Lennarz W.J., Lane M.D. (eds.) Encyclopedia of Biological Chemistry. Four-Volume Set . V. 4

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FURTHER READING

Holmes, F. L. (1991). Hans Krebs, the Formation of a Scientiﬁc Life,

1900 to 1933. Oxford University Press, Oxford.

Holmes,F.L.(1993).Hans Krebs, Architect of Intermediary

Metabolism, 1933 to 1937. Oxford University Press, Oxford.

Krebs, H. A. (1954). The citric acid cycle. In Les Prix Nobel en 1953,

pp. 139–150. Boktryckeriet P.A. Norstedt and Soner, Stockholm,

Kungl.

Krebs, H. A. (1981). Hans Krebs: Reminiscences and Reﬂections.

Clarendon Press, Oxford.

Krebs, H. A., and Henseleit, K. (1932). Untersuchungen u

ber die

Hamstoffbildung in Tierko

rper. Hope-Seyler’s Z. Physiol. Chem.

210, 33–66.

Krebs, H. A., and Johnson, W. A. (1937). The role of citric acid in

intermediate metabolism in animal tissue. Enzymologia 4,

148–156.

Krebs, H. A., and Veech, R. L. (1969). Pyridine nucleotide

interrelations. In The Energy Level and Metabolic Control in

Mitochondria (S. Papa, J. M. Tager, E. Quagliariello and E. C. Slater,

eds.) pp. 329– 382. Adriatica Editrice, Bari.

Morowitz, H. J., Kostelnik, J. D., Yang, J., and Cody, G. D. (2000).

The origin of intermediary metabolism. Proc. Natl. Acad. Sci USA

97, 7704– 7708.

Ogston, A. G. (1948). Mechanism of ﬁxation of carbon dioxide in the

Krebs cycle. Nature 162, 963.

Wood, H. G., Werkman, C. H., Hemingway, A., and Nier, A. O.

(1941). Interpretation of experiments on metabolic processes using

isotopic tracer elements. J. Biol. Chem. 139, 483.

BIOGRAPHY

Richard L. Veech is the Head of the Laboratory of Metabolic Control,

NIAAA, NIH, DHHS, in Rockville, Maryland. He is an M.D. from

Harvard Medical School, and has a D.Phil. in Biochemistry from

Oxford University where his supervisor was Hans Krebs. He works on

problems involving the control of the processes of intermediary

metabolism, particularly in disease states.

262 TRICARBOXYLIC ACID CYCLE

tRNA Synthetases

Karla L. Ewalt and Paul Schimmel

Scripps Research Institute, La Jolla, California, USA

Protein biosynthesis is the culmination of the transfer of genetic

information from DNA to proteins. It is a highly coordinated

process that requires many cellular factors including RNAs,

proteins, nucleotides, and amino acids. An essential component

for the translation of genetic information from genes to

proteins, is the group of enzymes known as aminoacyl-tRNA

synthetases. These enzymes covalently link amino acids to

transfer RNAs (tRNAs) and thereby establish the rules of the

genetic code that pair amino acids to codons. The translation of

codons on the messenger RNA into amino acids occurs at the

ribosome – the center of protein biosynthesis.

Enzymatic Reaction

Accurate protein biosynthesis requires a collection of

amino acids attached to tRNAs (aminoacyl-tRNAs),

which are typically generated by 20 amino acid-speciﬁc

enzymes called aminoacyl-tRNA synthetases. The ami-

noacylation reaction is carried out at the enzymes active

sites in two-steps. In the ﬁrst step, an amino acid (AA) is

condensed with ATP yielding an aminoacyl-adenylate

(AA-AMP) and pyrophosphate (PPi) (Scheme 1). In the

second step, the aminoacyl group is transferred to either

the 2

or 3

-ribose hydroxyl on the terminal adenosine of

tRNA (AA-tRNA). Certain tRNA synthetases contain a

second active site (editing active site) for the hydrolysis

of incorrectly paired amino acids and tRNAs. At this

site, the tRNA synthetase corrects its errors.

