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Posttranscription Initiation Control of
Tryptophan Metabolism in Bacillus subtilis
by the trp RNA-Binding Attenuation Protein
(TRAP), anti-TRAP, and RNA Structure
Paul Babitzke and Paul Gollnick
J. Bacteriol. 2001, 183(20):5795. DOI:
10.1128/JB.183.20.5795-5802.2001.
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JOURNAL OF BACTERIOLOGY, Oct. 2001, p. 5795–5802
0021-9193/01/$04.00⫹0 DOI: 10.1128/JB.183.20.5795–5802.2001
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Vol. 183, No. 20
MINIREVIEW
Posttranscription Initiation Control of Tryptophan Metabolism
in Bacillus subtilis by the trp RNA-Binding Attenuation
Protein (TRAP), anti-TRAP, and RNA Structure
responsible for the decision to terminate transcription in the
trp operon leader region or to allow transcription to proceed
into the trp structural genes by sensing the level of tryptophan
in the cell (e.g., 4, 9, 27, 33). TRAP is also responsible for regulating translation of trpE and trpG. In the case of trpE, TRAP
binding promotes formation of an RNA structure that sequesters the trpE Shine-Dalgarno (SD) sequence (14, 27, 31). Interestingly, the trpE SD sequence is more than 100 nucleotides
downstream from the TRAP binding site. In contrast, TRAP
regulates TrpG synthesis by binding to a segment of the trpG
message that contains the SD sequence (7, 16, 38, 44). In
addition to the tryptophan biosynthetic genes, TRAP regulates
expression of a putative tryptophan transport gene (yhaG) (Fig.
1). As with trpG, it appears that TRAP regulates YhaG synthesis
by binding to the cognate SD sequence (34). Thus, TRAP coordinately regulates tryptophan synthesis and transport by three
distinct mechanisms; transcription attenuation of the trpEDCFBA
operon, promoting formation of the trpE SD blocking hairpin,
and blocking ribosome access to the trpG and yhaG ribosome
binding sites. In these situations TRAP functions by binding to
RNA targets in a tryptophan-dependent manner.
Organisms utilize a wide range of regulatory mechanisms to
control gene expression. While regulation of transcription initiation is a common regulatory strategy, it is now apparent that
this is only the starting point. Bacteria have developed several
sophisticated regulatory mechanisms that allow the organism
to modulate gene expression after transcription has initiated.
In addition, several subtle mechanisms allow organisms to finetune the final level of any particular gene product. Several of
these mechanisms, which operate after transcription initiation,
are crucial for regulating tryptophan metabolism in Bacillus
subtilis.
The B. subtilis trpEDCFBA operon contains six of the seven
genes that are required for the biosynthesis of tryptophan from
chorismic acid, the common aromatic amino acid precursor
(Fig. 1). The trp operon is present within a histidine and aromatic amino acid supraoperon. In addition to the trp operon
promoter driving expression of this operon, the promoter from
the upstream aroFBH operon contributes to trp operon expression. Since the first terminator for the aro operon is the terminator in the trp leader, transcriptional readthrough results in
transcription of the trp operon structural genes (23). trpG, the
remaining tryptophan biosynthetic gene, is present in an operon primarily concerned with folic acid biosynthesis (Fig. 1)
(38). Since there is no evidence for regulation of initiation
from the trpEDCFBA promoter, it appears that the greater
than 1,000-fold regulation observed for TrpE synthesis occurs
after transcription has initiated. The trp RNA-binding attenuation protein (TRAP) plays a central role in controlling tryptophan metabolism by sensing the concentration of tryptophan
in the cell (for previous reviews, see references 3, 20, 23).
Another recently identified protein called anti-TRAP (AT)
antagonizes TRAP activity (41). Since expression of the gene
encoding AT responds to the accumulation of uncharged
tRNATrp, it is now apparent that B. subtilis regulates tryptophan biosynthesis by sensing the levels of both tryptophan and
uncharged tRNATrp in the cell (35, 41).
TRAP regulates tryptophan biosynthesis by participating in
transcription attenuation and translational control mechanisms. In the transcription attenuation mechanism, TRAP is
TRANSCRIPTION ATTENUATION OF
THE trp OPERON
Transcription of the trpEDCFBA operon initiates 203 nucleotides upstream of the trpE start codon (24, 36). The B. subtilis
trp leader transcript contains several inverted repeats that are
capable of forming three RNA secondary structures involved
in the transcription attenuation mechanism (Fig. 2). All three
of these structures are conserved in Bacillus pumilus, Bacillus
stearothermophilus, and Bacillus caldotenax.
