2076 Tr y pt op ha n Op e ron

Tryptophan Operon will describe the organization of the trp operon of

C Yanofsky
Copyright ß 2001 Academic Press
doi: 10.1006/rwgn.2001.1343
The trp Operon and Tryptophan
Biosynthesis
All organisms that synthesize tryptophan use the same
sequence of biochemical reactions. In Escherichia coli,
these reactions are catalyzed by enzymes formed from
polypeptide chains encoded in the structural genes of a
single transcriptional unit, the trp operon. This article
E. coli, the pathway and enzymes of tryptophan bio-
synthesis, and the regulatory mechanisms this organ-
ism employs to regulate trp operon expression.
Gene Arrangement in the trp Operon of
Escherichia coli
The trp operon of E. coli and some other enteric
microorganisms contains five major structural genes,
designated trpE through trpA (Figure 1). These five
genes encode five polypeptides bearing the seven
functional domains that are necessary for tryptophan
formation. The genetic segments corresponding to
two pairs of functional domains are fused, yielding

regulatory region structural genes

trpL trpE trpD trpC trpB trpA

gene segments: trpL trpE trpG • trpD trpC • trpF trpB trpA
op. attn. t t'
trpP1 trpP2

Anthranilate synthase(E) L-serine

hydro-lyase(B)
trp
polypeptides: } trp leader
peptide
Glutamine amidotransferase(G)-
APR transferase(D)
IGP synthase(C)-
IGP
aldolase(A)
PRA isomerase(F)
}

}
(C-F)
Anthranilate synthase,
Tryptophan synthase:
Glutamine amidotransferase-
IGP aldolase, L-serine hydro-lyase
enzyme complexes: APR transferase
(A2B2)
(E2G-D2)
+ L-glutamine +PRPP
reactions: chorismate anthranilate N-(5'-phosphoribosyl)- 1-(o-carboxyphenylamino)-
anthranilate 1-deoxyribulose-5-phosphate
TrpE,TrpG TrpD TrpF

TrpC
+L-serine
L-tryptophan indole indole-3-glycerol
phosphate
TrpB TrpA

Figure 1 The trp operon of Escherichia coli, its specified polypeptides, the enzyme complexes they form, the
reactions in tryptophan biosynthesis, and the polypeptide or polypeptide domain responsible for catalysis of each
reaction. The operon consists of a transcription regulatory region followed by five structural genes and tandem sites
of transcription termination (t and t0 ). The principal promoter (trpP1) overlaps multiple operators (op.) at which the
tryptophan-activated trp repressor can bind and inhibit transcription initiation. Following the promoter, there is a
transcribed regulatory leader region containing the coding region (trpL) for a 14-residue peptide. Transcription may
either terminate at a regulated site of transcription termination, the attenuator (attn.), located in this leader region,
or proceed into the structural genes of the operon. Two of the structural genes, trpD and trpC, consist of fused
genetic segments. Each genetic segment specifies a polypeptide domain that can catalyze one of the tryptophan
biosynthetic reactions. There is an internal promoter (trpP2) near the distal end of trpD. TrpA through TrpG (and A
through G) refer to the polypeptide domains responsible for catalysis of the indicated reactions. Four of the five trp
polypeptides form enzyme complexes. APR transferase, anthranilate phosphoribosyl transferase; PRA isomerase,
phosphoribosyl anthranilate isomerase; IGP synthase, indoleglycerol phosphate synthase; IGP aldolase, indoleglycerol
phosphate aldolase; PRPP, 5-phosphoribosyl-1-pyrophosphate.