Translational Accuracy

Aminoacyl-tRNA synthetases establish and determine

the accuracy of the genetic code and of protein

biosynthesis, as they pair amino acids with codons

through selection of amino acids and tRNAs. Accuracy

is achieved during selection of amino acids and tRNAs,

as well as through editing reactions that correct errors.

AMINO ACIDS

Each tRNA synthetase is speciﬁc for one of the 20 amino

acids. A selective binding site for the amino acid

accomplishes discrimination among the various amino

acid substrates. However, amino acid substrates for a

few enzymes are closely similar in size and functional

groups to other amino acids. For example, isoleucyl-

tRNA synthetase selects between isoleucine and valine,

which are both

-branched aliphatic amino acids

differing in length by only one methylene. The

misactivation of valine to valyl-adenylate by isoleucyl-

tRNA synthetase occurs at a low frequency. Threonyl-

tRNA synthetase must select between threonine and

serine, which differ by only a single methylene. The

active site is a “course sieve” that allows the correct

amino acid and occasionally similar, but slightly smaller

amino acids to be transferred to tRNA.

tRNAs

tRNAs are adapters that covalently connect anticodons

with amino acids. Sequences of tRNAs are 70–90

nucleotides (76 is most common) that form a common

cloverleaf secondary structure with four major arms.

This secondary structure in turn folds into an L-shaped

tertiary structure made up of two domains (Figure 1).

Amino acids are attached to tRNAs at the 3

-terminal

adenosine at the end of a CCA trinucleotide sequence

that is common to all tRNAs. In the second domain is a

trinucleotide sequence known as the anticodon, located

, 75A

from the amino acid attachment site. During

protein synthesis, the anticodon triplet binds through

complementary base pairing to a codon on mRNA

and thereby delivers an amino acid to the growing

polypeptide chain.

Since the genetic code is degenerate, more than one

tRNA may exist for a single amino acid. The 61 sense

codons of the code are recognized by a combination of

distinct tRNAs (one tRNA speciﬁc for each codon) and

tRNAs that employ wobble-base pairing (one tRNA that

recognizes sets of related codons). (A notably reduced

set of tRNAs is found in the mitochondria of eukaryotes,

where 22 tRNAs read the codons.) Although, trypto-

phan and methionine each have a single tRNA, there are

as many as ﬁve tRNAs speciﬁc for the six leucine codons.

The multiple tRNAs specifying a single amino acid are

termed isoacceptors, and are substrates for a single

aminoacyl-tRNA synthetase. Speciﬁc recognition of

tRNA is primarily through a nucleotide determinant

located in the acceptor stem. Nucleotides at other

positions including the anticodon stem are also import-

ant in many instances. The speciﬁc recognition of tRNAs

by synthetases based on the nucleotides present in the

acceptor stem, has led to the idea that these features

constitute a second genetic code that may be left over

from a smaller, historical RNA, which predated the

current tRNA structure.

EDITING

Several tRNA synthetases have an editing activity to

ensure correct pairing of amino acids with tRNAs

(Scheme 2). This editing, or proofreading, function

prevents misincorporation of amino acids into proteins

by hydrolyzing incorrectly aminoacylated tRNAs (post-

transfer editing). The tRNA synthetases for isoleucine,

leucine, valine, threonine, and alanine have been shown

to have speciﬁc domains with editing active sites that are

separate from the active site for aminoacylation. The

editing site operates as a “ﬁne sieve” to exclude the

correct aminoacyl-tRNA while binding the smaller and

incorrect aminoacyl-tRNA, which is then hydrolyzed. It

is currently thought that tRNA synthetases can also

hydrolyze misactivated amino acids prior to attachment

to the tRNA (pre-transfer editing). Through the combi-

nation of proper substrate selection and editing, the

general error rate for protein synthesis is estimated to

be # 1/3000.