In B. subtilis, an antiterminator structure can form just upstream of an intrinsic terminator. Since these two structures
overlap by four nucleotides, their formation is mutually exclusive (4, 27, 37). In cells growing in the presence of excess
tryptophan, TRAP is activated to bind to 11 (G/U)AG repeats
(7 GAG and 4 UAG) that overlap the 5⬘ portion of the
antiterminator (7). Inhibition of antiterminator formation by
bound TRAP favors formation of the terminator hairpin, and
transcription halts in the trp leader region. In limiting-tryptophan growth conditions, TRAP is not activated and does not
bind to the nascent trp leader transcript. Under these conditions the antiterminator forms and the operon is expressed.
* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802. Phone: (814) 865-0002. Fax: (814) 863-7024.
Email: [email protected].
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PAUL BABITZKE1* AND PAUL GOLLNICK2
Department of Biochemistry and Molecular Biology, The Pennsylvania State University,
University Park, Pennsylvania 16802,1 and Department of Biological Sciences,
SUNY at Buffalo, Buffalo, New York 142602
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J. BACTERIOL.
Thus, the ability of TRAP to modulate which of these two
alternative structures forms in response to changes in the intracellular tryptophan concentration serves as the basis for the
transcription attenuation mechanism of this operon (Fig. 2).
In addition to the antiterminator and terminator structures,
an RNA hairpin forms at the 5⬘ end of the trp leader transcript
(5⬘ stem-loop) and participates in the transcription attenuation
mechanism (Fig. 2). Disruption of this structure increases trp
operon expression in vivo and transcriptional readthrough in
vitro (40). TRAP–5⬘ stem-loop interaction increases the affinity of TRAP for trp leader RNA and reduces the number of
(G/U)AG repeats that are necessary for tight TRAP binding
(15). Thus, it is possible that TRAP–5⬘ stem-loop interaction
increases the rate of TRAP binding to the nascent trp leader
transcript. This would increase the probability that TRAP
binding to the triplet repeats takes place before the antiterminator forms, thereby increasing the likelihood that transcription termination will occur before RNA polymerase can reach
the trp operon structural genes. Several TRAP-RNA binding
studies and in vitro transcription experiments using B. subtilis
RNA polymerase have firmly established that TRAP binds to
trp leader RNA, that binding is tryptophan dependent, and
that TRAP enhances termination in the leader region of the trp
operon (e.g., see references 4 and 33). Similar in vitro transcription results have also been obtained using T7, SP6, or
Escherichia coli RNA polymerase (33), consistent with the
model in which TRAP functions by interacting with the RNA
rather than with RNA polymerase.
TRAP is encoded by the second gene of the mtrAB operon
(Fig. 1) (21). While it is not known how expression of this
operon is regulated, mtrAB is not regulated in response to
tryptophan nor is TRAP involved in controlling its own expression (20, 29). Since mtrA encodes GTP cyclohydrolase I, an
enzyme involved in folic acid biosynthesis (9), it is possible that
folic acid or an intermediate in its biosynthesis is involved in
regulating expression of the mtr operon by activating an unidentified repressor protein. A potential candidate would be
chorismic acid since this compound is an intermediate in the
biosynthesis of both folic acid and tryptophan (3). The 75amino-acid TRAP polypeptide does not show significant similarity to the sequences of other characterized RNA binding
motifs. The only known protein that shows significant sequence
homology to TRAP is SplA, a regulator of the spore photoproduct lyase gene (splB) in B. subtilis (19).
TRANSLATIONAL CONTROL OF trpE
In addition to regulating transcription of the trpEDCFBA
operon, TRAP regulates translation of trpE. In vivo studies
indicate that TRAP regulates transcription termination in
the trp leader approximately 100-fold and that TRAP inhibits
translation of trpE an additional 13-fold (14, 27, 31). When
TRAP binds to a trp operon readthrough transcript, the trp
leader RNA adopts a structure in which the trpE SD sequence
is sequestered in a stable RNA hairpin (trpE SD blocking
hairpin) (Fig. 3) (14, 27, 31). Translational control of trpE
expression requires a higher intracellular concentration of
tryptophan than that required for attenuation (43a). This recent finding adds to the significance of previous results where
it was shown that the affinity of TRAP for trp leader RNA
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FIG. 1. Folate, mtr, trp, yhaG, and yczA-ycbK operons. trpG is located within the folate operon, while the rest of the trp genes are clustered in
the trpEDCFBA operon. yhaG encodes a putative tryptophan transport protein. mtrB encodes TRAP, while yczA encodes the AT protein (41).
TRAP is responsible for regulating expression of the trp operon by transcription attenuation and a translational control mechanism. TRAP
regulates translation of trpG, yhaG, and probably ycbK. P⬎ marks the position of the promoters for each operon, while the black boxes show the
positions of the TRAP binding sites. The positions of terminator hairpins (open circles) and antiterminator hairpins (filled circles) are shown.
