ABSTRACT
tRNA in which uracil is completely replaced by 5-nitro-uracil was prepared by substituting 5-nitro-UTP for UTP in an
in vitro
transcription reaction. The rationale was that the 5-nitro substituent activates the 6-carbon of the Ura heterocycle towards nucleophiles, and hence could provide mechanism-based inhibitors of enzymes which utilize this feature in their catalytic
mechanism. When assayed shortly after mixing, the tRNA analog, NO
2
Ura-tRNA, is a potent competitive inhibitor of tRNA-Ura methyl transferase (RUMT). Upon incubation, the analog causes a time-dependent inactivation of RUMT which could be reversed by dilution into a large excess of tRNA substrate. Covalent RUMT-NO
2
Ura-tRNA complexes could be isolated on nitrocellulose filters or by SDS-PAGE. The interaction of RUMT and NO
2
Ura-tRNA was deduced to involve formation of a reversible complex, followed by
formation of a reversible covalent complex in which Cys 324 of RUMT is linked to the 6-position of NO
2
Ura 54 in NO
2
Ura-tRNA.
Escherichia coli
tRNA (uracil-5-)-methyltransferase (RUMT) catalyzes the S-adenosylmethionine (AdoMet)-dependent methylation of a specific Urd residue to form the m
5
U residue found in the T-loop of most prokaryotic and eukaryotic tRNA. The catalytic mechanism of
RUMT is analogous to the mechanisms of thymidylate synthase (TS) and DNA-(m
5
C)-methyltransferase (
1
-
3
). The mechanism involves initial formation of a covalent Michael adduct between
the thiol of Cys 324 of the enzyme and the 6-carbon of U54 of tRNA (
2
,
4
) which serves to activate the 5-position of U54 for the subsequent one-carbon transfer. We have previously reported that, in the absence of
AdoMet, RUMT forms binary covalent complexes with unmodified tRNA
Phe
or synthetic tRNA
Phe
containing 5-fluoroUra substituted for Ura (FUra-tRNA), which may be isolated on nitrocellulose filters or by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (
5
).
The inhibitory and substrate properties of a number of 5-substituted dUMPs with TS suggested that electron withdrawing groups at the 5-position increase the affinity of such analogs for the enzyme,
probably through increasing the reactivity of the 6-carbon toward nucleophiles (
6
-
8
). Indeed, C-6 of the analog 5-nitro-2-deoxyuridine monophosphate (NO
2
dUMP) forms a sufficiently strong covalent attachment with thiols, that a
covalent TS-NO
2
dUMP complex can be physically isolated (
9
). Although covalent, the TS-NO
2
dUMP complex is reversible, and free enzyme and inhibitor can be restored by [beta]-elimination of the enzyme from C-6 of the pyrimidine. It was reasonable to believe that other
enzymes whose mechanisms involve covalent attachment of a nucleophilic catalyst
to C-6 of pyrimidine substrates would likewise be inhibited by substrates
containing the NO
2
Ura substituent. In this paper, we describe the synthesis and characterization of tRNA containing NO
2
Ura, and its potent mechanism-based inhibition of RUMT.
5-Nitro-uridine triphosphate (NO
2
UTP) was prepared by a slight modification of the method of Huang and Torrence (
10
). Briefly, the trisodium salt of UTP (Yamasa, Choshi, Japan; 550 mg, 1 mmol) was mixed with a solution of nitronium tetrafluoroborate (Aldrich, 0.5 M, 10 ml) in sulfolane. The solution was stirred for 48 h at room
temperature. Chloroform (50 ml) was added to the mixture, and the resulting
precipitate was collected by centrifugation, washed with chloroform (2 * 20 ml), and dissolved in water (20 ml). The pH of the solution was
adjusted to ~7 by the addition of concentrated ammonium hydroxide. The solution was applied to a DEAE-cellulose A-200m column (Chisso, Japan; 2 * 30 cm), which was washed with 0.05 M triethylammonium
bicarbonate (pH 7.9, 500 ml) and eluted with a linear gradient (0.05-0.25 M) of triethylammonium bicarbonate (pH 7.9, total volume 3 l).
Appropriate triphosphate fractions were pooled, evaporated, and co-evaporated several times with water
in vacuo
to give NO
2
UTP (~20% yield as a yellow glass).
Plasmid p67YF0 used for preparation of unmodified yeast tRNA
Phe
was a gift from O. C. Uhlenbeck (Department of Chemistry and Biochemistry,
University of Colorado, Boulder, CO). T7 RNA polymerase was isolated from
E.coli
BL21 harboring the plasmid pAR1219 (J. J. Dunn, Brookhaven National Laboratory,
Upton, New York). [5'-
32
P]pCp (3000 Ci/mmol) was from Amersham. [
32
P]RNA was purified on urea-PAGE (
11
). RUMT was purified as previously described (
12
,
13
).
