From the
A filter binding assay was used to determine the structural
features of L-tryptophan required for activation of TRAP, the
trp RNA-binding attenuation protein of Bacillus
subtilis. We examined the ability of L-tryptophan and 26
of its analogs to activate TRAP. Our findings show that TRAP activation
by L-tryptophan is highly cooperative. We also observed that
TRAP activation is stereospecific; D-tryptophan did not
activate. Our results further indicate that the
The trp RNA-binding attenuation protein
(TRAP)
The one unlinked trp gene, trpG, is part of the folic acid biosynthetic
operon
(9) . TrpG is a bifunctional polypeptide involved in the
biosynthesis of folic acid as well as
L-tryptophan
(10) . TRAP binds to a segment of the
trpG transcript that includes the trpG ribosome-binding site
(8, 9) , suggesting that TRAP
binding regulates trpG translation by physically blocking
ribosome access to the trpG ribosome-binding site.
Results
from studies characterizing the RNA-binding targets of TRAP revealed
that the TRAP-binding site in trp leader RNA consists of 11
equivalently spaced (G/U)AG repeats (7 GAG and 4 UAG), whereas the
trpG-binding site contains 8 such repeats (7 GAG and 1 UAG),
although the spacing between adjacent repeats is more variable in the
trpG transcript
(8) .
TRAP was recently crystallized
in the presence of L-tryptophan and shown to be a complex
composed of 11 identical 8328-Da subunits
(11) . Electron
microscopic studies indicate that the TRAP subunits are arranged in a
single toroid ring.
In previous
studies using an in vitro transcription attenuation assay, we
observed that L-tryptophan, 5-fluoro-DL-tryptophan,
and 5-methyl-DL-tryptophan could activate TRAP, whereas
D-tryptophan, 1-methyl-L-tryptophan,
7-aza-DL-tryptophan, tryptamine, and indole-3-propionic acid
could not
(3) . In the present study we performed filter binding
experiments with L-tryptophan and 26 of its analogs to
determine the features of ligand required for TRAP activation.
A filter binding assay that measures
TRAP-trp leader RNA interaction
(8) was used to analyze
the effect on RNA binding as a function of increasing
L-tryptophan concentration. The results of this analysis
(Fig. 1) demonstrated that the ability of TRAP to bind RNA
approached saturation at an L-tryptophan concentration of 10
µM with half-activation at approximately 5
µM. Under our conditions 2 µML-tryptophan was the lowest concentration that allowed TRAP to
bind to the test transcript (Fig. 1). The sigmoidal shape of the
activation curve indicates that L-tryptophan association with
TRAP is highly cooperative. When taken together with previous findings
demonstrating that one molecule of L-tryptophan can bind per
TRAP subunit in a cooperative manner, these results suggest that one
molecule of L-tryptophan/TRAP subunit may be required for full
activation. The number of bound L-tryptophan molecules
required for partial activation is not known (compare the activity at 2
and 10 µML-tryptophan in Fig. 1).
Results with the transcription attenuation assay indicated that the
hydrogen atom at position 5 of the L-tryptophan benzene ring
could be replaced by a fluoro (5-fluoro-L-tryptophan) or a
methyl (5-methyl-L-tryptophan) group and still allow TRAP
activation
(3) . These and other analogs with benzene ring
substitutions at positions 4, 5, or 6 were tested in the filter binding
assay. Each of these analogs at a concentration of 1 mM
activated TRAP (). The modifications included the
replacement of a hydrogen with a methyl group at position 4
(4-methyl-L-tryptophan), a hydroxyl, fluoro, or methyl group
at position 5 (5-hydroxy-L-tryptophan,
5-fluoro-L-tryptophan, or 5-methyl-L-tryptophan), or
a fluoro or methyl group at position 6 (6-fluoro-L-tryptophan
or 6-methyl-L-tryptophan). Of these,
5-hydroxy-L-tryptophan, 5-fluoro-L-tryptophan, and
6-fluoro-L-tryptophan activated TRAP at a concentration of 50
µM (). Notably, the level of TRAP activation
by these analogs correlates with the physical size of the chemical
substituent replacing the hydrogen; however, the relative polarity of
the various substituents may effect TRAP activation ( Fig. 2and
).