VOL. 183, 2001
MINIREVIEW
5797
increases with increasing tryptophan concentrations (33, 43)
and that the dissociation rate of TRAP-trp leader RNA complexes decreases as the concentration of tryptophan increases
(10). In the transcription attenuation mechanism, only transient TRAP interaction with the (G/U)AG repeats is necessary
to block formation of the antiterminator. In contrast, for translational control TRAP must remain associated with the triplet
repeats to maintain formation of the trpE SD blocking hairpin
(14). In the absence of bound TRAP the trp leader transcript
adopts a structure in which the trpE SD sequence is single
stranded (14). Studies carried out in vitro support the TRAP-dependent trpE translational control model. TRAP binding promotes formation of the trpE SD blocking hairpin, and formation of this structure inhibits ribosome binding and, as a
consequence, TrpE synthesis (14). Thus, TRAP regulates TrpE
synthesis by promoting the formation of a trp leader RNA
secondary structure that is more than 100 nucleotides downstream from the 3⬘ end of the TRAP binding site. The ability
to form the trpE SD blocking hairpin is conserved in B. pumilus, B. stearothermophilus, and B. caldotenax.
For example, the coding sequences of trpE and trpD overlap by
29 nucleotides. Recent results establish that translation of trpD
is coupled to translation of trpE and that formation of the trpE
SD blocking hairpin regulates expression of trpD via translational coupling (43a). Thus, it is possible that TRAP-dependent formation of the trpE SD blocking hairpin coordinately
regulates translation of the entire operon via translational coupling. The overlapping trp genes, and presumably translational
coupling, are conserved in B. stearothermophilus as well (12).
Another interesting finding is that Rho, a transcription termination factor, influences expression of the trp operon when cells
are grown under conditions that promote translational control of
trpE expression. When cells are grown under tryptophan excess
conditions, expression of the trp operon increases severalfold in
Rho-deficient strains compared to isogenic wild-type strains. It is
likely that the increase in translational inhibition that occurs in the
presence of excess tryptophan leads to the appearance of a relatively ribosome-free transcript segment downstream from the
trpE SD blocking hairpin. Thus, it appears that Rho gains access
to the nascent trp transcript downstream from this structure,
thereby causing transcriptional polarity of the trp operon (43a).
TRANSLATIONAL COUPLING AND TRANSCRIPTIONAL
POLARITY IN THE trp OPERON
TRANSLATIONAL CONTROL OF trpG
With the exception of trpC and trpF, all of the coding sequences in the trp operon overlap by several nucleotides. This
sequence arrangement suggested that translational coupling, a
process in which translation of an upstream gene in a polycistronic transcript influences translation of the gene immediately
downstream, plays a role in trp operon expression (24, 30, 32).
trpG is the second gene in an operon primarily concerned
with folic acid biosynthesis (38) (Fig. 1). The TrpG polypeptide
functions as a glutamine amidotransferase in the biosynthesis
of tryptophan (TrpG-TrpE) and folic acid (TrpG-PabB) (26).
Translation of trpG is regulated by TRAP in response to tryptophan, whereas translation of pabB is not (44). TRAP binds to
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FIG. 2. Transcription attenuation model of the trp operon. When tryptophan is limiting (⫺tryptophan) TRAP is not activated. During
transcription, antiterminator formation (A and B) prevents formation of the terminator (C and D), which results in transcription of the trp operon
structural genes. When tryptophan is in excess (⫹tryptophan) TRAP is activated. Tryptophan-activated TRAP can bind to the (G/U)AG repeats
and promote termination by preventing antiterminator formation. The overlap between the antiterminator and terminator structures is shown.
Numbering is from the start of transcription.
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FIG. 3. trpE translational control model. Under tryptophan-limiting conditions, TRAP is not activated and is unable to bind to the trp leader
transcript. In this case the trp leader RNA adopts a structure such that the trpE SD sequence is single stranded and available for translation. Under
excess tryptophan conditions, TRAP is activated and binds to the (G/U)AG repeats. As a consequence, the trpE SD blocking hairpin forms, which
prevents ribosome binding and translation. The overlap between the two alternative structures is shown. Numbering is from the start of
transcription.
VOL. 183, 2001
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5799
nine triplet repeats that surround and overlap the trpG SD
sequence and inhibits TrpG synthesis (7 GAG, 1 UAG, and 1
AAG) (7, 16). Deletion of the five triplet repeats located just
upstream from the trpG SD sequence abolishes TRAP-dependent regulation (44). These findings indicate that binding of
tryptophan-activated TRAP to the nine triplet repeats present
in the trpG message regulates translation of trpG by blocking
ribosome access to the trpG ribosome binding site. In addition,
TRAP binding to the folate transcript reduces the steady-state
level of the message that encodes all eight of the polypeptides,
but not the mRNA segment that corresponds only to the first
two genes of the operon (13). Since the steady-state level of
folate transcripts is increased when cells are transferred to
fresh medium, it also appears that the folate operon is influenced by the growth phase of the cells (13). Neither the mechanism by which TRAP regulates folate operon transcript levels
nor the mechanism of growth phase control is understood.