T7 RNA polymerase-catalyzed
in vitro
synthesis of tRNA
Phe
containing 5-nitroUra substituted for Ura (NO
2
Ura-tRNA)
was performed using
Bst
NI-digested p67YF0 and 100 [mu]M NO
2
UTP in place of 1 mM UTP as described (
14
). Products were purified using 7 M urea-20% PAGE. Unmodified tRNA, FUra-tRNA and m
5
U54-tRNA were prepared as described (
5
). The concentrations of tRNA and NO
2
Ura-tRNA were calculated using 1.6 nmol per 1 A
260
(
15
).
In vitro
synthesized NO
2
Ura-tRNA was labeled at the 3'-end with T4 RNA ligase (New England Biolabs) and [5'-
32
P]pCp (
16
). The 3'-end-labeled tRNA was purified by electrophoresis on 7 M urea-12% PAGE.
The thermal denaturation profiles of tRNA were obtained using a Cary 3E
spectrophotometer with a temperature controller and interfaced to a
microcomputer. The temperature was increased from 25 to 95oC at a rate of 1oC/min. Ten data points were collected/min. The tRNA samples in 23 mM
potassium phosphate, pH 7.45, 45 mM KCl and 10 mM spermine (
14
), were heated to 95oC, then slowly cooled to room temperature prior to obtaining the thermal
profiles.
Reaction mixtures (20 [mu]l) containing 50 nM [
32
P]NO
2
-Ura-tRNA and varying concentrations of RUMT (0.05-4 [mu]M) in binding buffer [50 mM N-tris (hydroxymethyl)-methyl-2-aminoethane sulfonic acid (TES),
pH 6.6, 5 mM dithiothreitol, 2 mM MgCl
2
, 1 mM EDTA and 50 mM NaCl] (
2
) were incubated at 15oC for 60 min, and assayed by nitrocellulose filtration (
5
,
17
). With saturating RUMT, ~50% of the NO
2
Ura-tRNA was trapped, which we assume represents the filtration efficiency for
the combination of noncovalent and covalent complexes. The apparent
dissociation constant (K
app
) for NO
2
Ura-tRNA was obtained by nonlinear least-squares fit of the binding data to the equation:
RNA
bound
/RNA
total
= 1/(1 + K
app
/E
total
) (
5
).
The nitrocellulose binding assay was used to determine the bimolecular rate
constant (k
1
) for association of RUMT and NO
2
Ura-tRNA to form isolable binary complexes. The [
32
P]NO
2
Ura-tRNA (1 nM) was incubated with specified concentrations of RUMT (50-150 nM) at 15oC in binding buffer. The initial rate was obtained from plots
of bound RNA versus time over the first 10% of the reaction (
5
). The dissociation of total noncovalent plus covalent bound complexes was
monitored by adding a large excess of unlabelled, unmodified tRNA to
equilibrated RUMT-[
32
P]NO
2
Ura-tRNA complexes, and measuring the bound radioactivity by the
nitrocellulose filtration assay with time. The rate constants were calculated
from the half lives for dissociation of total noncovalent plus covalent
complexes obtained from a plot of bound RNA versus time (
5
).
K
app
was obtained by non-linear least squares fit of the binding data in Figure
4
to an equation that calculates free ligand concentration after correction for
depletion of NO
2
Ura-tRNA by complex formation (
18
).
Gel shift assays were performed as previously described (
2
,
5
). Reaction mixtures (400 [mu]l) containing 50 nM [
32
P]NO
2
-Ura-tRNA (2 * 10
5
c.p.m.) and 8 [mu]M RUMT in binding buffer, were incubated at 15oC. Aliquots (20 [mu]l) were removed at the indicated times and the components were
separated on SDS-12% PAGE. The
32
P-containing bands were excised, extracted and counted in 5 ml Aquasol II (
5
). Covalent complexes were quantitated as the fraction of total tRNA that was
covalently bound.
Typically, reaction mixtures (20 [mu]l) containing 1 [mu]M unmodified tRNA, 25 [mu]M [
3
H-Me]AdoMet (2 Ci/mmol) and 0.2 [mu]M RUMT in binding buffer were incubated at 15oC. An aliquot (18 [mu]l) was removed at a specified time for DEAE-paper disk assay (
5
). The assay efficiency was ~60% for
3
H-methyl incorporation into tRNA (data not shown).
Data points in the figures represent duplicate determinations.
NO
2
Ura-tRNA was prepared by T7 RNA polymerase-catalyzed
in vitro
transcription of the yeast tRNA
Phe
gene contained within p67YFO using NO
2
UTP instead of UTP. With NO
2
UTP, the reaction mixture yielded 0.5 A
260
of transcript/ml, which corresponds to 20 mol RNA/mol template. After the
transcript was labeled with 5'-
32
pCp, analysis on 7 M urea-20% PAGE showed a major radioactive RNA product that migrated as did
unmodified tRNA. Complete RNase T2 digestion of this labeled product followed by separation of the products on two-dimensional TLC showed that ~90% of the transcripts had the expected 3' terminal adenosine (
19
). NO
2
Ura-tRNA showed a sharp T
m
of 84
o
C, which was 14oC higher than unmodified tRNA (Fig.
