 fold decrease in V .
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Nucleic Acids Research
U
(
lt"0.02r
i
u
c
o
0.01-
A
II
o rr
T
N 1
lU'U1 L
5
10
25
c
k
acms2C
50
75
Elution volume (ml)
Fig. 3: Nucleoside analysis of the radioactive Ti-fragment (Fig. 2A)
Aminoacylation
K
M
max
rel
pmoles/A 26 o-unit
tRNA species
tRNAAr9
1680
75
100
tRNAArg (acm s2C32)
1720
37
52
tRNA Arg (SL S2C32)
1650
35
33
1630
70
100
tRNA Arg (acm s 2 C32) +
1640
66
94
tRNA Arg (SL S2C32)+
1605
73
95
tRNA
Arg
+
Table 1: Kinetic parameters for the enzymatic aminoacylation of native
and modified tRNA$r9.
+
tRNA treated with g-mercaptoethanol as described under Materials and
Methods.
Fig. 4A, B show how gel f i l t r a t i o n on an AcA44-column can be used to separate free tRNA and tRNA bound to EF-Tu-GTP. Carbomoylmethylation and spin
l a b e l l i n g of Arg-tRNAj1"9 does not prevent formation of the ternary complex
with EF-Tu-GTP ( F i g . 4C, D). I t i s evident from F i g . 4 that neither the
1U
C-
a l k y l group nor the spin label is released under the conditions of analysis,
as that would give r i s e to
ir
*C-counts and spin label e l u t i n g together with
the small amount of free arginine eluting about f r a c t i o n 32. I t is also e v i dent that a small proportion (<10%) of the modified tRNAT1"9 has not been
aminoacylated giving r i s e to shoulders in the e l u t i o n p r o f i l e of I '*C-carbamoylmethyl group and ESR-signal around f r a c t i o n 26 ( F i g . 4C, D), and that
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Nucleic Acids Research
c
A
•H
cpm
A
\
/\
10002
5OQ2
"c
J
1
v
,
«'\
I
'H
cpm
"
B
i \
/
5000
A
\\
10000
400.5000
x O
200-
D
A
lOOQfi
c pm
o—o
600-
o
•
/
\
/
-.-„-.-,-<'"
ESR
rel
•—•
3-
A
•1
\
"C
c pm
2.500
1-
30
fraction
Fig. 4: Ultrogel AcA44-gel f i l t r a t i o n
A)
B)
3
3
H-Arg-tRNAArg + EF-Tu-GTP
H-Arg-tRNA^rg
3
C) H-Arg-tRNA^rg ('"C-acm s 2 C32) + EF-Tu-GTP
D)I1(C-Arg-tRNAAr9 (SL s 2 C32) + EF-Tu-GTP
Experimental conditions are described under Materials and Methods.
deacylation
n sec
%
tRNA? r g (SL s C32)
1.87
-
Arg-tRNA Arg (SL S2C32)
1.91
30
GDP
1.88
45
(SL S2C32) + EF-Tu •GTP
2.71
<10
Sample
2
Arg-tRNA^ rg (SL S 2 C32) + EF-Tu •
Arg-tRNA^
rg
Table 2: Correlation times of spin labelled t R N A i 9 under different conditions and the extent of deacylation during the time used for recording
the ESR-spectra.
this uncharged tRNAAl"9 does not bind to EF-Tu-GTP.
The rotational correlation time of the spin label is the same when attached to the s2C-residue of uncharged tRNA Arg , charged tRNA Arg , or arginyl887
Nucleic Acids Research
9
in the presence of EF-Tu-GDP (Table 2 ) , indicating that no s t r u c t u -
ral changes occur in that part of the anticodon loop upon aminoacylation.