TRANSLATIONAL CONTROL OF A PUTATIVE
TRYPTOPHAN TRANSPORT GENE
A computer search of the B. subtilis genome identified a
gene of previously unknown function (yhaG) that appears to
contain a TRAP binding site that overlaps the yhaG SD sequence and translation initiation region (34). This presumed
TRAP target contains as many as nine triplet repeats (5 GAG, 3
UAG, and 1 AAG). In vivo expression studies demonstrated
that translation of yhaG is regulated by TRAP. The findings
that YhaG is predicted to be a transmembrane protein and
that YhaG-deficient strains are more resistant to the tryptophan analog 5-fluorotryptophan than are wild-type strains suggest that YhaG functions in tryptophan transport (34).
REGULATION OF trp GENE EXPRESSION BY tRNATrp
AND THE ANTI-TRAP PROTEIN
In E. coli and many other bacterial species, the well-characterized transcription attenuation mechanism for the trp operon
senses the availability of charged tRNATrp (28). It is now
known that the availability of charged tRNATrp also influences
expression of the B. subtilis trp genes (29). In vivo studies
examining the effect of a mutant tryptophanyl-tRNA synthetase gene (trpS) suggested that B. subtilis has a mechanism
for recognizing an increase in the level of uncharged tRNATrp
and of responding to this accumulation by increasing expression of the trp genes (29, 39). A search of the B. subtilis genome
sequence identified an additional operon (yczA-ycbK) with a
presumed TRAP binding site (35). This site contains as many
as 11 triplet repeats (7 GAG, 3 AAG, and 1 CAG) that overlap
the ycbK SD sequence and translation initiation region. Furthermore, the features of this operon suggested that it could be
regulated by uncharged tRNATrp (35). yczA is preceded by a
leader regulatory region with the characteristics of operons
regulated by uncharged tRNA via the T-box antitermination
mechanism (reviewed in reference 22). Indeed, transcription of
this operon was shown to be regulated by antitermination in
response to changes in the level of uncharged tRNATrp (35).
Moreover, overexpression of yczA abolished TRAP-mediated
regulation of trp operon expression, whereas replacing the yczA
start codon with a stop codon eliminated this effect (41). In
addition, it was shown that purified YczA inhibits TRAP binding to trp leader transcripts via protein-protein interaction.
Since these results indicate that YczA functions as an inhibitor
of TRAP activity, this protein is now referred to as AT, for
anti-TRAP protein (41). While the function of yczA has been
determined, the function of ycbK is unknown.
TRAP-RNA RECOGNITION
Numerous in vitro (4, 6–8, 11) and in vivo (2) studies have
established that TRAP recognizes RNAs containing multiple
GAG and UAG (and rarely AAG) trinucleotide repeats.
These triplet repeats are separated from each other by nonconserved spacer nucleotides. Two nucleotide spacers are optimal (8), with pyrimidines generally favored over purines (11,
42). The TRAP binding site in the B. subtilis trp leader RNA
consists of 11 repeats (7 GAG and 4 UAG), each separated by
two or three nucleotide spacers (Fig. 4). The B. pumilus trp
leader also contains 11 (G/U)AG repeats (25), whereas this
region from B. stearothermophilus and B. caldotenax contains
12 triplet repeats, including 1 CAG in B. stearothermophilus
(12). The TRAP binding site in trpG of B. subtilis contains nine
triplet repeats (7 GAG, 1 UAG, and 1 AAG) (14, 38), while
the presumed TRAP binding sites in yhaG (34) and yczA-ycbK
(35) of B. subtilis contain as many as 9 or 11 triplet repeats,
respectively (Fig. 4). In these last three binding sites there are
a few exceptionally long spacers separating some of the repeats. Thus, it is apparent that these binding sites are suboptimal. A plausible reason for the suboptimal trpG TRAP target
is that it would allow a basal level of TrpG synthesis which
would maintain folate synthesis in the presence of tryptophanactivated TRAP (16). The finding that TrpG synthesis is reg-
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FIG. 4. Comparison of the TRAP binding sites. The (G/U)AG repeats are shown in bold type. The positions of the SD sequences and the start
codons are shown for trpG, ycbK, and yhaG.