Two Classes of Enzymes

STRUCTURES

Aminoacyl-tRNA synthetases are divided into two

groups of ten enzymes – class I and class II – based

upon the sequences and structures of the active site

domains. Ten different class I enzymes activate and

transfer the amino acids Arg, Cys, Glu, Gln, Ile, Leu,

Met, Tyr, Trp, and Val to tRNAs. All class I enzymes

contain a Rossmann nucleotide-binding domain and an

active site with two signature sequences, an 11 amino

acid element that ends in HIGH and a KMSKS

pentapeptide. The active site is interrupted by a

polypeptide insertion called CP1 that encodes an editing

domain. (Figure 2). Similarly, there are ten class II

enzymes that activate and transfer the amino acids Ala,

Asn, Asp, Gly, His, Lys, Phe, Pro, Ser, and Thr to tRNAs.

These ten enzymes share an active site architecture

formed by three conserved motifs (motif 1, 2, and 3) in

an overall structure having a seven-stranded antiparallel

-sheet ﬂanked by

-helices. Enzymes in both classes

have additional motifs or domains that are idiosyncratic

to each enzyme. Some of these domains provide RNA

binding elements, while others have functions such as

signaling in cytokine pathways.

MECHANISMS

The enzymes in the two classes are distinguished by the

orientation with which they bind tRNA. Class I enzymes

generally bind tRNA on the minor groove side of the

RNA helix and attach the amino acid to the 2

-ribose

hydroxyl of the terminal adenosine. Most class II

enzymes bind tRNA on the major-groove side and

attach the amino acid to the 3

-ribose hydroxyl. While

aminoacylation is speciﬁc to a particular hydroxyl, the

amino acid rapidly migrates between the 2

-and

-ribose hydroxyl groups.

Alternative Pathways and Enzymes

In a few cases, amino acids are modiﬁed while attached

to speciﬁc tRNAs in order to generate different

aminoacyl-tRNA species. This type of mechanism has

evolved as a biosynthetic pathway for glutamine,

asparagine, and selenocysteine.

tRNA DEPENDENT AMIDATION

Bacteria and archea have developed an alternate two-

step route for producing aminoacylated tRNAs for

glutamine and asparagine. In these organisms, glutamic

acid is attached to the glutamine speciﬁc tRNA

SCHEME 1 Two-step aminoacylation reaction catalyzed by amino-

acyl tRNA synthetases (E). Amino acid (AA) and ATP react to form an

activated aminoacyl-adenylate (AA-AMP) and pyrophosphate (PPi).

The aminoacyl group is transferred to either the 2

or 3

ribose

hydroxyl of the terminal adenine on tRNA.

FIGURE 1 The cloverleaf secondary and L-shape tertiary structure

of tRNA.

264 tRNA SYNTHETASES

(tRNA

Gln

) by glutamyl-tRNA synthetase. This system

requires that the glutamyl-tRNA synthetase have a

relaxed speciﬁcity that permits the acylation of glutamic

acid onto both tRNA

Glu

and tRNA

Gln

. In the second

step, glutamine is produced as Gln-tRNA

Gln

from Glu-

tRNA

Gln

, by the enzyme glutamyl-tRNA

Gln

amidotrans-

ferase. In these two kingdoms, this system is the

predominant mechanism for producing Gln-tRNA

Gln

Although some bacteria and archea have the gene

encoding asparaginyl-tRNA synthetase, an analogous

system also exists for the amidation of Asp-tRNA

Asn

produce Asn-tRNA

Asn

SELENOCYSTEINE

Sometimes called the 21st amino acid, selenocysteine is

incorporated into proteins at the UGA codon that

normally is a stop codon. However, a special tRNA

(tRNA

Sec

) exists for the incorporation of selenocysteine

at this position. Selenocysteyl-tRNA

Sec

is produced by

modiﬁcation of Ser-tRNA

Sec

. Serine is attached to the

tRNA

Sec

by seryl-tRNA synthetase, and is then modiﬁed

by selenocysteine synthase. The enzyme incorporates

selenide through a pyridoxal 5-phosphate-mediated

reaction. Incorporation of selenocysteine into proteins

at UGA is dependent on the presence of a selenocysteine-

insertion-sequence in the mRNA that is immediately

downstream of the codon.

