(Received for publication, March 1, 1995; and in revised form, May 19, 1995)
From the
Previously, we have demonstrated that the tRNA-guanine
transglycosylase (TGT) from Escherichia coli is capable of
utilizing an in vitro generated minihelix consisting of the
anticodon stem and loop sequence of E. coli tRNA
A key enzyme involved in the post-transcriptional modification
of tRNA with queuine
(7-(4,5-cis-dihydroxy-1-cyclopenten-3-ylaminomethyl)-7-deazaguanine) is
tRNA-guanine transglycosylase (TGT,
Figure 1:
The primary nucleotide sequences and
possible secondary structures of the E. coli tRNA
Thermal denaturation curves can be
utilized to elucidate the solution conformation adopted by nucleic
acids(6) . Such studies in our system of tRNA analogues
indicate that ECY2-A1 has a T
We have pursued several routes to a stable framework upon
which to base our sequence mutation studies. Kinetic parameters were
determined for several analogues at 20 °C, a temperature at which
ECY2-A1 will adopt a stable minihelix conformation. A non-helical
analogue of ECY2-A1 was also generated and evaluated to investigate the
possibility that the enzyme required sequence but not conformational
information for recognition. The anticodon arm of ECY2-A1 was extended
by 4 base pairs to provide a minihelix with enhanced stability. A yeast
tRNA
Figure 2:
The primary nucleotide sequences and
probable secondary structures of minihelices derived from yeast
tRNA
Figure 3:
Thermal denaturation curves for several
tRNA analogues. The relative absorbances at 260 nm for ECY2-A1 (1), ECY2-A1NH (2), ECY2 (3), ECY1 (4), ECYMH (5), and SCDMH (6) are plotted versus temperature. The profiles were measured in 10 mM sodium phosphate buffer, pH 7, 0.1 mM EDTA, and 150
mM NaCl. The temperature was increased at the rate of 1
°C/min with absorbance readings taken through a range from 10 to 90
°C.
Figure 4:
Michaelis-Menten plots of E. coli tRNA
In previous studies we have demonstrated that the major
elements utilized by TGT in the recognition of tRNA substrates are
found within the anticodon arm of E. coli tRNA
The UV thermal denaturation curves in Fig. 3indicate that the modified full-length tRNA (ECY1) has a
higher T
Our earlier results
indicated that the anticodon arm alone is sufficient for binding and
catalysis to occur but did not eliminate the possibility that TGT
recognizes oligoribonucleotides containing the UGU sequence in the
absence of a defined tertiary structure. The fact that there is no
energy coupling for this enzymic reaction suggests that binding energy
is used to catalyze this base exchange. If so, it is reasonable to
assume that a significant amount of tRNA tertiary structure may be
required to provide this binding energy. To address this question we
generated ECY2-A1NH. In this analogue, we have replaced the 3` side of
the helix (5 bases) with the reverse of the 5` sequence yielding an
analogue incapable of helix formation (Fig. 1). We attempted to
determine the kinetic parameters for this analogue at both 20 and 37
°C and found that in concentrations up to 100 µM the
analogue had no detectable activity at either temperature. The analogue
also does not seem to interact with TGT via a previously published
bandshift assay (5) (data not shown). Finally the analogue is
incapable of inhibiting the incorporation of guanine into the
full-length cognate tRNA (data not shown). These results suggest that
the non-helical analogue does not, to any significant extent, interact
with TGT and that some degree of loop/helix structure is required for
TGT catalysis to occur.
The relative activities of the tRNA
analogues with TGT observed at 20 °C are very much like the trend
we previously reported at 37 °C (Table 1). It is important to
note that each analogue displays a V
We have selected a yeast tRNA
The sequence mutation studies began in the anticodon
loop focusing on the U
G
We have
reached a very similar conclusion to Nakanishi et al.(8) that the U
It should be noted that these
studies are designed to investigate positive recognition elements only
and do not address the possibility that negative recognition elements
for TGT exist elsewhere in the non-cognate tRNA structure.
We thank Dr. David Giegel (Warner-Lambert/Parke-Davis
Pharmaceutical Research Division, Ann Arbor, MI) for assistance with
the UV thermal denaturation experiments.
Note Added in Proof-Further support for our conclusions comes
from recent footprinting experiments by Mueller and Slany (Mueller, S.