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MINIREVIEW
ulated only sevenfold in response to tryptophan is consistent
with this hypothesis (44). As mentioned above, the 5⬘ stemloop also participates in the attenuation mechanism (40). This
structure is located two nucleotides upstream from the first
triplet repeat (Fig. 2). Exactly how this structure acts is not
firmly established, but it appears to interact with TRAP to
increase the affinity of TRAP for trp leader RNA (15).
TRAP STRUCTURE
The structures of TRAP complexed with tryptophan from
B. subtilis (2) and B. stearothermophilus (12) have been determined by X-ray crystallography. The amino acid sequences of
the two proteins are 77% identical, and their structures are
also very similar, consisting of 11 identical subunits arranged in
a ring around a central hole (Fig. 5). The TRAP oligomer is
composed of 11 seven-stranded antiparallel ␤-sheets, each containing four ␤-strands from one subunit and three strands from
the adjacent subunit. This structural arrangement generates
extensive interfaces between adjacent subunits that greatly stabilize the oligomer structure (2, 11, 12). TRAP is activated to
bind RNA by the cooperative binding of tryptophan (2, 5). The
crystal structure reveals that each tryptophan molecule binds
between adjacent subunits with its indole ring buried in a
hydrophobic pocket. Nine hydrogen bonds are formed between the amino group, the carboxyl group, and the indole
nitrogen of each bound tryptophan with amino acids residing
in two loops on adjacent subunits. The hydrogen bond formed
between Thr30 and the amino group of tryptophan, as well as
the hydrogen bond between Thr49 and the carboxyl group of
tryptophan, appears to be essential for TRAP function. Con-
versely, the hydrogen bond between Ser53 and the carboxyl
group of tryptophan is dispensable for TRAP activity (43). The
mechanism by which tryptophan binding activates TRAP to
bind RNA is not known. While the structure of TRAP in the
absence of bound tryptophan has not yet been determined,
several biochemical studies have established that TRAP remains as an 11-subunit complex in the absence of bound tryptophan.
The discoveries that TRAP contains 11 subunits and that its
RNA binding sites contain multiple trinucleotide repeats, including 11 in the B. subtilis and B. pumilus trp operon leaders,
suggested a model in which the bound RNA wraps around the
TRAP protein (Fig. 5) (2). Further support for this model
came from the identification of three amino acid residues from
each subunit (Lys37, Lys56, and Arg58), whose replacement by
alanine specifically interferes with RNA binding (45). These
three residues are aligned on the perimeter of the TRAP ring,
suggesting that the (G/U)AG repeats interact with the 11 KKR
patches on the protein. Nucleoside analog studies identified
several important functional groups for interaction with TRAP
on the second A and third G of each triplet repeat. These
studies also suggested that the bases in the first position (G or
U), as well as the spacer bases, are not crucial for TRAP
recognition and binding (17).
Several unusual properties of the TRAP-RNA complex may
be relevant to the mechanism by which TRAP binds RNA to
regulate gene expression in vivo. Several studies of TRAP
binding to RNAs containing various numbers of NAG repeats
(where N is any nucleotide) showed that the stability of the
TRAP-RNA complex is not directly proportional to the number of triplet repeats in the RNA (8, 18). Measurable affinity
for TRAP was seen with RNA containing as few as three GAG
repeats. The affinity then increased as the number of repeats
was raised from three to six GAGs. However, raising the number of repeats from 6 to 11 repeats resulted in only a slight
increase in the stability of the complex with TRAP. These
studies suggested a cooperative mechanism of RNA binding to
TRAP. Additional studies using nucleoside analogs (18) as
well as studies of heteromeric TRAP proteins containing mixtures of wild-type and mutant subunits (P. Li and P. Gollnick,
submitted for publication) further support these findings. Together these results suggest that the mechanism of TRAP
binding to RNA involves initial interaction with a small subset
of the repeats followed by a cooperative wrapping of the remainder of the RNA binding site around the protein (see
below).
The crystal structure of B. stearothermophilus TRAP complexed with a 53-base RNA consisting of 11 GAG repeats
separated by AU spacers was recently solved (1). As predicted,
the RNA wraps around the outside of the protein ring (Fig. 5).
The phosphodiester backbone is on the outside of the RNA
ring, and the bases point in toward the protein. Nearly all of
the contacts with RNA are to groups on the bases; there are no
contacts to phosphates. The only direct hydrogen bond to the
phosphodiester backbone is a hydrogen bond to the 2⬘ OH of
the ribose of the third G in each repeat. This contact, which
was predicted by deoxyribonucleoside substitution studies (17),
explains how TRAP distinguishes RNA from DNA. As predicted, Lys37, Lys56, and Arg58 all form hydrogen bonds with
the RNA. Lys37 hydrogen bonds to the second A of each
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FIG. 5. Ribbon diagram of TRAP complexed with an RNA containing 11 GAG repeats separated by AU spacers. TRAP binds to the
linear transcript and wraps the RNA around the periphery of the
TRAP complex. The TRAP subunits are shown in various shades of
gray, and the RNA is shown in ball-and-stick models. The bound
L-tryptophan molecules are shown as van der Waals spheres.