Novel Functions of

tRNA Synthetases

In addition to their essential role in protein biosynthesis,

several tRNA synthetases are involved in other cellular

processes. The novel functions that appear in tRNA

synthetases are not common features to all of the

enzymes for a particular amino acid, but rather are

specialized features of the enzymes in selected organ-

isms. For examples, mitochondrial leucyl- and tyrosyl-

tRNA synthetase promote splicing in certain fungi.

Bacterial threonyl-tRNA synthetase regulates its own

translation by binding to an upstream region of the

SCHEME 2 Pretransfer and post-transfer editing reactions catalyzed by aminoacyl-tRNA synthetases (E). Pretransfer editing occurs prior to

the aminoacyl group transfer to tRNA. Posttransfer editing occurs after the aminoacyl group has been transferred to tRNA. The net result of

both pathways is the enzymatic hydrolysis of the aminoacyl-group (AA-AMP or AA-tRNA) to the free amino acid (AA) with release of AMP

and tRNA.

FIGURE 2 The crystallographic structure of a class I enzyme, leucyl-tRNA synthetase, and a class II enzyme, threonyl-tRNA synthetase,

indicating the active sites and editing domains.

tRNA SYNTHETASES 265

mRNA, and a histidyl-tRNA synthetase related protein

called GCN2 regulates translation in eukaryotes.

Increasingly, roles for tRNA synthetases as cell signaling

factors are being discovered in higher eukaryotes.

Fragments of tyrosyl- and tryptophanyl-tRNA synthe-

tase are mediators of angiogenesis, while histidyl- and

asparaginyl-tRNA synthetase speciﬁcally stimulate

immune cells. These extra functions demonstrate how

tRNA synthetases have been adapted and utilized for a

wide variety of functions in addition to their enzymatic

role in protein biosynthesis.

FURTHER READING

Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., and Watson, J. D.

(2002). Molecular Biology of the Cell. Garland Publishing,

New York.

Schimmel, P., and Ribas de Pouplana, L. (2000). Footprints of

aminoacyl-tRNA synthetases are everywhere. Trends Biochem.

Sci. 25(5), 207–209.

Soll, D., and RajBhandary, U. L. (1995). tRNA Structure, Biosynthesis,

and Function. ASM Press, Washington, DC.

Voet, D., and Voet, J. G. (1995). Biochemistry. Wiley, New York.

BIOGRAPHY

Paul Schimmel is the Ernest and Jean Hahn Professor and member

at the Skaggs Institute for Chemical Biology at the Scripps

Research Institute. He holds a Ph.D. from the Massachusetts Institute

of Technology, where he went on to become the John D. and Catherine

T. MacArthur Professor of Biochemistry and Biophysics in the

Department of Biology prior to joining the Scripps Research Institute.

His major research activities have concentrated on the decoding of

genetic information, with an emphasis on the rules of the universal

genetic code established by aminoacyl-tRNA synthetases.

Karla L. Ewalt is a Staff Scientist in the Department of Molecular

Biology at the Scripps Research Institute. She holds a Ph.D. from the

University of California, San Diego. Her research focus is on the

functional specializations found in components of eukaryotic protein

translation machinery.

266 tRNA SYNTHETASES

trp Operon and Attenuation

Paul Gollnick

State University of New York, Buffalo, New York, USA

Transcription attenuation can be deﬁned as any mechanism

that utilizes transcription pausing or termination to control

expression of downstream genes. This mechanism of regu-

lation is used to control expression of many genes in bacteria in

responses to changes in their environment. Moreover, bacteria

have evolved several elaborate and elegant variations on the

attenuation theme, mainly based on the mechanism used to

control formation the RNA structures that signal RNA

polymerase to pause or terminate.