O., and Slany, R. K.(1995) FEBS Lett.361, 259-264) that indicate the E. coli TGT protects only
the anticodon stem-loop of tRNA. Our results are also consistent with
those of Carbon et al. (Carbon, P., Haumont, E., Fournier, M.,
de Henau, S., and Grosjean, H.(1983) EMBO J.2, 1093)
where they found that the UGU sequence was the main determinant for
queuine modification in Xenopus oocytes.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(Curnow, A. W., Kung, F. L., Koch, K. A., and Garcia, G. A.(1993) Biochemistry 32, 5239-5246). This suggests that the tRNA
structural motifs necessary for recognition comprise a loop at the end
of a short helix. To gain further insight into the structural
requirements for TGT recognition, we have investigated the conformation
of this minimal substrate. Thermal denaturation studies and kinetic
analyses at 20 and 37 °C indicate that this minihelix is
predominantly melted at 37 °C and that the melted conformation is
not a substrate for TGT. This is confirmed by the determination that a
non-helical analogue of the minihelix is not a substrate for TGT at
either temperature. Two additional minihelices designed to be stable at
37 °C, ECYMH (a 4-base pair extension of the previous minihelix)
and SCDMH (a yeast tRNA
analogue of ECYMH), were
generated and characterized. Finally, several sequence mutants of
SCDMH, focusing on the G
U
base pair and
U
G
U
loop sequence, have been
produced, and kinetic parameter determinations have been performed at
37 °C. Our results are consistent with a recent report (Nakanishi,
S., Ueda, T., Hori, H., Yamazaki, N., Okada, N., and Watanabe, K.
(1994) J. Biol. Chem. 269, 32221-32225) indicating that
a UGU sequence in a 7-base loop is the minimal requirement for TGT
recognition.
(
)EC
2.4.2.29)(1) . In Escherichia coli, TGT catalyzes the
replacement of guanine 34 in the tRNA anticodon with a queuine
precursor, preQ
(7-aminomethyl-7-deazaguanine). preQ
is, in turn, further modified to queuine. The overall process
results in the substitution of guanine 34 with queuine and maturation
of the tRNA molecule. The tRNAs (tRNA
,
tRNA
, tRNA
and tRNA
), which
act as substrates for this enzyme, contain the anticodon sequence, GUN,
where N may be any base(2, 3) . Our laboratory has
initiated the evaluation of the molecular recognition of tRNA by TGT
from E. coli using in vitro generated tRNA analogues
based upon E. coli tRNA
(Fig. 1).
structural analogues. Guanine 34 is underlined in each
analogue.
Comparison of the activity of totally unmodified tRNA (Fig. 1, ECY2) to that for Q-deficient but
otherwise modified tRNA
isolated from a tgt-deficient clone of E. coli revealed that the
other modified bases do not appear to play a significant role in the
recognition of tRNA by TGT (4) . We subsequently found that a
dimeric conformer of tRNA
has a slightly higher K
than monomeric tRNA
(ECY2) but has an equal maximum velocity (5) . Generation
and evaluation of truncated analogues of tRNA
indicated
that ECY2-A1, an analogue corresponding to the 17-base anticodon arm of
tRNA
, was found to have a K
value only 6-fold higher than full-length tRNA
and V
/K
only
40-fold lower (4) . This suggests that, other than the
anticodon arm, the majority of the tRNA structure has very little
effect upon recognition by TGT.
33
°C(7) . Thus, at 37 °C ECY2-A1 is mostly denatured,
suggesting that the specificity constant (V
/K
) calculated
for ECY2-A1 at this temperature may be artificially low since the
analogue will exist in a predominantly melted conformation. A recent
report by Nakanishi et al.(8) suggests that a YUGU
sequence in the anticodon loop of cognate tRNAs is the minimal
requirement for recognition by TGT. These analyses were carried out at
37 °C using 5-base pair stem minihelices similar to ECY2-A1.
Presumably, the minihelices studied will also be predominantly melted
under the conditions of the assay, leaving the results subject to
question.
analogue of this minihelix was used as a framework
for several sequence mutations (Fig. 2) and kinetic evaluation
at 37 °C.
. A, SCDMH includes the yeast tRNA
anticodon domain with a 3-base pair extension of the helical
stem. There are seven sequence mutants whose names are derived by
adding the site of the mutation, using the standard numbering system
for tRNAs, to SCDMH. Guanine 34 has been underlined, and the
site of mutation is indicated with an arrow pointing to the
mutation. The mutant indicated by the parentheses is a double
mutant. B, there are two loop conformation analogues. In
SCDMH6BL cytosine 38 has been deleted to form a 6-base loop, and in
SCDMH8BL a cytosine residue has been added between positions 38 and 39
to form an 8-base loop.
Materials
Chemical reagents were purchased from
Sigma, Boehringer Mannheim, and Life Technologies, Inc. Nucleoside
triphosphates and RNasin were from Boehringer Mannheim. BstNI
restriction endonuclease was from Stratagene.
