(Received for publication, September 5, 1995; and in revised form, March 10, 1996)
From the
In Bacillus subtilis, expression of the tryptophan
biosynthetic genes is regulated in response to tryptophan by an
RNA-binding protein called TRAP (trp RNA-binding attenuation
protein). TRAP has been shown to contain 11 identical subunits arranged
in a symmetrical ring. Kinetic and thermodynamic parameters of the
interaction between tryptophan-activated TRAP and trp leader
RNA were studied. Results from glycerol gradients and mobility shift
gels indicate that two TRAP 11-mers bind to each trp leader
RNA. A filter binding assay was used to determine an apparent binding
constant of 8.0 ± 1.3 10
M
(K
=
0.12 ± 0.02 nM) for TRAP and an RNA containing residues
+36 to +92 of the trp leader RNA in 1 mML-tryptophan at 37 °C. The temperature dependence of K
was somewhat unexpected demonstrating that the
H of the interaction is highly unfavorable at +15.9
kcal mol
. Therefore, the interaction is completely
driven by a
S of +97 cal mol
K
. The interaction between tryptophan-activated TRAP
and trp leader RNA displayed broad salt and pH activity
profiles. Finally, the rate of RNA dissociation from the
RNA
TRAP
tryptophan ternary complex was found to be very slow
in high concentrations of tryptophan (>40 µM) but
increased in lower tryptophan concentrations. This suggests that
dissociation of tryptophan from the ternary complex is the
rate-limiting step in RNA dissociation.
In Bacillus subtilis, the genes for tryptophan biosynthesis are regulated in response to tryptophan by an RNA-binding protein called TRAP (trp RNA-binding attenuation protein; (1, 2, 3) ). TRAP regulates expression of the trp genes in three ways, all of which involve TRAP binding RNA in a tryptophan-dependent manner(3) . The trp operon (trpEDCFBA), which contains 6 of the 7 genes required for L-tryptophan biosynthesis, is regulated by transcription attenuation within a 204-nucleotide leader region preceding the first structural gene, trpE(1, 2) . In the presence of L-tryptophan, TRAP binds the nascent trp leader transcript (4) at a series of 11 G/UAG repeats between residues +36 and +91 (Fig. 1; (5, 6, 7) ). This binding prevents formation of an anti-terminator secondary structure which allows a transcription terminator to form and transcription halts in the leader region(1, 8) . When L-tryptophan is limiting, TRAP does not bind, the anti-terminator forms, and the operon is expressed.
Figure 1: Representation of residues +14 to +139 of trp leader RNA. The proposed terminator and anti-terminator structures are shown. Numbering is based on (1) and refers to the start of transcription. Residues in bold are the GAG and UAG repeats involved in TRAP binding. Asterisk indicates the residues proposed to be involved in the RNA-induced dimerization described under ``Discussion.''
TRAP also regulates translation of two trp genes, trpE (2, 9) and trpG(10) , apparently by two different mechanisms. Control of trpE translation is believed to be mediated by TRAP binding to the same series of G/UAG repeats in the leader segment of trp mRNAs that have escaped termination at the attenuator. This binding is thought to alter the RNA secondary structure so as to sequester the trpE ribosome binding site in a stem-loop structure, thus reducing translation initiation(2) .
trpG is the only tryptophan biosynthetic gene not located within the trp operon and is located within a folic acid biosynthetic operon(11) . Regulation of trpG translation occurs when TRAP binds a series of nine trinucleotide repeats which overlaps the putative ribosome binding site of trpG(5, 10) . Although the exact mechanism of control is not yet known, the data are consistent with a model involving direct competition between tryptophan-activated TRAP and ribosomes for trpG mRNA.
Recently, the three-dimensional
structure of TRAP complexed with L-tryptophan has been solved
by x-ray crystallography and refined to 1.8-Å
resolution(6, 12) . TRAP is an endecamer, with the 11
identical subunits arranged in a symmetrical ring. The secondary
structure of TRAP is formed entirely of -sheets,
-turns, and
random coils; no
-helices are present. TRAP is activated to bind trp leader RNA by binding 11 molecules of L-tryptophan in a highly cooperative manner(6) . The
crystal structure reveals that each tryptophan molecule binds between
two loops contributed by adjacent subunits; these loops contain
residues 25-33 of one subunit and residues 49-52 of the
other (6) .
