3 March
5 Number
1978
Volume
Volue
5
umbr
3
arch197
Nucleic Acids Research
Nuceic
cid
Resarc
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-Institut fUr Experimentelle Medizin, Abteilung Chemie, G8ttingen, GFR
Received 5 January 1978
ABSTRACT
Escherichia coli arginine tRNA, has been modified at position s2C32 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-tRNA, decodes all 4
codons beginning with CG. The modified Arg-tRNA, 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
tRNAI'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. 1B) containing a spin label3,4
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
C Information Retrieval Limited 1 Falconbeg Court London WI V 5FG England
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Nucleic Acids Research
also been used in a companion study I on yeast tRNAPhe in which s2C has been
enzymically introduced.
Our general approach is to test the modified tRNA for several biological
functions. The E. coli tRNA used in these experiments is tRNAl 9 5,6,7 (Fig.
1A) because it contains one 2-thiocytidine (s2C32) residue two bases before
the anticodon so this provides our specific modifiable site. The biological
activities tested are charging with an amino acid by E. coli arginyl-tRNA
synthetase, formation of a ternary complex of modified Arg-tRNA with elongation factor EF-Tu, and GTP, and binding to ribosomes induced by oligonucleotides of defined sequence.
MATERIALS AND METHODS
Purification of tRN Arg
Unfractionated tRNA from E. coli K12 strain CA 265 was obtained from Microbiological Research Establishment, Porton Down. A four-step procedure was
used to purify tRNAArg: 1) BD-cellulose column-chromatogra;hy 8 at 40C using
a linear NaCl-gradient from 0.45 M to 0.75 M in a buffer containing 10 mM
Tris HCl pH 7.6, 10 MM MgCl2, 2 mM NaN3 and 1 nM Na2S203; tRNAArg was eluted
at approx. 0.6 M NaCl, 2) column-chromatography on Sepharose 4B I applying
a decreasing (NH4)2SO4- gradient from 1.3 M to 0.7 M in a buffer containing
20 mM NaOAc pH 4.5, 10 mM MgCl2, and 1 flM Na2S203; tRNAArg was eluted at
approx. 1.0 M (NH4)2SO4, 3) reverse phase chromatography (on RPC-5) 10 applying a linear NaCl gradient from 0.5-0.6 M in a buffer containing 10 mM
Tris HC1 pH 7.5, 10 mM MgCl2 and 2 nmM Na2S203; tRNAArg was eluted at approx.
0.54 M NaCl, 4) rechromatography on RPC-5. Fractions were assayed for arginine
acceptor activity according to Holladay et al.11. The major tRNAArg was obtained at a purity of 1500-1600 pmoles/A260-unit, and it reacted with approx.
1500 pmoles 14C-iodoacetamide per one A260-unit tRNA, indicating one mole of
s2C per mole tRNA. The sequence of E. coli tRNA
is shown in Fig. A and is
consistent with the major tRNAArg purified by us although no full sequence
analysis was carried out.
Cel 1 extracts
Ribosomes were prepared 12 from E. coli MRE 600 cell paste obtained from
Microbiological Research Establishment, Porton Down. The S 100 from ribosome
preparation was either used as crude E. coli enzyme mixture after 1% streptomycin cut, 70% ammonium sulphate precipitation and batchwise removal of nucleic acids on Sephadex - A 50, or it was used for the preparation of puri880
Nucleic Acids Research
A
c
C
A
pG * C
C * G
A * U
U * A
C * G
C * G
G * C
c c u CC
s4U
C G A
D
G
G
A D A
NH2
*
*
A
cu c G
..A.
*
*
GGAGG
C
U A A
0
TU)p
0
OH
x
cr m7G
%a
AAU
A
C
U
C
G
G
U
U2c
*
*
*
*
*
G
G
A
G
C
C
RI = -CH2-CO-NH2
R2 = -CH2-CO -NH
¶N-O
A
I
m2A
A
Fig. 1A: Nucleotide sequences of E. coli
tRNAArg 5956_7 * The modification sTte
s2C32 -is marked by an arrow.
Fig. 1B: Structure of the s2C-residue after alkylation with iodoacetamide or spin label.
fied ariginyl-tRNA synthetase according to F. von der Haar 13 * The purified
synthetase was used for kinetic measurements, the crude enzyme mixture for
other assays.
Alkyation of
tRNAI-9
14C-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. 1B) was obtained from SYVA
(Palo Alto, Ca., USA).