Previous results suggested that substitution of a
pyridine ring for the benzene ring of L-tryptophan
(7-aza-L-tryptophan) eliminated TRAP activation (3). However,
this compound gave significant TRAP activation at a concentration of 1
mM when it was tested in the more sensitive filter binding
assay (). This result suggests that TRAP can tolerate a
significant distortion of the heterocyclic ring of the indole moiety of
L-tryptophan and still be activated. However, loss of the
heterocyclic ring, as in L-phenylalanine and
L-tyrosine, could not be tolerated ( Fig. 2and
).
Several other L-tryptophan analogs were also
tested for the ability to activate TRAP in the filter binding assay. Of
these, only substitution of the
The results of these
studies indicate that TRAP activation by L-tryptophan is
highly cooperative and that TRAP activation is stereospecific. We
determined that the
Concentrations of compounds used are indicated. The number
of counts/min (cpm) obtained with 1 mML-tryptophan
was arbitrarily set at 100. Values are the averages of four
experiments. See ``Materials and Methods'' for experimental
details. ND, not determined.
The number of counts/min (cpm) obtained with 5 µML-tryptophan alone was arbitrarily set at 100. The indole
and 5-methylindole concentrations used are indicated. Values are the
averages of four experiments. See ``Materials and Methods''
for experimental details.
We thank Bob Matthews and Peter Margolis for critical
reading of the manuscript. We also thank Virginia Horn for technical
assistance.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-amino group and
the carbonyl moiety of the
-carboxyl group of the ligand are
required for TRAP activation and that the heterocyclic amino nitrogen
of L-tryptophan greatly enhances TRAP activation. We also
found that changes at several positions of the indole ring of
L-tryptophan resulted in reduced TRAP activation. In addition,
indole and 5-methylindole were shown to be effective competitors of
L-tryptophan activation of TRAP.
(
)
of Bacillus subtilis is
responsible for regulating expression of the tryptophan biosynthetic
genes of this organism in response to changes in the intracellular
level of L-tryptophan (Ref. 1 and references therein).
Attenuation of the trpEDCFBA operon is mediated by the binding
of TRAP, the product of mtrB, to the nascent trp leader
transcript
(2, 3, 4, 5, 6, 7) .
In the presence of L-tryptophan, TRAP binds to a segment of
the nascent trp transcript and prevents formation of or
disrupts an RNA secondary structure, the antiterminator, thereby
promoting formation of an overlapping rho-independent terminator that
causes transcription termination at a site just preceding the trp structural genes
(3) . In the absence of
L-tryptophan, TRAP does not bind to the trp leader
transcript
(3, 6, 8) ; this allows the
antiterminator to form, thereby preventing formation of the terminator
and permitting transcriptional read-through into the trp structural genes
(3) .
(
)
In addition, several lines
of evidence suggest that the TRAP subunit composition is identical in
the presence or absence of L-tryptophan
(3, 8) .
Previous results demonstrated that the addition of
L-tryptophan to TRAP altered the gel mobility of TRAP,
suggesting that multiple L-tryptophan molecules can bind to
TRAP
(8) . These results are consistent with the results of
equilibrium dialysis studies that showed that association of
L-tryptophan and TRAP was cooperative, with one molecule of
L-tryptophan bound per TRAP subunit (11).
RNA Synthesis
Labeled transcripts were
synthesized in vitro using T7 RNA polymerase according to the
manufacturer's specifications (New England Biolabs Inc.). The
labeling nucleotide was [5,6-H]UTP (approximately
8 Ci/mmol). Transcripts were separated from unincorporated nucleotides
by using a Sephadex G-50 Quick Spin column (Boehringer Mannheim). The
template used in the transcription reaction was pPB77 that had been
linearized with BamHI; the resulting transcripts contained all
11 of the (G/U)AG trinucleotide repeats present in the wild type
trp leader transcript
(8) . The presence of a single RNA
species of the appropriate size was confirmed by polyacrylamide gel
electrophoresis.