Attenuation

The ﬁrst attenuation mechanism to be described

was for the Escherichia coli tryptophan biosynthetic

(trpEDCBA) operon. When Charles Yanofsky and his

co-workers elucidated this mechanism, it was the ﬁrst

demonstration that organisms can utilize changes in

RNA structure to regulate gene expression. Sub-

sequently many other examples of transcription attenu-

ation have been discovered. In each case the cis-acting

genetic information is contained within a 150–300 bp

region located after the start of transcription and prior

to the start of the coding sequence for the ﬁrst struc-

tural gene of the operon. This region is called the

leader region. In this article, the features of transcrip-

tion attenuation control of the E. coli trp operon are

discussed, followed by the modiﬁcations in this classic

system that allow it to be adapted to control other amino

acid biosynthetic operons. Thereafter attenuation con-

trol of the trp operon in the gram-positive bacterium

Bacillus subtilis, which involves a larger variation on the

attenuation theme, is described. There are several recent

reviews that cover the attenuation in more depth as well

as describe attenuation control of other bacterial genes.

The E. coli trp Operon: Attenuation

Based on Translation of a

Leader Peptide

The E. coli trpEDCBA operon encodes the enzymes

required to synthesize L-tryptophan from chorismic

acid. Transcription of the trp operon is regulated in

response to changes in intracellular tryptophan

levels. When the cells contain adequate amounts of

tryptophan, for example when it is present in the

growth medium, transcription of the operon is down-

regulated. In contrast, when tryptophan is limiting,

the trp operon is actively transcribed in order to

express the enzymes required for its synthesis.

Initiation of transcription is regulated by the trp

repressor, a DNA-binding protein encoded by the

trpR gene. In addition, after transcription has

initiated, the elongating transcription complex is subject

to regulation by attenuation. Together, repression (80-

fold) and attenuation (8-fold) serve to allow , 600-

fold overall control of transcription of the trp operon

in response to various levels of tryptophan

availability.

ATTENUATION CONTROL OF THE

E. COLI TRP OPERON

The E. coli trp operon contains a 162 bp leader region

prior to the start of the trpE coding sequence. The trp

leader transcript contains several inverted repeats,

composed of the segments labeled 1–4 in Figure 1,

that can form three different overlapping base-paired

RNA secondary structures. These structures include an

intrinsic transcription terminator (3:4), an overlapping

antiterminator (2:3), and a pause structure (1:2).

In addition, the leader transcription contains a small

open reading frame (ORF) that encodes a 14-amino

acid leader peptide, which contains two critical tandem

UGG Trp codons. The cell’s ability to efﬁciently

translate these two Trp codons determines which RNA

structure forms in the nascent leader transcript, which

in turn controls whether transcription halts in the

leader region or continues into the structure genes of

the operon.

Shortly after transcription initiates from the trp

promoter, the 1:2 pause hairpin forms (Figure 1).

This structure signals RNA polymerase to pause

transcription after nucleotide 92. This pausing of RNA

polymerase is a critical feature of the attenuation

mechanism because it allows time for a ribosome to

initiate translation of the leader peptide. When the

ribosome begins translating the leader peptide this

releases the paused RNA polymerase to resume tran-

scription. Transcription and translation are now

coupled, with the ribosome closely following RNA

polymerase. This situation is essential to allow events

involving the ribosome to affect transcription by the

associated RNA polymerase.

At this point there are two possible pathways for

the attenuation mechanism to follow depending on

the level of tryptophan in the cell. The choice depends

on how efﬁciently the tandem Trp codons in the

leader peptide are translated. This efﬁciency reﬂects

the availability of aminoacylated tRNA

Trp

in the cell.

Under conditions where tryptophan is limiting, the

amount of charged tRNA

Trp

is low. As a result of this

low concentration of tryptophanyl-tRNA

Trp

trans-

lation of the tandem Trp codons is inefﬁcient and

the ribosome stalls at one of these two codons. The

associated RNA polymerase continues to transcribe

through the trp leader region and transcription and

translation become uncoupled. As RNA polymerase

proceeds through segments 2 and 3 of the leader

region, the antiterminator structure (2:3) forms,

which prevents formation of the overlapping intrinsic

terminator (3:4) structure. Hence transcription con-

tinues through the leader region and into the trp

structural genes.

FIGURE 1 Model of transcription attenuation of the E. coli trp operon. RNA polymerase pauses following formation of the pause

structure. This provides time for a ribosome to initiate translation of the leader peptide. Under tryptophan-limiting conditions the ribosome

stalls at the tandem Trp codons, resulting in transcription read through. Under conditions of tryptophan excess the ribosome reaches

the leader peptide stop codon. This ribosome position blocks formation of the antiterminator leading to terminator formation and

transcription termination.