Based on the structure of the TRAP endecamer and the multiple trinucleotide repeats in its RNA targets, we have proposed that upon binding, the RNA wraps around the protein such that each G/UAG repeat interacts with one subunit or a combination of two adjacent subunits of the TRAP 11-mer(6) . By wrapping around the protein, trp leader RNA would bind TRAP in a manner different from that observed in other characterized RNA-protein interactions(13) . Moreover, TRAP contains no sequences with similarity to other characterized RNA-binding motifs(14) .
Given the novel structure of TRAP and its RNA target, as well as the
unusual manner by which tryptophan-activated TRAP is believed to bind trp leader RNA, it was of interest to examine the
stoichiometry and thermodynamics of this interaction. Our results show
that the activated form of TRAP binds trp leader RNA with a
stoichiometry of two TRAP 11-mers to each RNA and an apparent binding
constant of 8.0 ± 1.3 10
M
(K
= 0.12 ± 0.02 nM). The free energy of
interaction is driven by a large favorable change in entropy
(
S = +97 cal mol
K
) which is necessary to compensate for the
substantial unfavorable change in enthalpy (
H =
+15.9 kcal mol
). The lack of any significant
salt dependence of the TRAP-RNA interaction (-
log K
/
log
[K
]
0) indicates that
electrostatic contacts do not play a major role in the stability of the
complex and that the release of ions upon complex formation is not the
major source of the favorable change in entropy. Previous work
demonstrated that in the presence of excess tryptophan, the RNA in the
tryptophan
TRAP
RNA ternary complex shows no significant
dissociation after 30 min(5, 7) . Since TRAP functions
as a regulatory protein by binding transcripts produced from the
apparently unregulated trp promoter(1) , it seems
unlikely that in vivo, the tryptophan
TRAP
RNA
complex is this stable. To address this apparent paradox, we have
studied the rate of RNA dissociation from the
RNA
TRAP
tryptophan ternary complex as a function of
tryptophan concentration and have shown that the rate of dissociation
of the ternary complex increases rapidly as the concentration of L-tryptophan is lowered.
For
dissociation rate experiments, 0.8 nM TRAP was equilibrated
with 40 µML-tryptophan for 5 min at room
temperature in FBB. Then, 4 fmol of RNA 36-92 (4 10
dpm) were added to a total volume of 25 µl and incubated for
15 min at 37 °C. 975 µl of FBB containing various amounts of L-tryptophan were then added to yield final concentrations of L-tryptophan between 1 and 10.75 µM. 75-µl
aliquots were removed at the indicated time points, filtered, washed,
and counted as described above.
The sedimentation profile of a
glycerol gradient containing 5 pmol of S-labeled TRAP and
1 pmol of
P-labeled RNA 14-92 is shown in Fig. 2.
Nearly all of the RNA (Fig. 2, solid line, closed
circles) was in fractions 14-19 which contained the
TRAP
RNA complex, whereas TRAP (which was present in a 5-fold
molar excess) was found in two peaks (Fig. 2, solid line,
closed squares), one corresponding to the TRAP
RNA complex
(fractions 14-19) and the second corresponding to the uncomplexed
TRAP (fraction 20-25). Within the fractions containing the
complex (Fig. 2, dashed line), the molar ratio of TRAP
to RNA was 2.1 ± 0.2, n = 19. Similar results
were obtained using 2.5-10 pmol of TRAP, as well as using an RNA
containing the motif GAGUU repeated 11 times (data not shown). Control
experiments showed that the free RNA and TRAP ran as single peaks
within fractions 21-25 and 25-30, respectively, and were
well separated from the TRAP
RNA complex (data not shown).
Figure 2:
Glycerol gradient sedimentation of S-TRAP
P-trp leader RNA
complex. Data shown were obtained with a trp leader RNA
containing residues +14 to +92 relative to the transcription
start site and the gradients contained 1 mML-tryptophan. Solid lines show the amount
(femtomoles) of TRAP (closed squares) and RNA (closed
circles) in each fraction. Dashed line shows the molar
ratio of TRAP to RNA. y1 axis is the ratio of TRAP to RNA. y2 axis is the amount in femtomoles of TRAP and RNA. x axis is the fraction number with the top of the gradient being on
the right.
