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Journal of Bacteriology, August 2004, p . 5157-5159, Vol . 186,
No . 15
Cellular Levels of trp RNA-Binding Attenuation Protein in Bacillus
subtilis
Barbara C . McCabe and Paul Gollnick*
Department of Biological Sciences, University at Buffalo, The State
University of New York, Buffalo, New York 14260
Received 14 April 2004/ Accepted 28 April 2004
Expression of the Bacillus subtilis trp genes is negatively
regulated by an 11-subunit trp RNA-binding attenuation protein
(TRAP), which is activated to bind RNA by binding L-tryptophan .
We used Western blotting to estimate that there are 200 to 400
TRAP 11-mer molecules per cell in cells grown in either minimal or
rich medium .
In Bacillus subtilis and several related bacilli, expression
of genes involved in tryptophan metabolism is regulated in response
to changes in intracellular L-tryptophan levels by an
RNA-binding protein called TRAP (trp RNA-binding attenuation
protein) (4, 5, 12,
13) . TRAP regulates transcription of the trpEDCFBA
operon through an attenuation mechanism (6,
18), as well as translation of trpE by
altering the trp mRNA structure to sequester the trpE
Shine-Dalgarno sequence in a stem-loop (10, 18,
21) . TRAP also regulates translation of trpG
(pabA) (11, 30), trpP (yhaG)
(23, 29), and ycbK (24)
through direct competition with ribosomes for binding to these mRNAs .
In addition, the activity of TRAP is regulated by the anti-TRAP (AT)
protein (26, 27) . AT binds to
tryptophan-activated TRAP and inhibits it from binding to its RNA
targets, thereby increasing expression of the trp genes .
TRAP is composed of 11 identical subunits arranged in a ring
structure (3) . Each 75-amino-acid subunit is encoded by the
mtrB gene (14) . TRAP is activated to bind RNA by
binding up to 11 molecules of L-tryptophan in
pockets between adjacent subunits (20) .
Single-stranded RNA binds to TRAP by wrapping around the outside of
the protein ring (2) . The RNA targets of TRAP
consist of multiple GAG, UAG, and occasionally AAG repeats, which are
separated from each other by several nonconserved spacer nucleotides .
The TRAP binding sites in the trp operon and in ycbK
contain 11 repeats, whereas there are 9 triplet repeats in the
trpG- and trpP-binding sites .
We used Western blotting to estimate the number of TRAP 11-mers
per B . subtilis cell to be approximately 200 to 400 . This number
varies only slightly with growth phase or in the absence or
presence of tryptophan in the growth medium .
Cell growth and preparation of protein extracts. One-liter
cultures of B . subtilis BG2087 (argC4) or BG4233 (argC4
 mtrB)
cells were grown in Luria-Bertania (LB) or minimal medium (28)
at 37°C either overnight or to mid-log phase (A600
of 0.8) . The number of cells per milliliter of culture was determined
by plating 25, 50, or 100 µl of a 1:106 dilution on LB
agar plates . Counting of cells microscopically showed no significant
differences with viable cell counts . Cells were harvested by
centrifugation at 5,000 x g for 10
min . Cell pellets were resuspended in 5 ml of 10 mM Tris-HCl (pH
7.6)-1 mM EDTA, and the cells were broken by three passages through a
French pressure cell at 12,000 lb/in2 . The cell lysate was
cleared by centrifugation at 30,000 x
g for 20 min . TRAP is heat stable (7); therefore,
the extract was heated at 65°C for 15 min to denature most of
the Escherichia coli proteins and cleared by centrifugation as
described above . Total protein in the extract was determined by using
the Bio-Rad protein assay with bovine serum albumin standards .
Electrophoresis and western blotting. Protein samples were
run on sodium dodecyl sulfate-Tris-Tricine-15% polyacrylamide gels by
using a Bio-Rad mini-Protean II system run at 40 mA . Samples were
mixed with an equal volume of loading dye (0.1 M Tris HCl [pH 6.8],
25% glycerol, 1% sodium dodecyl sulfate 0.02% Coomassie G-250) at
room temperature and loaded onto the gel without heating . Standard
curves were generated by mixing 0 to 25 ng of purified B . subtilis
TRAP (1) with protein extract from BG4233 (mtrB)
cells, which do not produce TRAP . The amount of control extract
protein was equivalent to that used for TRAP determination from
BG2087 .
Proteins were transferred to polyvinglidene difluoride membrane
(Amersham Hybond-P) by submerged wet transfer at 40 V for 10 h in 25
mM Tris-glycine-20% methanol . After being blocked with 5% bovine
serum albumin, the membranes were incubated for 1 h with rabbit AT
antibodies (1:10,000) . Blots were then washed and incubated for 1 h
with goat anti-rabbit immunoglobin G conjugated with horseradish
peroxidase (Cappel) . TRAP was visualized on the blots with the use of
an Amersham ECL+ kit and a Storm PhosphorImager (Molecular Dynamics)
and quantitated with ImageQuant software .