the bifunctional polypeptides TrpG-TrpD and TrpC-
TrpF. Dissection studies with these fused polypeptides
have established that each domain is a more or less
independent functional unit.
The Pathway and Enzymes of
Tryptophan Biosynthesis
The pathway of tryptophan biosynthesis proceeds
from chorismate, the common precursor of the three
aromatic amino acids. Chorismate also serves as pre-
cursor of several minor aromatic metabolites, includ-
ing p-aminobenzoic acid, a component of folic acid.
The biochemical reactions proceeding from chorismate
totryptophan,andthe seven polypeptide domains cata-
lyzing these reactions, are illustrated in Figure 1. The
synthesis of anthranilate from chorismate, and phos-
phoribosyl anthranilate from anthranilate, are cataly-
zed by a tetrameric enzyme complex consisting of two
TrpE and two TrpG-TrpD polypeptides. Although
l-glutamine is the preferred amino group donor during
the synthesis of anthranilate (o-aminobenzoate) from
chorismate, ammonia may be used as alternative source
of this amino group by the complex or by the TrpE
polypeptide alone. Glutamine utilization requires
the TrpG glutamine amidotransferase domain. In
the conversion of anthranilate to phosphoribosyl
anthranilate, by the TrpD domain, 5-phosphoribo-
syl-1-pyrophosphate (PRPP) contributes the side
chain of phosphoribosyl anthranilate. Phosphoribosyl
anthranilate is then rearranged by anthranilate
phosphoribosyl transferase, the TrpF domain, to
form 1-(o-carboxyphenylamino)-1-deoxyribulose-
5-phosphate (CdRP). The carboxyl group of CdRP
is then removed and the pyrrole ring of the indole
moiety is formed, yielding the next intermediate in
the pathway, indole-3-glycerol phosphate. The latter
reaction is catalyzed by indoleglycerol phosphate
synthase, the TrpC domain. Indole glycerol phosphate
is then converted to indole by the TrpA polypeptide of
the tryptophan synthase enzyme complex; this tetra-
meric complex consists of two molecules each of TrpA
and TrpB. Finally, indole is condensed with a pyridoxal
phosphate derivative of l-serine, to form l-tryptophan;
the final reaction is catalyzed by the TrpB polypeptide
of the tryptophan synthase complex.
Synthesis of tryptophan from chorismate requires
the products of four additional biosynthetic path-
ways, the compounds l-glutamine, phosphoribosyl-
1-pyrophosphate, l-serine, and pyridoxal phosphate.
Glutamine provides the amino group of anthranilate,
phosporibosyl pyrophosphate is the source of two
carbon atoms of the pyrrole ring of indole, l-serine
provides the alanyl side chain of tryptophan, and
pyridoxal phosphate is the coenzyme essential for
Tr yp t oph an Op eron 2077
activation of l-serine during catalysis of the final reac-
tion in tryptophan formation.
Structure/Function Studies with the
Tryptophan Biosynthetic Enzymes
The mechanism of enzymatic catalysis of each of the
tryptophan biosynthetic reactions has been investi-
gated and appreciable information has been gathered
on the key active site residues in each biosynthetic
protein or protein domain. The three-dimensional
structure of the tryptophan synthase enzyme complex
of Salmonella typhimurium has been determined, as
well as the structures of complexes containing mutant
protein variants. The three-dimensional structure ofthe
bifunctional phosphoribosyl anthranilate isomerase-
indoleglycerol phosphate synthase of E. coli has also
been determined. These structures have revealed that
the TrpA, TrpC, and TrpF polypeptide domains have
similar structures of the a/b TIM barrel type, raising
the possibility that they evolved from one another or
from a common ancestor. Structural studies with the
tryptophan synthase enzyme complex have shown
that a tunnel connects the active site of the TrpA
polypeptide to the active site of the TrpB polypeptide.
Indole, generated in the TrpA active site, travels
through this tunnel to the TrpB active site, where it
is condensed with serine. Studies with this enzyme
complex have also revealed features of the complex
that explain the mutual activation of each polypeptide
upon complex formation with the heterologous poly-
peptide.
Regulation of Expression of the trp
Operon of Escherichia coli
The five structural genes of the trp operon are pre-
ceded by a transcription regulatory region consisting
of a promoter/operator, at which transcription init-
ation is regulated, and a transcribed leader segment,
within which transcription termination is regulated.
Initation at the trp promoter is regulated by the
tryptophan-activated trp repressor protein; the extent
of repression varies in response to changes in the intra-
cellular concentration of free tryptophan. Repression
regulates operon expression over about an 80-fold
range. Polymerase molecules that have initiated tran-
scription at the trp promoter and escaped repression
are subject to a second regulatory mechanism, tran-
scription attenuation. The latter mechanism deter-
mines whether or not transcription will terminate at
a site located in the distal portion of the leader region.
This decision is influenced by the intracellular con-
centration of tryptophan-charged tRNATrp. When the
Trp-tRNATrp concentration is high, transcription