We
also used mobility shift gels to separate the TRAPRNA complex
from the free species of each (Fig. 3). Bands containing the
complex were excised from the gel and the molar ratio of TRAP:RNA
determined as above. In this case, RNA 36-92 was used and 18 bands were
analyzed. The average ratio of TRAP to RNA in the complex was 2.8
± 1.8. Although the variability seen in this case was higher
than seen with the velocity gradients, the results are consistent with
a dimer of 11-mers interacting with a single RNA. Taken together, these
results indicate that under the conditions tested, two TRAP 11-mers
form a complex with one molecule of trp leader RNA. Therefore,
all binding data in this paper were analyzed using this stoichiometry.
Figure 3:
Mobility shift gel using P-labeled RNA 36-92 and
S-labeled TRAP. Each
lane contained 1 pmol of RNA 36-92 and the indicated amount of TRAP.
Experimental conditions were as described under ``Materials and
Methods.'' Bands labeled as Complex correspond to the
RNA
TRAP complex and were excised from the gel, dissolved in 1 M dithiothreitol, and the amount of RNA and TRAP in each band
was determined as described in the text.
Figure 4:
Equilibrium binding curve with trp leader RNA. The indicated concentration of TRAP was mixed with
10 pM
P-labeled trp leader RNA
containing residues +36 to +92 (RNA 36-92) relative to the
start of transcription(1) . The curves shown are the
best fit of the filter binding data in the presence of 1 mML-tryptophan (open circles, closed triangles, and closed stars) and in the absence of L-tryptophan (closed squares) using a nonlinear least
squares fitting algorithm. Control experiments, where the complex was
allowed to form for an additional hour (closed triangles) and
where equilibrium was reached by dissociation (closed stars)
are shown and described in the text. Background retention in the
absence of TRAP was subtracted from the binding data and was usually
less than 2% of the input counts. The y axis is normalized to
the total counts retained on the filter at saturation which, under
standard buffer conditions, was greater than 65% of the input counts.
Data are the average of at least four individual
experiments.
Based on the best fit of the binding
data, the apparent binding constant (K) for the
TRAP-RNA interaction was determined to be 8.0 ± 1.3
10
M
(K
= 0.12 ± 0.02 nM). Based on the procedure
of Witherell and Uhlenbeck (21) , we found that at least 65% of
our TRAP preparation was active to bind RNA (data not shown).
Therefore, the K
reported above must be
considered as a lower limit since less than 100% of the protein may be
active. As shown previously(4, 5) , the interaction
between TRAP and trp leader RNA is completely dependent on the
presence of L-tryptophan since apo-TRAP, in the absence of
tryptophan, showed no significant binding to trp leader RNA at
any protein concentrations tested (Fig. 4, closed
squares). Furthermore, a nonspecific RNA showed no binding to TRAP
above background levels within the protein concentrations tested (data
not shown).
Figure 5:
Temperature dependence of K. Apparent binding constants for TRAP binding
to RNA 36-92 obtained in FBB at the indicated temperatures (5-47
°C) and plotted as a van't Hoff plot. The slope of the best
fit line is -8.0 and the y intercept = 48.7
yielding
H = +15.9 kcal mol
and
S = +97 cal mol
K
based on the van't Hoff equation; ln K
=
(
H/R)(1/Temp) +
(
S/R), where R = 1.987
10
kcal mol
K
.
Figure 6:
Ionic strength dependence of K. Apparent binding constants of the
TRAP-trp leader RNA 36-92 interaction determined in FBB with
the indicated potassium glutamate concentration (100-700
mM) plotted as a log-log plot. Solid circles, with 4
mM MgCl
; open circles, without
MgCl
.
Figure 7:
Effect of pH on K.
Apparent binding constants with RNA 36-92 obtained at 37 °C in FBB
adjusted to the indicated pH. The following buffers were used: MES, pH
5 and 6; HEPES, pH 8; CAPS, pH 10. Data are the average of at least
four experiments.
Figure 8:
Effect of L-tryptophan
concentration of k. Dissociation experiments
were performed by incubating TRAP (0.8 nM) and 0.16 nM
P-labeled trp leader RNA 36-92 in FBB at 37
°C containing 40 µM tryptophan. At time 0, the complex
was diluted 40-fold in FBB containing various amounts of tryptophan to
yield the final concentration shown. Final concentration of tryptophan
was: 10.75 µM (closed circles), 5.9 µM (closed squares), 3.4 µM (closed
triangles), 1 µM (closed stars). Samples
were filtered at various times and the counts retained were normalized
to the counts at time 0, which were defined as 1. Data are the average
of two independent experiments.