Filter Binding Assays
TRAP was purified as
described previously
(3) . Filter binding assays were performed
by a modification of an established procedure
(8) . Standard
filter binding reaction mixtures (0.1 ml) contained approximately 0.27
pmol (25 ng) of TRAP, 0.5 pmol of [H]RNA, 10
units of RNasin (Promega), 1 mML-tryptophan or one
of its analogs, and 1 mM dithiothreitol in TKM buffer (40
mM Tris-HCl, pH 8.0, 250 mM KCl, 4 mM
MgCl
). TRAP dilution buffer was 50 mM Tris-HCl, pH
8.0, 50 mM KCl, and 15% glycerol
(3) . Mixtures were
incubated for 20 min at 37 °C and then filtered through BA85
nitrocellulose membranes (Schleicher & Schuell). The filters were
washed twice with 0.1 ml of TKM buffer, dried, and counted in a liquid
scintillation counter. Control experiments were performed in the
absence of L-tryptophan or its analogs. Filter binding assays
were performed in RNA excess. Modifications of the filter binding assay
are described in the text, figure legends, and tables.
L-Tryptophan and various tryptophan analogs were obtained from
Sigma, Aldrich, or other commercial sources, except for
1-methyl-L-tryptophan, which was generously provided by Robert
Phillips of the University of Georgia.
TRAP Activation by L-Tryptophan Is
Cooperative
Previous results demonstrated that
L-tryptophan is responsible for TRAP activation (3, 6). In the
presence of L-tryptophan, TRAP binds to a segment of the
trp leader transcript containing 11 closely spaced (G/U)AG
repeats and to a segment of the trpG message containing 8 such
repeats
(8) . Recent results have established that the TRAP
multisubunit complex is composed of 11 identical subunits
(11) .
It was shown that one molecule of L-tryptophan can bind per
TRAP subunit and that association of ligand with TRAP is
cooperative
(11) .
Figure 1:
Cooperative activation of TRAP by
L-tryptophan. TRAP was incubated with labeled RNA in the
presence of increasing concentrations of L-tryptophan, and the
mixtures were filtered. Background counts obtained in the absence of
L-tryptophan were subtracted from each value. The number of
counts/min (cpm) obtained following incubation with 1
mML-tryptophan was arbitrarily set at 100. Wild type
trp leader RNA containing 11 (G/U)AG repeats was used in these
analyses. See ``Materials and Methods'' for experimental
details.
Cooperative activation suggests that association of the first
molecule of L-tryptophan to unliganded TRAP might exert a
conformational change in the TRAP complex that increases the likelihood
that a second molecule of L-tryptophan will be bound. This
process may be repeated until 11 L-tryptophan molecules are
bound. Whatever the mechanism, however, the cooperative activation of
TRAP by L-tryptophan differs from the mechanism of activation
of Escherichia coli trp aporepressor (TrpR) by
L-tryptophan. In the E. coli system, one molecule of
L-tryptophan binds to each subunit of the TrpR repressor dimer
noncooperatively
(12, 13, 14, 15) .
Binding of TRAP to trp Leader RNA in the Presence of
L-Tryptophan Analogs
Using an in vitro transcription attenuation assay, we previously observed that
L-tryptophan, 5-fluoro-DL-tryptophan, and
5-methyl-DL-tryptophan could activate TRAP, whereas
D-tryptophan, 1-methyl-DL-tryptophan,
7-aza-DL-tryptophan, tryptamine, and indole-3-propionic acid
could not
(3) . Subsequently a more sensitive filter binding
assay was developed that allows us to examine TRAP activation by
analogs directly
(8) . To determine the features of
L-tryptophan that are required for TRAP activation we used the
filter binding assay to compare the ability of L-tryptophan
and 26 of its analogs to activate TRAP (Fig. 2). Several analogs
activated TRAP at a concentration of 1 mM; however, in no case
was activation as effective as with L-tryptophan
(). Analogs that resulted in appreciable TRAP activation at
1 mM were also tested at 50 and 5 µM. Five of the
analogs were found to activate TRAP at 50 µM; however,
none of these analogs activated appreciably at 5 µM
().
Figure 2:
Structures of L-tryptophan
analogs. Structures of L-tryptophan and 26 analogs were
examined in these studies. The name of each compound is given below each structure.