2078 Tu m o r An t i g e ns En c o d ed b y S im i a n V i r u s 4 0
terminates in the leader region. When tRNATrp is
mostly uncharged, which occurs when cells experi-
ence a severe tryptophan deficiency, termination is
avoided and transcription proceeds to the end of the
operon. Transcription attenuation in the trp operon of
E. coli regulates transcription of the structural genes of
the operon over about an eightfold range. The com-
bined action of repression and attenuation regulates
transcription of the structural genes of the operon
over about a 600-fold range. There is an internal pro-
moter located in the distal portion of trpD (Figure 1).
Transcription initiation at this promoter is unregu-
lated and proceeds at a frequency less than 10% that
attributable to the principal promoter. Tandem sites of
transcription termination are located following the
trpA structural gene; the first is protein-factor-inde-
pendent, a so-called intrinsic terminator, while the
second required the protein Rho. Completion of tran-
scription of the operon yields a polycistronic messen-
ger RNA. Ribosomes can initiate translation at any of
the five major ribosome binding sites on this polycis-
tronic messenger.
The trp promoter region of E. coli contains three
operators that can bind trp repressor. Operator-bound
repressor inhibits transcription initiation. The trp
repressor also regulates transcription initiation in sev-
eral other operons concerned with tryptophan metab-
olism. The three-dimensional structures of the trp
aporepressor (aporepressor lacks bound tryptophan),
the trp repressor, and the trp repressor±operator com-
plex, have been determined. These structures have
revealed the features of this protein that are respon-
sible for its activation by tryptophan and its recogni-
tion of specific operators.
The transcribed leader region of the trp operon of
E. coli is about 160 bp in length. As mentioned, this
genetic segment encodes an mRNA segment that can
cause transcription termination in the leader region.
The transcript of the leader region can fold to form
three RNA structures, termed terminator, antitermin-
ator, and transcription pause structure. The terminator
and antiterminator are alternative RNA structures,
i.e., they have a sequence of nucleotides in common,
thus either, but not both, can exist at one time. When
cells are deficient in charged tRNATrp the antitermin-
ator forms; this precludes formation of the terminator.
When cells have adequate levels of charged tRNATrp,
the terminator forms and transcription terminates in
the leader region. A deficiency of charged tRNATrp is
sensed during attempted translation of tandem Trp
codons in a 14-residue leader peptide coding region,
trpL, located near the 50 end of the trp operon tran-
script. Coupling of transcription and translation,
essential to this mechanism of attenuation, is achieved
by the formation of the transcription pause structure,
located near the 50 end of the transcript. Polymerase
pausing allows a ribosome to bind to the transcript
and initiate synthesis of the leader peptide. The move-
ment of this ribosome then releases the paused tran-
scription complex, and transcription and translation
proceed in unison.
Two of the trp polypeptides, the products of genes
trpE and trpA, lack tryptophan, therefore they are
synthesized preferentially during severe tryptophan
starvation. An additional regulatory feature, transla-
tional coupling, insures equimolar synthesis of the
polypeptide products of two pairs of adjacent genes,
trpE and trpD, and trpB and trpA. As mentioned, the
products of these genes form enzyme complexes. The
enzyme complex catalyzing the first two reactions in
the pathway is feedback-inhibited by tryptophan. The
tryptophan binding site is located in the TrpE poly-
peptide.
The use of two transcription regulatory mechanisms
and feedback inhibition of anthranilate synthase activ-
ity allows E. coli to regulate tryptophan biosynthesis
efficiently in response to changes in the availability of
tryptophan and the rate of protein synthesis.
Further Reading
Crawford IP (1989) Evolution of a biosynthetic pathway: the
tryptophan paradigm. Annual Review of Microbiology 43: 567±
600.
Miles EW (1995) Tryptophan synthase: structure, function, and
protein engineering. Subcellular Biochemistry 24: 207±254.
Yanofsky C (1984) Comparison of regulatory and structural
regions of genes of tryptophan metabolism. Molecular Biology
and Evolution 1: 143±161.
Yanofsky C and Crawford IP (1989) The tryptophan operon of
Escherichia coli. In: Neidhardt FC, Ingraham JL, Low KB et al.
(eds) Escherichia coli and Salmonella: Cellular and Molecular
Biology, vol. 2, pp. 1453±1472. Washington, DC: American
Society for Microbiology Press.
Yanofsky C, Platt T, Crawford IP et al. (1981) The complete
nucleotide sequence of the tryptophan operon of Escherichia
coli. Nucleic Acids Research 9: 6647±6668.
See also: Escherichia coli; Operon
Tumor Antigens Encoded
by Simian Virus 40
J M Pipas
Copyright ß 2001 Academic Press
doi: 10.1006/rwgn.2001.1624
Simian virus 40 (SV40) is a small (45 nm) DNA-
containing virus that establishes a lifelong, harmless