[8-C]Guanine was from Moravek Biochemicals. TGT
was isolated from an overexpressing clone as described
previously(9, 10) . T7 RNA polymerase was isolated
from E. coli BL21/pAR1219 following the procedure of Grodberg
and Dunn(11) .
Preparation and Purification of the tRNA
Transcript
Unmodified E. coli tRNA and
minihelix RNA analogues were produced via the transcription of a
double-stranded DNA template in an in vitro system and
purified as described previously(4) .
Thermal Denaturation Curves
Thermal denaturation
profiles were determined on a Cary Cotinua Varian spectrophotometer
following the method suggested by Groebe and
Uhlenbeck(12, 13) . The absorbance was monitored at
260 nm for 2 µM RNA solutions in 10 mM sodium
phosphate buffer, pH 7, 0.1 mM EDTA, and 150 mM NaCl.
The temperature was increased at the rate of 1 °C/min with
absorbance readings taken through a range from 10 to 90 °C. The
denaturation curves for ECY1 and ECY2 (the full-length modified and
unmodified analogues of E. coli tRNA), ECY2-A1
(the anticodon microhelix), ECY2-A1NH (the nonhelical analogue of
ECY2-A1), and ECYMH and SCDMH (the extended minihelix analogues) are
presented in Fig. 3. The total change in absorbance for SCDMH is
less than that for the other analogues, most likely due to the fact
that SCDMH contains no adenosine, which contributes largely to the UV
absorbance change.
Kinetic Assays
The kinetic parameter
determinations were performed in a buffer consisting of 100 mM HEPES at pH 7.5, 10 mM MgCl, and 10 mM dithiothreitol. The concentration of
[8-
C]guanine was maintained at 10 µM (about 10
K
(4) )
while the concentration of the tRNA substrates was varied from 0.5 to
100 µM. The general procedure included incubating a
300-µl mixture of buffer, substrate, and 12.6 µg/ml TGT at
either 20 or 37 °C. At 3-min intervals 70-µl aliquots were
removed from the reaction mixture. For the longer tRNA sequences,
>30 nucleotides, the aliquots were quenched in 5% trichloroacetic
acid and filtered onto glass fiber filters (Whatman, GF/C). The filters
were then washed 3 times with 5% trichloroacetic acid and once with
ethanol, dried, and quantitated via liquid scintillation counting. For
the shorter sequences, <30 nucleotides, 10 µl of 3 M NaOAc, pH 5.3, was added to the aliquots, which were then
precipitated with 1.5 ml of ethanol and collected on glass fiber
filters (Whatman, GF/C). The filters were then washed 3 times with
ethanol, dried, and quantitated via liquid scintillation counting.
Initial velocities were determined by linear regression, and from that
data Michaelis-Menten parameters were determined by nonlinear
regression and are displayed in Fig. 4and 5. The kinetic
parameters (K
, V
,
and V
/K
) along
with the standard errors of the fits for the structural analogues at 37
and 20 °C are shown in Table 1and for the sequence mutants
are shown in Table 2. Analogues for which the guanine
incorporation was below the limit of detection (about 0.1% of
full-length tRNA activity) at 100 µM analogue are denoted
as having no detectable activity.
structural analogues. A, at 37 °C:
ECY2 (solidtriangles), ECYMH (solidcircles), and ECY2-A1 (soliddiamonds). B, at 20 °C: ECY2 (opentriangles),
ECYMH (opencircles), and ECY2-A1 (opendiamonds). Solidcurves are calculated
fits of the data by nonlinear regression.
(4, 5) . This was demonstrated
by employing RNA substrates varying widely in gross structure while
maintaining a common motif, the anticodon arm. Such structural
analogues have included full-length E. coli tRNA
, a dimeric form of E. coli tRNA
, and the anticodon arm itself. For example, at
37 °C the anticodon arm minihelix has a V
/K
that is
40-fold lower and a K
that is only 6-fold
higher than that for the full-length tRNA
, suggesting
that the predominate effect of the loss of tRNA structure is upon
catalysis. This is similar to the conclusion drawn by Gu and
Santi(14) , who reported that an 11-base oligoribonucleotide
corresponding to the T
C arm of tRNA
has a k
/K
that
is 300-fold lower and a K
that is 5-fold
higher than that for the full-length tRNA
for the tRNA
(m
US4) methyltransferase and concluded that the effect was
primarily upon catalysis. A recent report from Nakanishi et
al. (8) has probed further into tRNA substrate recognition
by the E. coli TGT. In their study, the kinetic parameters for
several sequence mutants of an RNA minihelix, similar to ECY-A1,
derived from the anticodon arm of E. coli tRNA
have been determined. The conclusion drawn by the authors was
that the YUGU sequence in the anticodon loop is the minimum requirement
for recognition.
than the unmodified tRNA (ECY2)
(61 versus 56 °C). These results are consistent with
earlier findings from studies with tRNA
and
tRNA
, which indicate that base modifications enhance the
stability of the tRNA native conformation (15, 16) .