TRAP Activation by L-Tryptophan Is
Stereospecific
In a previous study using an in vitro transcription attenuation assay we found that
D-tryptophan did not activate TRAP
(3) . We tested
D-tryptophan in the more sensitive filter binding assay to
substantiate our previous result. D-Tryptophan did not
activate TRAP at a concentration of 1 mM ( Fig. 2and
). This result, combined with the observations that 10
µML-tryptophan almost fully activated TRAP
(Fig. 1) and that 5 µML-tryptophan
resulted in 50% activation ( Fig. 1and ),
demonstrates that TRAP activation is highly stereospecific, suggesting
that positioning of the -carboxyl and amino groups of tryptophan
is crucial for TRAP activation. This finding contrasts with results
obtained for the E. coli trp repressor (TrpR), for which it
was observed that, once bound, D-tryptophan is a more
effective corepressor than L-tryptophan
(16) .
The
We previously reported that
indole-3-propionic acid did not activate TRAP in the transcription
attenuation assay
(3) . This result suggested that the
-Amino Group of L-Tryptophan Is
Required for TRAP Activation
-amino group might be crucial for TRAP activation (Fig. 2).
To examine this possibility more thoroughly, we used the filter binding
assay to test analogs of L-tryptophan in which the
-amino
group of L-tryptophan was replaced by a hydrogen atom
(indole-3-propionic acid) or a hydroxyl group (indole-3-lactic acid) or
was methylated (N-methyl-L-tryptophan or
L-abrine) (Fig. 2). Each of these changes eliminated
TRAP activation (). These results suggest that the hydrogen
donor capabilities and/or the positive charge of the
-amino group
is required for TRAP activation. Note that the simple presence of a
positive charge is not sufficient for TRAP activation because both
L-tryptophan and N-methyl-L-tryptophan
(L-abrine) carry a positive charge (Fig. 2). These
results are similar to findings with E. coli TrpR that
indicated that the positive charge of the
-amino group is required
for corepressor activity. However, with TrpR L-abrine was
found to be a better corepressor than
L-tryptophan
(16) .
The Carbonyl Moiety of the
We previously observed, using
a transcription attenuation system, that tryptamine did not activate
TRAP (3). This result suggested that the -Carboxyl Group Appears
to Be Required for TRAP Activation
-carboxyl group of
L-tryptophan was involved in the activation of TRAP
(Fig. 2). To substantiate this conclusion we used the filter
binding assay to examine L-tryptophan analogs in which the
-carboxyl group was replaced by a hydrogen (tryptamine) or a
hydroxyl group (L-tryptophanol) or in which the negative
charge was removed but the carbonyl moiety of the carboxyl group was
retained (L-tryptophanamide and L-tryptophan methyl
ester) (Fig. 2). The negative charge associated with the carboxyl
group does not appear to be required for TRAP activation, because both
L-tryptophan methyl ester and L-tryptophanamide
activated TRAP at a concentration of 1 mM (). In
fact, the methyl ester derivative resulted in substantial TRAP
activation at a concentration of 50 µM ().
However, removal of the carbonyl moiety and the negative charge
(L-tryptophanol) or the entire carboxyl group (tryptamine)
prevented TRAP activation (). These results suggest that
the hydrogen acceptor capability of the carbonyl moiety is involved in
TRAP activation and that the negative charge of the carboxyl group
enhances but is not required for TRAP activation. These results are
similar to findings with E. coli TrpR, for which it was shown
that the
-carboxyl group enhances but is not essential for
corepressor activity
(16) .
The Heterocyclic Amino Nitrogen of L-Tryptophan
Greatly Enhances TRAP Activation
In studies with E. coli TrpR it was proposed that a substituent other than hydrogen at the
N-1 position of L-tryptophan would sterically interfere with
the protein-DNA contact surface and deprive the repressor-operator
interaction of a stabilizing hydrogen bond
(16) . We previously
reported that 1-methyl-L-tryptophan did not activate
TRAP
(3) . This result seemed to indicate that the hydrogen donor
capability of the indole ring's secondary amine was required for
TRAP activation (Fig. 2). Using the more sensitive filter binding
assay, we found that 1-methyl-L-tryptophan at a concentration
of 1 mM resulted in low level TRAP activation ().