Alkylations were carried out at 370C in 10 mM KPO4 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 p1l 7.0, 10 mM MgCl2, 50 mM KC1, 50 mM NH4Cl and 28 mM S-mercaptoethanol
or 1,2-ethanedithiol as indicated at 37°C for 4 hr. The tRNA was desalted by
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gel filtration and precipitated by addition of 2 vol. 96% ethanol before use.
Anal,ysis of -alkyl ation
Carbamoylmethylation of tRNA,rg was followed by use of '4C-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 scintillation.
The specificity of the reaction was analysed by three different methods:
1) Nucleoside analysis by cation exchange column chromatography as described
earlier 14, 2) the extent of alkylation of the s4U-residue in tRNAArg was determined by following the ratio between UV-absorption at 335 nm (characteristic for s4U) and absorption at 260 nm during reaction with iodoacetamide, and
3) native and alkylated tRNA, was digested with RNase T1 and analysed by
chromatography on an RPC-5-column (see Fig. 2). 2.25 A260
or 3 A2 6p
tRNA
(4C-acm s2C32) were incubated at 37°C for 3 hr. with 250 units RNase
T1 in 100 pl 100 mM Tris HC1 pH 7.6. The digests were applied to an RPC-5-column (0.5 x 80 cm), and eluted with first 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 NH4CO3, 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 ISCO-UA5
monitor with a Type 6-optical unit attached. In the case of tRNAArg (14C-acm
s2C32) 0.5 ml samples were taken from each fraction and counted in Aquasol.
tRNAArg
Assay for determining kinetic_parameters
of the
aminoacylation
of
I
Arg
Different amounts of tRNAArg were incubated at 370C 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/mr,;ol and 1.46 x 10-4
A280 purified arginine-tRNA-synthetase in a volume of 100 p1. The reaction
was started by the addition of tRNAArg 20 p1 aliquots were taken after 1',
2', 3' and 4', spotted on 2.5 cm Whatman 3 MM filter discs, that were washed
2 x 10' in 5% TCA and 1 x 2' in EtOH, dried and counted in toluene-scintillation liquid. The extent of aminoacylation was calculated using a specific activity 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 initial rate
of aminoacylation, VO, at the specific tRNA concentration. In all cases less
than 25% of the tRNAArg was aminoacylated within 4'. KM- and Vmax-values were
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determined from Eadie-Hofstee-plots, i.e. V0 plottet against
Formation of
Vo/[tRNA].
ternary com2l2ex. EF-Tu.GTP-arginyl:tRNAIrg
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 15: 600 pmoles EF-Tu.GDP (1.5 x 10 5M) were incubated for 15' at
270C with 5 mM phosphoenolpyruvate, 5 x 10 5M GTP, 0.25 mg/ml pyruvate kinase, 50 mM HEPES pH 7.0, 10 mM MgCl2, 50 mM NH4Cl and 50 mM KCI in 40 pl 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 40C with elution buffer:
10 mM Tris HCl pH 7.5, 10 mM MgCl2 and 100 mM NH4Cl, at X' 60 cm hydrostatic
pressure giving a flowrate of 7 ml/hr. 8-drop-fractions were colklcted and
4 ml of BBS-3-scintillation liquidwereadded to each fraction before counting. The sample for measuring the ESR-spectra of EF-Tu bound tRNA contained
in 22 }l of the same buffer 0.4 mg ("s 9 nmoles) EF-Tu.GDP, 10 pg pyruvate
kinase, 50 nmoles GTP, 400 nmoles PEP and 2.3 A260 (ni 3.5 nmoles) 14C-arginyl-tRNAAr9 labelled with the spin label; all components except the tRNA
were incubated at 37°C for 15' before addition of tRNA. In control/txperiments 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
pl were counted in BBS-3-scintillation liquid, the rest was dried in desiccator at room temperature, taken into 25 pl H20 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 tRNAArg (SL s2C32) were
recorded at the same tRNA-concentration and the same buffer as above.
Ribosomal
binding
assay
Non-enzymatic triplet dependent binding of native and alkylated arginyltRNA,9 was tested according to Leder 16. 150 pmoles 3H-Arg-tRNAAr9 were incubated with 2.4 A260 ('\, 60 pmoles) ribosomes and 0.15 A260 (". 5000 pmoles)
triplet in 100 mM Tris HCl pH 7.2, 50 mM NH4Cl and 15 mM MgCl2 in 50 pl, 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+ concentrationsand different incubation conditions were tested. 15 nM MgCl2 and incubation for 20 min. at 230C were found
to give the best results.
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Trinucleoside diphosphates C-G-C, C-G-G and C-G-U were prepared according
to Leder 17 C-G-A-A was a generous gift from Dr. J.H. van Boom, Leiden.