In addition, the curves for the full-length analogues have gradual
transitions possibly due to the melting of the tertiary base
interactions followed by melting of the base pairs of the four helices,
which make up the secondary structure. The transitions for the smaller
analogues, ECYMH, SCDMH, and ECY2-A1, which presumably have no tertiary
interactions, are much sharper and most probably simply reflect the
melting of the base pairing in the helices. Perhaps the most important
aspect of the thermal denaturation profiles for the minihelices is that
the T
for ECY2-A1 is
33 °C and
the T
for ECYMH
75 °C. If TGT
requires an intact helix leading into the anticodon loop for
recognition, then the kinetic parameters that were calculated for
ECY2-A1 at 37 °C must be artificially low since at this temperature
this analogue will be predominantly denatured.
approximately 5-fold lower at 20 than at 37 °C, reflecting an
overall lower activity for TGT at 20 °C. At the lower temperature
ECYMH has a larger K
than at 37 °C,
while ECY2 has an identical K
at both
temperatures. The change in K
for ECYMH
can be partially explained by the larger error for the values at 20
°C where the enzyme is less active. The relative V
/K
ratio for
ECYMH is similar at both temperatures, suggesting that the analogue is
stable at both temperatures. The relative V
/K
ratio for
ECY2-A1 decreases from a factor of 40 at 37 °C to a factor of 10 at
20 °C, suggesting that at 37 °C only 25% of the analogue is in
the stable helical conformation required for TGT recognition. This is
consistent with the thermal denaturation curve for ECY2-A1 shown in Fig. 3. We conclude that an RNA minihelix with an extended stem,
stable at 37 °C, is the minimum stable framework suitable for
sequence specificity studies.
minihelix framework, SCDMH, in which to carry out site-specific
sequence mutations to investigate the specific bases involved in
substrate recognition by TGT. The x-ray crystal structure for the
full-length yeast tRNA
has been
determined(17, 18, 19) . Future modeling
studies will use this information to generate structures for our
minihelices, which will be used as a framework in which to interpret
our kinetic results. Prior to performing sequence mutations, it is
necessary to determine the kinetic parameters for SCDMH with TGT.
Comparing the results for ECYMH (Table 1) to the results for
SCDMH (Table 2) we find that the analogues have similar kinetic
parameters.
G
U
sequence, which was inferred to be the major determinant for TGT
recognition in earlier sequence homology studies(3) . The only
loop sequence mutant that has any detectable activity is SCDMHU33C,
which has an 80-fold loss in substrate specificity relative to SCDMH.
The other mutants showed no detectable activity in concentrations up to
100 µM. The inactivity of the one-base insertion,
SCDMH8BL, and one-base deletion, SCDMH6BL, mutants (Table 2)
suggests that the size of the loop, and presumably its conformation, is
also vital for recognition of tRNA substrates by TGT.
is conserved among the TGT cognate tRNAs, being base paired
either with C
or U
. The establishment of a
canonical base pair, G
C
in SCDMH, results in
a modest increase in substrate specificity while mutation to
A
U
results in a 2-fold loss in substrate
specificity. Mutation of the G
U
pair to give
C
G
yields a 7-fold loss in substrate
specificity relative to SCDMH. These results suggest that the enzyme
does not have a specific amino acid-base interaction at position 30;
however, it may be that the conformation imparted in the loop by this
base pair plays a role in the recognition of tRNA by TGT.
G
U
sequence found in tRNAs, which are active as substrates for TGT,
is the major recognition determinant within the anticodon domain.
However, we conclude that the 5 base pairs (three G-C and two A-U) in
the anticodon stem investigated by Nakanishi et al. are
inadequate to maintain the stable helical structure necessary for TGT
catalysis to occur optimally at 37 °C. Thus, additional structural
stabilization is necessary in order to form a true minimal substrate.
This stabilization may be provided by: 1) lowering the assay
temperature, 2) additional canonical base pairs and the tertiary
interactions of distant bases, in the instance of the full-length tRNA,
or 3) by extending the anticodon stem by 3-4 base pairs, in the
instance of a minihelix analogue.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.