When the charged tRNA Arg is bound to EF-Tu-GTP some immobilization of the
label is observed from the increase in i - v a l u e , and the ester linkage between
tRNA and amino acid is protected as expected. Gel f i l t r a t i o n experiments show
t h a t about 90% of the arginyl-tRNA^ 1 " 9 (SL s2C32) is s t i l l bound to the prot e i n after the ESR-spectra have been recorded.
tRNA^ rg , tRNA^rg (C32), tRNA*rg (acm s2C32) and tRNA^rg (SL s2C32) were
tested for t h e i r a b i l i t y to bind to ribosomes in response to the codon-cont a i n i n g - t r i p l e t s and tetranucleoside triphosphate : C-G-C, C-G-U, C-G-G and
C-G-A-A. In a l l cases the background binding (codon independent) was between
2 and 3 pmoles arginine bound. The results from the binding experiments
(Table 3) show that tRNA^rg recognizes the codons CGC, CGU and CGA, as expected when the anticodon is ICG. Surprisingly CGG is recognized too i n cont r a d i c t i o n with the wobble-hypothesis that forbids I-G base pairing
to C-conversion at position 32 of tRNAi
g
23
. s2C
does not change the binding charac-
t e r i s t i c s s i g n i f i c a n t l y . Alkylation of the s 2 C-residue reduces the codon dependent binding to ribosomes in response to C-G-C, C-G-U and C-G-A-A, and
destroys the "forbidden" C-G-G dependent binding completely.
tRNA species
Codon-dependent binding
CGU
CGG
CGC
Arg-tRNA^rg
5.7
9.5
4.1
6.9
4.4
8.5
2.8
6.4
Arg-tRNA^ " (acm s C32)
1.0
6.3
0.3
1.9
Arg-tRNA^rg (SL s 2 C32)
1.5
6.6
0.2
2.6
1 9
Arg-tRNA^ " (C32)
1 9
2
(pmoles)
CGA+
Table 3: Non-enzymatic ribosomal binding. Experimental conditions are
described under Materials and Methods.
+
For these experiments C-G-A-A was used because C-G-A was not available.
DISCUSSION
We have been able to show that i t i s possible to modify s p e c i f i c a l l y s2C
residues at d i f f e r e n t positions i n tRNA structures. Not only i s i t possible
to use iodoacetamide and i t s spin labelled derivative f o r the modification
of yeast tRNAPhe containing s2C75 near i t s 3-end (see companion paper ••) but
we have shown in t h i s work that the anticodon loop of £ . c o l i tRNA/ 9 can be
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Nucleic Acids Research
modified likewise at s*C32. The specificity of the reaction can be controlled so that less than 4% of the s 1 *^ in Ec. tRNA'i 9 is simultaneously modi3'-end
fied. In comparison with the rate of modification of the yeast tRNA
Ar 9
we have observed that the rate is somewhat slower for £. coli tRNA consistent with the proposal of a more restricted stacked structure for the anticodon loop position 32 than the 3'-end assuming that £. coli tRNA Arg has the
same tertiary structure as determined for yeast tRNA . Interestingly the
2-position of s 2 C32 is accessible enough to allow full modification at this
position whereas other workers have indications that the 5,6 positions of residue 32 are not very accessible because of reaction with methoxyamine is restricted 2<t and with busulphite is prevented 25 in the case of yeast tRNA Phe .
It is difficult to interpret meaningfully the result of charging the modified tRNA/ 9 by the arginyl-tRNA synthetase. Although the maximum rate V m
of charging and the Km of the reaction are influenced both the modified
tRNA Arg (acm s 2 C32) and tRNA Arg (SL s 2 C32) are fully chargeable. Clearly the
modification size does not radically change the specificity of the enzyme for
charging with arginine. However, the changes measured after modifications indicate tighter binding to the enzyme resulting in a slower rate. Possibly
this reflects a role for the anticodon loop in synthetase binding but an alternative explanation could be that the change in binding to the synthetase
is brought about by a conformational change in the tRNA structure due to the
chemical modification causing a transmitted effect somewhere else.