ESR-spectra were recorded on a Varian E3-spectrometer using a microwave
powerof5 mWor less to avoid saturation. The samples having a tRNA concentration of approx. 1.5 x 10 4 M were introduced in glass capillaries 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 quadratic term of the nitrogen quantum number m 18,19.
The following tensor elements are used Az=87 MHZ, Ax=Ay=14 MHz, gx=
2.0089, gz=2.0027 '-, which gives the following expression for T (involving
the quadratic term in m):
T = 6.5 x 10-1 X ( /ii +
; 2) x AS(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.
All spectra were recorded four times at 24-260C with scan times of 8-16
min., and the mean values from the four runs were used in the calculations.
In no case could any systematic change in the spectra be detected during the
four runs.
RESULTS
As sihown earlier 2-thiocytidine can be alkylated by alkylhalides, e.g.
iodomethane or iodoacetamide 21, in neutral or slightly acidic aqueous solutions at low temperatures, (Fig. 1B). E. coli tRNAlAr , was alkylated with iodoacetamide in a pseudo - 1st order reaction having a second order rate constant k=2.2 min-1 M 1. For comparison yeast tRNAPhe, in which the penultimate nucleoside at the 3'-end normally a cytidine is enzymatically replaced
by 2-thiocytidine 22 is modified under the same conditions with a rate constant k=3.1 min-1 M 1.
Nucleoside analysis showed the disappearance of s2C and appearance of
a new nucleoside with the same chromtographic behaviour as acm s2C after preparative alkylation. It was also shown that in agreement with the literature
the modification can be removed by certain nucleophiles, e.g. ethanedithiol,
giving rise to s2C again, or a-mercaptoethanol, giving rise to C.
The specificity of the reaction was analysed by RNase Tl-digestion and
column Chromatography on RPC-5, as shown in Fig. 2. Except for a small amount
of unreacted '4C-iodoacetamide eluted at the break-through volume, the only
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Nucleic Acids Research
A
0
°
05
-~
~
~ ~
OL03
10
20
30
~
~
50
~
60
70
80
90
100
110
120
130
f ract ion
Fig. 2: Analysis of RNase Ti-digests of A) tRNAArg modified by
14C-iodoacetamide, and B) native tRNAArg 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 T1-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 tRNA9
that is not removable by 0-mercaptoethanol treatment, it can be concluded
that the extent of alkylation of S4U under standard conditions is about 4%.
Aminoacylation
of modified tRNAAr_
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 KM and a 2 to
of s2C to C in
Ar has no
1). The s2C to C-conversion
either, whereas alkylation of
3 fold decrease in Vmax'
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Nucleic Acids Research
.0a.
QOlL
0~~~~~~~~
C4
~~5
i
10
25
50
75
Elution volume (ml)
Fig. 3: Nucleoside analysis of the radioactive Ti-fragment (Fig. 2A) 14.
Aminoacylation
pmoles/A260-unit
KM
nM
Vma,
rel
1680
75
100
1720
37
52
tRNA,9 (SL s2C32)
1650
35
33
tRNAArg
tRNAArg
+
1630
70
100
(acm s2C32)+
1640
66
94
tRNAIArg
(SL S2C32)+
1605
73
95
tRNA species
tRNA,r9
|tRNAArg
(acm s2C32)
Table 1: Kinetic parameters for the enzymatic aminoacylation of native
and modified tRNA rg.
+tRNA treated with S-mercaptoethanol as described under Materials and
N2thods.
Formation of the
ternary
complex
EF-Tu-GTP.arginXl:tRN Arg
Fig. 4A, B show how gel filtration on an AcA44-column can be used to separate free tRNA and tRNA bound to EF-Tu-GTP. Carbomoylmethylation and spin
labelling of Arg-tRNAArg does not prevent formation of the ternary complex
with EF-Tu.GTP (Fig. 4C, D). It is evident from Fig. 4 that neither the "4Calkyl group nor the spin label is released under the conditions of analysis,
as that would give rise to 14C-counts and spin label eluting together with
the small amount of free arginine eluting about fraction 32. It is also evident that a small proportion (<10%) of the modified tRNAIrg has not been
aminoacylated giving rise to shoulders in the elution profile of 14C-carbamoylmethyl group and ESR-signal around fraction 26 (Fig. 4C, D), and that
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Nucleic Acids Research
Fig. 4: Ultrogel AcA44-gel filtration
A) 3H-Arg-tRNA Ar9 + EF-Tu.GTP
H-Arg-tRNA1rr
C) 3H-Arg-tRNAAr9 (14C-acm s2C32) + EF-Tu-GTP
D)14C-Arg-tRNAAr9 (SL s2C32) + EF-Tu.GTP
B)B)
3
Experimental conditions are described under Materials and Methods.