This result indicates that the presence of the secondary amine, though
not absolutely required, greatly enhances TRAP activation. Perhaps
L-tryptophan donates the hydrogen atom at the N-1 position to
a suitable hydrogen bond acceptor in TRAP.
TRAP Activation Is Reduced by Changes in the
L-Tryptophan Benzene Ring
Several mtrB mutations have been isolated that are resistant to the
L-tryptophan analogs 5-fluoro-L-tryptophan
(4) and 5-methyl-L-tryptophan
(17) . Because
these analogs can activate TRAP
(3) but are not suitable for
protein synthesis
(11) and because the drugs depress
L-tryptophan synthesis, wild type cells presumably are
inhibited. Thus, analog resistance arises by a loss of TRAP activity.
-hydrogen with a methyl group
(
-methyl-L-tryptophan) still permitted TRAP activation (
Fig. 2
and ). Taken together, the results of our TRAP
activation studies indicate that TRAP can tolerate relatively modest
changes in the structure of the indole ring of L-tryptophan;
however, changes that disrupt the functional groups that may form
hydrogen bonds with TRAP tend to have more serious consequences. Even a
modest change such as methylation of the
-amino group of
L-tryptophan (N-methyl-L-tryptophan)
completely abolished TRAP activation ( Fig. 2and ).
In addition, changes that eliminate the carbonyl moiety of the
-carboxyl group (L-tryptophanol and tryptamine) rendered
the analog ineffective in TRAP activation, whereas analogs that retain
the carbonyl moiety (L-tryptophan methyl ester and
L-tryptophanamide) still can activate TRAP, albeit at
substantially reduced levels in the case of L-tryptophanamide
( Fig. 2and ). Furthermore, disruption of the
hydrogen bonding capability of the heterocyclic amino group of
L-tryptophan (1-methyl-L-tryptophan) resulted in a
dramatic decrease in its ability to activate TRAP ( Fig. 2and
).
Indole and 5-Methylindole Are Effective Competitors of
L-Tryptophan
Previously, using the transcription
attenuation assay, we found that D-tryptophan,
1-methyl-L-tryptophan, 7-aza-DL-tryptophan,
tryptamine, and indole-3-propionic acid could not prevent TRAP
activation when the analogs were present in a 50-fold molar excess over
L-tryptophan
(3) . We repeated and extended these
L-tryptophan competition experiments using the filter binding
assay with analogs that were unable to activate TRAP ().
An L-tryptophan concentration of 5 µM was
selected for analog competition analysis; at this concentration even
slight competition will result in a substantial change in activation of
TRAP (Fig. 1). Competition experiments were initially performed
at an analog concentration of 1 mM (200-fold molar excess over
L-tryptophan). Indole and 5-methylindole were very effective
competitors; other analogs that were unable to activate TRAP showed
only modest competition (data not presented). Competition by indole and
5-methylindole was examined at a range of concentrations, and both
analogs were found to be much less effective at a 20-fold molar excess
(100 µM) than at a 200-fold excess (1 mM) (
Fig. 2
and ). This requirement for excess competitor
may reflect a weaker affinity for TRAP and/or the analogs may not
induce cooperative binding. The fact that these analogs bind to TRAP
and compete with L-tryptophan suggests that the indole ring is
sufficient to confer attachment to the tryptophan-binding site(s) of
TRAP. However, the indole moiety is insufficient to activate TRAP; as
seen with other L-tryptophan analogs, the -carboxyl and
-amino groups are required as well.
-amino group and the carbonyl moiety of the
-carboxyl group of the ligand are required for TRAP activation and
that the secondary amine of the indole ring of L-tryptophan
greatly enhances TRAP activation. We found that indole and
5-methylindole are effective competitors of L-tryptophan
activation of TRAP, suggesting that the indole ring is recognized by
the protein and that bound indole prevents L-tryptophan
binding. However, indole and 5-methylindole did not activate TRAP.
Table:
TRAP activation by various L-tryptophan
analogs
Table:
Inhibition of
L-tryptophan activation of TRAP by indole and 5-methylindole
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.