Other workers have found that modification of the anticodon itself in
tRNA/ 9 can either inhibit charging (at the second position) 26 or have no effect on charging (at the first position) 27 . Thus the role of the anticodon
loop in charging must await further experimentation. We do not feel that it
is too beneficial to compare the results obtained with other tRNAs because
it is possible that different aminoacyl-tRNA synthetases can recognize different parts of tRNA structure.
We found also that the modified arginyl-tRNAs form apparently normal ternary complexes but we have not measured the different binding constants. Unfortunately there is no completely satisfactory method for such measurements
at present. There is much irreproducibility in the assays for ternary complex
formation of aminoacyl-tRNA, GTP and EF-Tu but we used probably the most reliable gel filtration method since it is a direct, positive method. The other
Millipore binding method where only the binary complex EF-Tu:GTP binds to
Millipore filters necessitates measuring the ternary complex formation by difference, a negative method, is clearly not so reliable. Our assay was not
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Nucleic Acids Research
quantitative as an excess of EF-Tu i s necessary for the gel column assays
but our results indicate that the modified Arg-tRNAs formed detectable ternary complexes whose formation did not depend upon the size of the substituent on the s2C32 under the conditions used.
I t was apparent from the shapes of the curves of Fig. 4 that there is no
large difference in binding constants of the modified and native Arg-tRNAs
for EF-Tu and GTP because there was no obvious dissociation of the ternary
complex. Our results make i t likely that EF-Tu does not bind to the anticodon
arm in the region of s2C32, in agreement with the findings that cleavage of
28
the phosphodiester chain in the anticodon of Phe-tRNA
, cyanoethylation
r g 29
and removal of W-base (previously
of anticodon inosine of Arg-tRNA
30
called Y-base) next to the anticodon of yeast Phe-tRNA
seem to have no
effect on ternary complex formation.
Our ESP. results concerning the ternary complex formation with the spin
labelled modified Arg-tRNA (SL szC32) yielded the new interesting finding
that the Arg-tRNA conformation apparently changes on complexing with GTP and
EF-Tu.-we determined a small increase in the correlation time reflecting a
small immobilisation of the label on the semi-invariant residue 31 . The results referred to above make i t likely that the position s2C32 is not involved in interaction with EF-Tu but some long range effect of EF-Tu binding as
an effector has caused this immobilisation. Clearly this finding needs more
investigation to be meaningful.
Another apparent effect on the anticodon loop structure by the modifications was seen in the t r i p l e t dependent ribosomal binding assay. Interestingl y , similar to the results recently reported by other workers working with
yeast 32 and rabbit liver 33 valine tRNAs we found that Arg-tRNA bound to the
70S ribosome significantly with triplets C-G-N (N is any of the 4 standard
nucleosides) reflecting decoding a b i l i t y for the codons-CGN although the anticodon ICG according to the wobble hypothesis 22 should decode only CGU, CGC
and CGA. Thus we have another apparent case in vitro where the decoding uses
only 2 out of 3 positions of the codon in selecting specificity. When we
checked the decoding ability of the modified Arg-tRNAs we observed a reduction in the number of codons possibly reflecting a restriction in the anticodon conformation, perhaps due to the positive charge on the s2C32 residue generated by the modification. This interesting finding must be checked by a
more comprehensive study using the modified tRNAs to decode natural or synthetic mRNAs containing arginine codons.
Finally we wish to point out that we have established the basic features
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of a system which by using further appropriately bifunctional chemical reagents has the potential for crosslinking the £. coli tRNAi 9 to proteins interacting in the neighbourhood of the anticodon loop.
ACKNOWLEDGEMENTS
In addition to generous support from our institutes we are grateful for
support from Aarhus University's Forskningsfond and from EMBO for the award
of a short term fellowship to T.A.K. During part of this work M.S. was a Visiting Professor at Aarhus University.
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