J. BACTERIOL.
VOL. 183, 2001
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FUTURE CONSIDERATIONS
While several mechanistic features concerning the regulation of tryptophan biosynthesis in B. subtilis and its relatives
are understood, many fundamental questions have not been
answered. Since the structure of TRAP in the absence of
bound tryptophan has not been determined, the mechanism of
TRAP activation by tryptophan is not known. Furthermore,
the reason that G or U is preferred over A or C in the first
position of triplet repeats in TRAP binding sites is unresolved.
In addition, structural information will be required to understand how AT inhibits TRAP activity. It will also be important
to elucidate the mechanism of 5⬘ stem-loop function and to
determine if translational coupling influences expression of the
entire trp operon. Finally, it will be necessary to investigate the
mechanisms responsible for regulating expression of yhaG,
yczA, and ycbK and to determine the functions of YhaG and
YcbK.
ACKNOWLEDGMENTS
We thank Angela Valbuzzi and Charles Yanofsky for sharing data
prior to publication. We also thank Fred Antson of York University for
making the TRAP-RNA structure figure.
This work was supported by grant GM52840 from the National
Institutes of Health to P.B. and grant GM62750 from the National
Institutes of Health and grant MCB 9982652 from the National Science Foundation to P.G.
REFERENCES
1. Antson, A. A., E. J. Dodson, G. Dodson, R. B. Greaves, X. Chen, and
P. Gollnick. 1999. Structure of the trp RNA-binding attenuation protein,
TRAP, bound to RNA. Nature 401:235–242.
2. Antson, A. A., J. Otridge, A. M. Brzozowski, E. J. Dodson, G. G. Dodson,
K. S. Wilson, T. M. Smith, M. Yang, T. Kurecki, and P. Gollnick. 1995. The
structure of the trp RNA attenuation protein. Nature 374:693–700.
3. Babitzke, P. 1997. Regulation of tryptophan biosynthesis: Trp-ing the TRAP
or how Bacillus subtilis reinvented the wheel. Mol. Microbiol. 26:1–9.
4. Babitzke, P., and C. Yanofsky. 1993. Reconstitution of Bacillus subtilis trp
attenuation in vitro with TRAP, the trp RNA-binding attenuation protein.
Proc. Natl. Acad. Sci. USA 90:133–137.
5. Babitzke, P., and C. Yanofsky. 1995. Structural features of L-tryptophan
required for activation of TRAP, the trp RNA-binding attenuation protein of
Bacillus subtilis. J. Biol. Chem. 270:12452–12456.
6. Babitzke, P., D. G. Bear, and C. Yanofsky. 1995. TRAP, the trp RNA-binding
attenuation protein of Bacillus subtilis, is a toroid-shaped molecule that binds
transcripts containing GAG or UAG repeats separated by two nucleotides.
Proc. Natl. Acad. Sci. USA 92:7916–7920.
7. Babitzke, P., J. T. Stults, S. J. Shire, and C. Yanofsky. 1994. TRAP, the trp
RNA-binding attenuation protein of Bacillus subtilis, is a multisubunit complex that appears to recognize G/UAG repeats in the trpEDCFBA and trpG
transcripts. J. Biol. Chem. 269:16597–16604.
8. Babitzke, P., J. Yealy, and D. Campanelli. 1996. Interaction of the trp
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
RNA-binding attenuation protein (TRAP) of Bacillus subtilis with RNA:
effects of the number of GAG repeats, the nucleotides separating adjacent
repeats, and RNA secondary structure. J. Bacteriol. 178:5159–5163.
Babitzke, P., P. Gollnick, and C. Yanofsky. 1992. The mtrAB operon of
Bacillus subtilis encodes GTP cyclohydrolase I (MtrA), an enzyme involved
in folic acid biosynthesis, and MtrB, a regulator of L-tryptophan biosynthesis.
J. Bacteriol. 174:2059–2064.
Baumann, C., J. Otridge, and P. Gollnick. 1996. Kinetic and thermodynamic
analysis of the interaction between TRAP (trp RNA-binding attenuation
protein) of Bacillus subtilis and trp leader RNA. J. Biol. Chem. 271:12269–
12274.
Baumann, C., S. Xirasagar, and P. Gollnick. 1997. The trp RNA-binding
attenuation protein (TRAP) from Bacillus subtilis binds to unstacked trp
leader RNA. J. Biol. Chem. 272:19863–19869.