The x-ray crystal structure of TRAP complexed with L-tryptophan shows that TRAP is a homopolymer of 11 identical
subunits arranged in a ring or ``doughnut''
structure(6) . It has also been shown that tryptophan-activated
TRAP binds its RNA targets at a series of multiple G/UAG
repeats(5, 6, 7) . From these findings, we
have proposed that TRAP binds its RNA targets by the RNA forming a
matching circle with the protein in which each trinucleotide repeat
interacts with one subunit (or a combination of adjacent
subunits)(6) , suggesting a 1:1 stoichiometry of TRAP and RNA
in the complex. However, our results using glycerol gradients and
mobility shift gels with S-labeled TRAP and
P-labeled RNA suggest that two TRAP 11-mers bind each RNA.
Interestingly, in crystals, TRAP was found to be a dimer of 11-mers
lying ``face to face''(6, 12) . Furthermore,
results from laser light scattering experiments (
)indicate
that while the majority of TRAP exists as an 11-mer in solution, a
small percentage is found as a higher molecular mass complex of
approximately 190,000 daltons, which is consistent with the predicted
molecular mass of a dimer of 11-mers. Thus, it seems that the TRAP
11-mer may dimerize before or upon binding RNA. It remains to be seen
if the TRAP dimer present in the crystals is the same as that which
binds RNA.
Using a filter binding assay, an apparent binding
constant of 8.0 ± 1.3 10
M
(K
=
0.12 ± 0.02 nM) was determined for the interaction
between TRAP and trp leader RNA in FBB containing 1 mML-tryptophan at 37 °C. The data fit well to a simple
bimolecular association curve indicating that only one complex is
formed between L-tryptophan activated TRAP and this segment of trp leader RNA under these conditions. Analysis of this
interaction by mobility shift assay also detected only a single complex (Fig. 3; (4) ). Similar results were obtained using an
artificial TRAP binding site containing the motif GAGUU repeated 11
times which yielded a K
of 8.6
10
M
(K
=
0.12 nM) (data not shown). It is interesting to note that the
binding constant for the E. coli trp repressor for its DNA
target is 0.2 nM(23) which is similar to the value we
have observed for TRAP binding to its RNA target.
When several trp leader RNAs were used that contained 22 nucleotides 5` of
the first UAG repeat (RNA 14-92 or RNA 14-115; see Fig. 1) a
second inflection in the binding curve was observed (data not shown).
This observation suggests that a second complex forms at high
concentrations of TRAP with these RNAs. Since these 22 residues are not
predicted to interact with TRAP upon RNA binding, we believe that they
should be unconstrained in the complex. We propose that this second
complex may be due to an RNA induced dimerization of the TRAPRNA
complexes caused by base pairing of the 5` end of the RNA in one
complex with the 5` end of the RNA in the other (Fig. 1).
The
interaction between tryptophan-activated TRAP and trp leader
RNA differs in several ways from those observed for other RNA-protein
interactions. For most RNA-protein interactions studied, H is approximately 0(13, 24, 25) ,
although R17 coat protein is a notable exception exhibiting a
H = -19 kcal/mol(26) . However, the
interaction between tryptophan-activated TRAP and trp leader
RNA displays a very large unfavorable
H of +15.9
kcal mol
. In our model of the TRAP-RNA interaction
we propose that the single-stranded trp leader RNA wraps
around tryptophan-activated TRAP(6) . The observed
H may represent the energy required to disrupt base stacking
interactions within the RNA in order for it assume the proper
conformation to bind TRAP.
Since the H of the
interaction between tryptophan-activated TRAP and trp leader
RNA is unfavorable, binding is driven entirely by a large favorable
change in entropy.
S is favorable for most RNA-protein
interactions(13) , ranging from +13.1 cal mol
K
for TFIIIA (27) to +56 cal
mol
K
for ermC`
methyltransferase(28) . Again, R17 coat protein is an exception
with an unfavorable
S of -30 cal mol
K
(26) . To our knowledge, the
interaction between TRAP and trp leader RNA has the highest
S seen for an RNA-protein interaction, 97 cal
mol
K
, which is needed to offset
the highly unfavorable enthalpic contribution.