The results of Western blotting to quantitate cellular TRAP are
shown in Fig . 1 . (B) The standard curve for TRAP was generated
from Western blotting data (Fig . 1B) . Heat treatment of
the lysate does not precipitate TRAP (Fig . 1C) .
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FIG . 1 . (A and C) Western blots of TRAP from B . subtilis cells .
(A) Lanes: 1 to 6, various amounts of purified B . subtilis TRAP
mixed with extract from BG4233 (mtrB)
cells; 7 and 8, 3 µl of treated BG2087 extract . (B) Standard curve for
TRAP generated from the data from lanes 1 to 6 in panel A . In each case,
the background calculated from data from lane 1, with no added TRAP, was
subtracted from each value . (C) Effect of heat treatment in Western
blotting is shown . Lanes 1, 3 µl of treated extract from BG2087; 2 and
3, 2.5 and 5 µl, respectively, of resuspended pellet after the heat
treatment of the BG2087 extract.
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Table 1 shows the average number of TRAP 11-mers per B .
subtilis cell for growth under several conditions . The average
number for cells grown in LB harvested at mid-log phase was slightly
greater than for that for stationary-phase cells . The number
for cells grown overnight in minimal medium without tryptophan was
slightly lower than that for cells grown in the presence of
tryptophan . In all cases, the differences are less than twofold,
although they are significant based on a t test (P
0.99) . Studies have shown that the volume of bacterial cells changes
with growth conditions including growth rate (17);
therefore, the small changes in TRAP per cell that we observed likely
reflect changes in cell volume rather than regulation of mtrB
expression . The aqueous volume of an E . coli cell has been
measured to be 6.7 x 10–15
liters (22) . Because the dimensions of an average
B . subtilis cell are approximately 1.5-times larger than those
for E . coli (8), or approximately 1
x 10–14 liter the
concentration of TRAP for exponentially growing cells in LB is
approximately 80 nM .
| TABLE 1 . The number of TRAP 11-mer molecules per B . subtilis cella
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The level of TRAP protein in vivo is higher than those previously
determined for several DNA-binding repressor proteins, including
repressor (19) and the trp repressor from E . coli
(15) . This finding is appropriate given that TRAP
binds to at least four different binding sites in B . subtilis
(5) . Moreover, regulatory mechanisms based on an
RNA-binding protein introduce additional issues beyond those relevant
to DNA-binding proteins . Multiple copies of each mRNA are produced
from each promoter, and these levels may vary depending on the
physiological conditions of the cell . By contrast, the DNA copy
number varies by at most a factor of two . One of TRAP's target mRNAs,
trp mRNA, is produced from a strong promoter, and the level of
this mRNA is highly variable, resulting in the number of copies of
trp mRNA per cell varying . Our estimate of 200 to 400 TRAP 11-mer
copies per cell would appear to provide sufficient TRAP to cope with
all eventualities . However, since the TRAP protein requires
tryptophan for activation, the fraction of active TRAP molecules
available for mRNA binding will vary, depending on the intracellular
concentration of tryptophan . In addition, the availability of active
TRAP is also regulated by the AT protein (26,
27) .
When an RNA-binding regulatory protein is bound to an mRNA, it is
not available to bind other copies of this mRNA, or to other target
mRNAs, until the bound protein is released . Recently, it was shown
that mRNA degradation plays a role in recycling TRAP molecules to
maintain the cellular levels of free protein necessary to regulate
the trp operon (9) . Cells lacking the
degradative RNase polynucleotide phosphorylase overexpress the trp
operon structural genes when the cells are grown in the presence of
excess tryptophan, apparently due to insufficient free TRAP to bind
to newly synthesized trp mRNA . This effect was not observed in
cells with active RNase polynucleotide phosphorylase . This issue may
be a common concern regarding availability of RNA-binding proteins (16,
25) . It may be dealt with by predisposing specific
mRNAs that would be affected to susceptibility to attack by nucleases
that free the RNA-binding protein .
The antibodies used in this work were generated at the Department
of Laboratory Animal Resources Core facility, which is supported in
part by the Roswell Park Cancer Institute, National Cancer
Institute-funded Cancer Center Support Grant CA16056 . This work was
supported by grants GM62750 from the National Institutes of Health
and MCB 9982652 from the National Science Foundation .
* Corresponding author . Mailing address: Department of
Biological Sciences, University at Buffalo, The State University of New York,
Buffalo, NY 14260 . Phone: (716) 645-2363, ext.189 . Fax: (716) 645-2975 . E-mail: Gollnick@acsu.buffalo.edu.
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