Chen, X.-P., A. A. Antson, M. Yang, P. Li, C. Baumann, E. J. Dodson, G. G.
Dodson, and P. Gollnick. 1999. Regulatory Features of the trp operon and
the crystal structure of the trp RNA-binding attenuation protein from Bacillus stearothermophilus. J. Mol. Biol. 289:1003–1016.
de Saizieu, A., P. Vankan, C. Vockler, and A. P. G. M. van Loon. 1997. The
trp RNA-binding attenuation protein (TRAP) regulates the steady-state
levels of transcripts of the Bacillus subtilis folate operon. Microbiol. 143:979–
989.
Du, H., and P. Babitzke. 1998. trp-RNA binding attenuation protein-mediated long-distance RNA refolding regulates translation of trpE in Bacillus
subtilis. J. Biol. Chem. 273:20494–20503.
Du, H., A. V. Yakhnin, S. Dharmaraj, and P. Babitzke. 2000. trp RNAbinding attenuation protein-5⬘ stem-loop RNA interaction is required for
proper transcription attenuation control of the Bacillus subtilis trpEDCFBA
operon. J. Bacteriol. 182:1819–1827.
Du, H., R. Tarpey, and P. Babitzke. 1997. The trp-RNA binding attenuation
protein regulates TrpG synthesis by binding to the trpG ribosome binding
site of Bacillus subtilis. J. Bacteriol. 179:2582–2586.
Elliott, M. B., P. A. Gottlieb, and P. Gollnick. 1999. Probing the TRAP-RNA
interaction with nucleoside analogs. RNA 5:1277–1289.
Elliott, M. B., P. A. Gottlieb, and P. Gollnick. 2001. The mechanism of RNA
binding to TRAP: initiation and cooperative interactions. RNA 7:85–93.
Fajardo-Cavazos, P., and W. L. Nicholson. 2000. The TRAP-like SplA protein is a trans-acting negative regulator of spore photoproduct lyase synthesis
during Bacillus subtilis sporulation. J. Bacteriol. 182:555–560.
Gollnick, P. 1994. Regulation of the Bacillus subtilis trp operon by an RNAbinding protein. Mol. Microbiol. 11:991–997.
Gollnick, P., S. Ishino, M. I. Kuroda, D. J. Henner, and C. Yanofsky. 1990.
The mtr locus is a two gene operon required for transcription attenuation in
the trp operon of Bacillus subtilis. Proc. Natl. Acad. Sci. USA 87:8726–8730.
Henkin, T. M. 1996. Control of transcription termination in prokaryotes.
Annu. Rev. Genet. 30:35–57.
Henner, D., and C. Yanofsky. 1993. Biosynthesis of aromatic amino acids, p.
269–280. In A. L. Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus
subtilis and other gram-positive bacteria: biochemistry, physiology and molecular genetics. American Society for Microbiology, Washington, D.C.
Henner, D., L. Band, and H. Shimotsu. 1984. Nucleotide sequence of the
Bacillus subtilis tryptophan operon. Gene 34:169–177.
Hoffman, R. J., and P. Gollnick. 1995. The mtrB gene of Bacillus pumilus
encodes a protein with sequence and functional homology to the trp RNAbinding attenuation protein (TRAP) of Bacillus subtilis. J. Bacteriol. 177:
839–842.
Kane, J. F. 1977. Regulation of a common amidotransferase subunit. J. Bacteriol. 132:419–425.
Kuroda, M. I., D. Henner, and C. Yanofsky. 1988. cis-acting sites in the
transcript of the Bacillus subtilis trp operon regulate expression of the operon. J. Bacteriol. 170:3080–3088.
Landick, R., C. L. Turnbough, Jr., and C. Yanofsky. 1996. Transcription
attenuation, p. 1263–1286. In F. C. Neidhardt (ed.), Escherichia coli and
Salmonella: cellular and molecular biology, 2nd ed. ASM Press, Washington,
D.C.
Lee, A. I., J. P. Sarsero, and C. Yanofsky. 1996. A temperature-sensitive trpS
mutation interferes with TRAP regulation of trp gene expression in Bacillus
subtilis. J. Bacteriol. 178:6518–6524.
McCarthy, J. E. G., and C. Gualerzi. 1990. Translational control of prokaryotic gene expression. Trends Genet. 6:78–85.
Merino, E., P. Babitzke, and C. Yanofsky. 1995. trp RNA-binding attenuation protein (TRAP)-trp leader RNA interactions mediate translational as
well as transcriptional regulation of the Bacillus subtilis trp operon. J. Bacteriol. 177:6362–6370.
Oppenheim, D. S., and C. Yanofsky. 1980. Translational coupling during
expression of the tryptophan operon of Escherichia coli. Genetics 95:785–
795.