In general, a large
favorable entropy change may be due to the release of either ions or
water molecules upon complex formation(29) . To address the
former possibility, the ionic strength dependence of TRAP binding trp leader RNA was investigated. In most RNA-protein
interactions studied, the dependence of salt on binding is small with
the -log K
/
log
[M
] being between 1 and 5(13) . For
the TRAP-trp leader RNA interaction, K
is not dependent on the concentration of potassium glutamate in
the presence of MgCl
with -
log K/
log [K
]
0 (Fig. 6, closed circles). Without MgCl
,
there is only a slight dependence of the K
on
the concentration of potassium glutamate equivalent to
1 ionic
interaction per complex. If ionic interactions were involved in the
binding, there would likely be at least 11 ionic contacts being made.
This assumption is based on our model of the TRAP-RNA interaction where
each of the 11 subunits would contact one of the 11 trinucleotide
repeats(6) . Therefore, if ionic contacts were significantly
involved in stabilizing the complex, we would predict that the binding
would have a much greater ionic strength dependence than observed. Our
findings do not completely rule out the possibility that ion release is
involved in the favorable change in entropy but it does make it likely
that the release of water molecules is a major driving force in the
binding.
Previous work has suggested that TRAP binds single stranded
RNA(2, 5) . The lack of MgCl dependence on
complex formation is consistent with this hypothesis since
Mg
is often involved in stabilizing RNA secondary and
tertiary structures(30) . We have observed that RNA 36-92, RNA
14-92, and an artificial TRAP binding site containing the motif GAGUU
repeated 11 times, none of which are predicted to form any stable
secondary structures, all bind TRAP approximately 3-fold better than an
RNA containing residues +14 to +115 (data not shown), which
is capable of forming the anti-terminator structure (Fig. 1).
Furthermore, it has been shown that when the TRAP binding site is
placed in a perfect stem-loop, this RNA does not bind TRAP. (
)Taken together, these observations support the model that
TRAP binds trp leader RNA in a single stranded form.
We
have shown that the TRAPRNA complex dissociates rapidly when
diluted into lower concentrations of tryptophan. Along these lines, the
concentration of tryptophan where RNA dissociation is observed was
found to be subsaturating for the TRAP-tryptophan interaction under
similar conditions (data not shown). This observation suggests that
tryptophan is able to dissociate from the RNA
TRAP
tryptophan
ternary complex resulting in the destabilization of the remaining
TRAP
RNA complex. If the concentration of tryptophan is too high,
such that TRAP remains completely bound by tryptophan, the ternary
complex dissociate very slowly. Therefore, it is reasonable to propose
that in B. subtilis, the in vivo concentration of
free tryptophan should not be allowed to rise to a concentration where
TRAP will not dissociate from trp leader RNA since this would
lead to the deregulation of the trp operon due to the
titration of TRAP. This contrasts the situation in E. coli,
where at high concentrations of free tryptophan, the trp repressor simply remains bound to its DNA binding site and
prevents transcription initiation from the trp promoter.
Moreover, excess free tryptophan in E. coli can also be used
as a source of carbon and nitrogen due to the products of the
tryptophanase operon(31) , which is lacking in B.
subtilis.
In the crystal structure, 11 molecules of L-tryptophan bind TRAP in deep clefts between adjacent
subunits and are completely buried within the protein with no exposure
to the solvent. This binding is proposed to activate TRAP to bind RNA
by inducing conformational changes in two loops formed by residues
25-33 on one subunit and 49-52 on the other(6) . We
have proposed that the RNA binds to TRAP at regions affected by
tryptophan binding and these two loops seemed likely candidates for the
RNA binding domain in TRAP(6) . However, if the RNA bound to
these loops, it is unlikely that L-tryptophan would still be
able to freely dissociate from the ternary complex as we have seen.
This observation suggests that although these loops are important for
tryptophan binding and likely play a role in the activation of TRAP,
the RNA interacts with another region of the protein. Recent results
from our laboratory using site-directed mutagenesis support this
hypothesis, indicating that the RNA binding site of TRAP involves
residues on -sheets located near the outer edge of the TRAP
doughnut. (
)
In this report, we have characterized several
aspects of the interaction between tryptophan-activated TRAP and trp leader RNA. Binding is driven completely by changes in
entropy yet is not ionic strength dependent, suggesting that the
interaction may be driven by the release of ordered water molecules.
The lack of ionic strength dependence also implies that the
TRAPRNA complex is mediated mostly by hydrophobic interactions
and hydrogen bonds rather than ionic interactions. Finally, the
dissociation of the complex suggests that L-tryptophan may
play a role in the regulation of the trp operon in vivo by not only controlling the association of TRAP and RNA but by
also controlling the rate of dissociation of the TRAP
RNA complex.