T
Sample
tRNA'j9
n sec
(SL
Arg-tRNAAr9
Arg-tRNAAlrg
|Arg-tRNAArg
.
deacylation
%
s2C32)
1.87
-
(SL s2C32)
1.91
30
+ EF-Tu.GDP
1.88
45
+ EF-Tu.GTP
2.71
<10
(SL
(SL
s2C32)
s2C32)
.~~~~~~~~~~~r
Table 2: Correlation times of spin labelled tRNA9 under different conditions and the extent of deacylation during the time used for recording
the ESR-spectra.
this uncharged tRNA, 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 tRNAArg, charged tRNA/Ar9, or arginyl887
Nucleic Acids Research
tRNA I9 in the presence of EF-Tu GDP (Table 2), indicating that no structural changes occur in that part of the anticodon loop upon aminoacylation.
When the charged tRMArg is bound to EF-Tu-GTP some immobilization of the
label is observed from the increase in T-value, and the ester linkage between
tRNA and amino acid is protected as expected. Gel filtration experiments show
that about 90% of the arginyl-tRNA, (SL s2C32) is still bound to the protein after the ESR-spectra have been recorded.
Non-enzymtic ribosomal binding
(C32 tRNA
tNA9, tRNA
(acm s2C32) and tRNAI (SL s2C32) were
tested for their ability to bind to ribosomes in response to the codon-containing-triplets and tetranucleoside triphosphate: C-G-C, C-G-U, C-G-G and
C-G-A-A. In all cases the background binding (codon independent) was between
2 and 3 pmoles arginine bound. The results from the binding experiments
(Table 3) show that tRMArg recognizes the codons CGC, CGU and CGA, as expected when the anticodon is ICG. Surprisingly CGG is recognized too in contradiction with the wobble-hypothesis that forbids I-G base pairing 23. s2C
to C-conversion at position 32 of tRNAlArg does not change the binding characteristics significantly. Alkylation of the s2C-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
CGG
CGC
CGU
(pmoles)
CGA+
Arg-tRNAArg
Ar-t
rgA
5.7
9.5
4.1
6.9
Arg-tRNA,9 (C32)
4.4
8.5
2.8
6.4
Arg-tRNAArg (acm s2 C32)
LAr-tRNAArg (SL s2C32)
1.0
6.3
0.3
1.9
1.5
6.6
0.2
2.6
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 it is possible to modify specifically s2C
residues at different positions in tRNA structures. Not only is it possible
to use iodoacetamide and its spin labelled derivative for the modification
of yeast tRNAPhe containing s2C75 near its 3-end (see companion paper 4) but
we have shown in this work that the anticodon loop of E. coli tRNAArg can be
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Nucleic Acids Research
modified likewise at s2C32. The specificity of the reaction can be controlled so that less than 4% of the s4U8 in Ec. tIRNArg is simultaneously modified. In comparison with the rate of modification of the yeast tRNAPhe 3'-end
we have observed that the rate is somewhat slower for E. coli tRNAArg consistent with the proposal of a more restricted stacked structure for the anticodon loop position 32 than the 3'-end assuming that E. coli tRNAArg has the
same tertiary structure as determined for yeast tRNA e. Interestingly the
2-position of s2C32 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 24 and with busulphite is prevented 25 in the case of yeast tRNAPhe.
It is difficult to interpret meaningfully the result of charging the modified tRNAArg by the arginyl-tRNA synthetase. Although the maximum rate Vm
of charging and the Km of the reaction are influenced both the modified
tRNAIr9 (acm s2C32) and tRNArg (SL s2C32) 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
tRNAArg 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 dif-
ference, 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 is 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.
It 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 it likely that EF-Tu does not bind to the anticodon
arm in the region of s2C32, in agreement with the findings that cleavage of
the phosphodiester chain in the anticodon of Phe-tRNAPhe 28, cyanoethylation
of anticodon inosine of Arg-tRNA
29 and removal of W-base (previously
called Y-base) next to the anticodon of yeast Phe-tRNAPhe 30 seem to have no
effect on ternary complex formation.
Our ESR results concerning the ternary complex formation with the spin
labelled modified Arg-tRNA (SL s2C32) 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 it 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 triplet dependent ribosomal binding assay. Interestingly, 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 ability 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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Nucleic Acids Research
of a system which by using further appropriately bi-functional chemical reagents has the potential for crosslinking the E. coli tRNAArg 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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