Otridge, J., and P. Gollnick. 1993. MtrB from Bacillus subtilis binds specifically to trp leader RNA in a tryptophan dependent manner. Proc. Natl.
Acad. Sci. USA 90:128–132.
Sarsero, J. P., E. Merino, and C. Yanofsky. 2000. A Bacillus subtilis gene of
previously unknown function, yhaG, is translationally regulated by trypto-
Downloaded from http://jb.asm.org/ on February 27, 2014 by PENN STATE UNIV
repeat, while both Lys56 and Arg58 hydrogen bond with the
third G of each repeat. In addition, Glu36 forms hydrogen
bonds with the third G of each repeat, and Phe32 stacks on this
base. The AU spacer bases do not directly contact the protein.
This is consistent with the lack of sequence conservation in
spacers of natural TRAP binding sites (12) and the observations that their composition is generally not critical for TRAP
binding (8, 10, 42). The preference for G and U in the first
position of the triplet repeats is not clarified by the crystal
structure. This simple mechanism of wrapping the RNA
around the circular TRAP complex explains both the specificity of the protein for its RNA targets as well as how TRAP
binding alters RNA structure to function in regulating both
transcription and translation.
5801
5802
35.
36.
37.
38.
40.
phan-activated TRAP and appears to be involved in tryptophan transport.
J. Bacteriol. 182:2329–2331.
Sarsero, J. P., E. Merino, and C. Yanofsky. 2000. A Bacillus subtilis operon
containing genes of unknown function senses tRNATrp charging and regulates expression of the genes of tryptophan biosynthesis. Proc. Natl. Acad.
Sci. USA 97:2656–2661.
Shimotsu, H., and D. J. Henner. 1984. Characterization of the Bacillus
subtilis tryptophan promoter region. Proc. Natl. Acad. Sci. USA 43:85–94.
Shimotsu, H., M. I. Kuroda, C. Yanofsky, and D. J. Henner. 1986. Novel
form of transcription attenuation regulates expression of the Bacillus subtilis
tryptophan operon. J. Bacteriol. 166:461–471.
Slock, J., D. P. Stahly, C.-Y. Han, E. W. Six, and I. P. Crawford. 1990. An
apparent Bacillus subtilis folic acid biosynthetic operon containing pab, an
amphibolic trpG gene, a third gene required for synthesis of para-aminobenzoic acid, and the dihydropteroate synthase gene. J. Bacteriol. 172:7211–7226.
Steinberg, W. 1974. Temperature-induced derepression of tryptophan biosynthesis in a tryptophanyl-transfer ribonucleic acid synthetase mutant of
Bacillus subtilis. J. Bacteriol. 117:1023–1034.
Sudershana, S., H. Du, M. Mahalanabis, and P. Babitzke. 1999. A 5⬘ RNA
stem-loop participates in the transcription attenuation mechanism that con-
J. BACTERIOL.
trols expression of the Bacillus subtilis trpEDCFBA operon. J. Bacteriol.
181:5742–5749.
41. Valbuzzi, A., and C. Yanofsky. 2001. Inibition of TRAP, the B. subtilis trp
RNA-binding attenuation protein, by the anti-TRAP protein, AT. Science,
in press.
42. Xirasager, S., M. B. Elliott, W. Bartolini, P. Gollnick, and P. Gottlieb. 1998.
RNA structure inhibits the TRAP (trp RNA-binding attenuation protein)RNA interaction. J. Biol. Chem. 273:27146–27153.
43. Yakhnin, A. V., J. J. Trimble, C. R. Chiaro, and P. Babitzke. 2000. Effects of
mutations in the L-tryptophan binding pocket of the trp RNA-binding attenuation protein of Bacillus subtilis. J. Biol. Chem. 275:4519–4524.
43a.Yakhnin, H., J. E. Babiarz, A. V. Yakhnin, and P. Babitzke. 2001. Expression
of the Bacillus subtilis trpEDCFBA operon is influenced by translational
coupling and Rho termination factor. J. Bacteriol. 183:5918–5926
44. Yang, M., A. de Saizieu, A. P. G. M. van Loon, and P. Gollnick. 1995.
Translation of trpG in Bacillus subtilis is regulated by the trp RNA-binding
attenuation protein (TRAP). J. Bacteriol. 177:4272–4278.
45. Yang, M., X.-P. Chen, and P. Gollnick. 1997. Alanine-scanning mutagenesis
of Bacillus subtilis trp RNA-binding attenuation protein (TRAP) reveals
residues involved in tryptophan binding and RNA binding. J. Mol. Biol. 270:
696–710.
Downloaded from http://jb.asm.org/ on February 27, 2014 by PENN STATE UNIV
39.
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