1
).
NO
2
Ura-tRNA was not methylated by RUMT (data not shown), but did inhibit the
methylation of unmodified tRNA by RUMT. NO
2
Ura-tRNA was a competitive inhibitor with respect to tRNA when the reaction was initiated with enzyme (Fig.
2
). The following constants were calculated from Figure
2
: K
m
= 0.37 [mu]M, k
cat
= 1.9 min
-1
and K
i
= 1.0 [mu]M.
A minimal mechanism for the interaction of RUMT with NO
2
Ura-tRNA is proposed in Scheme
1
. First, there is reversible formation of the noncovalent RUMT-NO
2
Ura-tRNA binary complex
1
characterized by rate constants k
1
and k-
1
and dissociation constant K
1
. Secondly, there is unimolecular conversion of
1
to one or more covalent complexes
2
characterized by apparent rate constants k
2
and k-
2
and apparent dissociation constant K
2
(K
2
= [noncovalent complex]/[covalent complex] = k
-2
/k
2
). We assessed these constants as described for the analogous RUMT-tRNA binary complexes (
20
).
A nitrocellulose filter binding assay commonly used to trap protein-RNA complexes was used to measure native noncovalent plus covalent RUMT-NO
2
Ura-tRNA complexes (
5
). In this assay, free [
32
P]NO
2
Ura-tRNA is almost completely removed (counts <150 c.p.m.) (data not shown). At an 80-fold excess of RUMT, 50% of the added NO
2
Ura-tRNA was trapped, which represents the filtration efficiency of the assay
(
21
). This is slightly lower than the filtration efficiency (65%) for the RUMT-tRNA complex (
5
).
Rates of association of RUMT and NO
2
Ura-tRNA were measured by mixing excess RUMT with [
32
P]NO
2
Ura-tRNA, and counting the nitrocellulose-bound radioactivity. Initial rates were obtained over the first 10%
of the reaction from plots of bound RNA versus time (Fig.
5
), and k
1
values were calculated from the equation for a bimolecular reaction. The k
1
was 4-fold lower for NO
2
Ura-tRNA than for unmodified tRNA (Table
1
).
In the present work, we have described the
in vitro
synthesis of NO
2
Ura-tRNA, its partial characterization and its interaction with RUMT. The
synthesis of NO
2
Ura-tRNA with complete substitution of NO
2
Ura for Ura was accomplished by substitution of NO
2
UTP for UTP in a standard plasmid-template directed
in vitro
synthesis reaction. The NO
2
Ura-tRNA was characterized by PAGE, 3'-nucleotide analysis and melting profile. Interestingly, the
melting temperature of the analog was 14oC higher than tRNA which is probably due to the stability afforded by the higher acidity and
therefore higher hydrogen bond forming ability of the 3-NH of NO
2
Urd residues.
Our studies demonstrated that NO
2
Ura-tRNA was a mechanism-based inhibitor of RUMT. As depicted in Scheme
1
, the interaction is
proposed to involve the formation of a reversible, noncovalent enzyme-inhibitor complex, followed by the formation of a reversible covalent
bond between a nucleophile of the enzyme and the 6-carbon of NO
2
Ura-tRNA at position 54. The proposed mechanism of inhibition is directly
analogous to that previously established for interaction of NO
2
dUMP with thymidylate synthase (TS) (
9
).
The experimental evidence for the mechanism of interaction of NO
2
Ura-tRNA with RUMT is as follows. First, NO
2
Ura-tRNA initially inhibits the methylation activity of RUMT competitively
with respect to the substrate tRNA (Fig.
2
). This implicates formation of a reversible RUMT-NO
2
Ura-tRNA complex (Scheme
1
) analogous to the normal RUMT-tRNA binary complex. Secondly, upon preincubation of RUMT with NO
2
Ura-tRNA, there is a time-dependent loss of enzyme activity (Fig.
3
). The enzyme inactivation is consistent with conversion of the noncovalent
complex to a much tighter complex, such as covalent complex
2
(Scheme
1
). Scheme
2
shows the conversion of the reversible complex to the
covalent complex. Thirdly, the kinetics of dissociation of the RUMT-NO
2
Ura-tRNA complexes are biphasic (Fig.
6
), suggesting the presence of at least two distinct chemical species which we
have assigned to the non-covalent and covalent forms of the complex (
1
and
2
). Finally, SDS-PAGE allows isolation of a RUMT-NO
2
Ura-tRNA complex which is most consistent with covalent complex
2
. Since Cys 324 of RUMT has been identified as the covalent catalyst of the
normal reaction with unmodified tRNA, it is most reasonable to suggest that the
Cys 324 thiol is the enzyme nucleophile attached to NO
2
Ura-tRNA in the covalent complex.
This work was supported by USPHS Grant CA-14394 from the National Institutes of Health (awarded to D.V.S.).
REFERENCES
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