268 trp OPERON AND ATTENUATION

When tryptophan is plentiful, the level of

charged tRNA

Trp

in the cell is high. This allows efﬁ-

cient translation of the tandem Trp codons and hence

the ribosome proceeds rapidly to the end of the

leader peptide. When the ribosome reaches the leader

peptide stop codon, it covers part of RNA segment 2

and thus prevents formation of the 2:3 anti-

terminator structure as transcription proceeds. This

frees RNA segment 3 to base pair with segment 4

and form the terminator. Under these conditions

transcription terminates in the leader region prior

to the trp structural genes, which are therefore

not expressed.

In this attenuation mechanism the regulatory signal

is the level of charged tRNA

Trp

and the sensory event is

the efﬁciency with which the ribosome can translate

the tandem Trp codons in the leader peptide. This

system can easily be adapted to regulate other bacterial

amino acid biosynthetic operons. The only needed

modiﬁcation is to change the identity of the critical

codons in the leader peptide, which controls the amino

acid the system will respond to. There are numerous

examples of such adaptations of this attenuation

mechanism, particularly in enteric bacteria, including

the his, phe, and leu operons, which contain seven His,

seven Phe and four Leu codons in their respective

leader peptides.

The Bacillus subtilis trp

Operon: Attenuation Mediated

by an RNA-Binding Protein

The transcription attenuation mechanism that regulates

the trp operon in the gram-positive bacterium Bacillus

subtilis differs more dramatically from that the E. coli

trp operon than those described above for other amino

acid biosynthetic operons in gram-negative bacteria.

Most notably there is no leader peptide, and ribosomes

are not involved in this attenuation mechanism. Instead

an RNA-binding protein senses the level of tryptophan

in the cell and determines whether transcription

terminates in the leader region or continues into the

structural genes.

THE B. SUBTILIS TRP LEADER

Expression of the trpEDCFBA operon in B. subtilis and

several related bacilli is regulated by the trp RNA-

binding Attenuation Protein (TRAP). Transcription of

the operon initiates from the trp promoter 203

nucleotides upstream of the start codon of the ﬁrst

structural gene, trpE (Figure 2). There is no evidence

for any regulation of initiation of transcription from

this promoter. Hence there is no homologue, either

FIGURE 2 Model of transcription attenuation of the B. subtilis trpEDCFBA operon. The large blue letters indicate the complementary strands of

the terminator and antiterminator RNA structures. The TRAP protein is shown as a ribbon diagram with each of the 11 subunits as a different color

and the bound RNA is shown forming a matching circle upon binding to TRAP. The bound tryptophans are shown as space ﬁlling models. The GAG

and UAG repeats involved in TRAP binding are shown in ovals and are also outlined in the sequence of the antiterminator structure. Numbers

indicate the residue positions relative to the start of transcription. Nucleotides 108– 111 overlap between the antiterminator and terminator

structures and are shown as darkly outlined letters.

trp OPERON AND ATTENUATION 269

functional or based on amino acid sequence, of the

E. coli DNA binding trp repressor in B. subtilis.

The B. subtilis trp leader transcript also contains

inverted repeats that can form an intrinsic transcription

terminator (C:D) and an antiterminator (A:B) RNA

secondary structure (Figure 2). These two structures

overlap by four nucleotides and hence their formation is

mutually exclusive.

In the presence of excess tryptophan, TRAP binds to

a series of 11 trinucleotide repeats in the trp leader

transcript consisting of seven GAGs and four UAGs

(Figure 2). The (G/U)AG repeats are separated from

each other by two or three “spacer” nucleotides, whose

sequence is not conserved. These triplet repeats in part

overlap the 5

portion of the antiterminator structure

and hence TRAP-binding interferes with form-

ation of the antiterminator (A:B) thus favoring form-

ation of the terminator (C:D) hairpin. Therefore under

these conditions transcription halts in the

leader region prior to the trp structural genes, which

are not expressed.

When tryptophan is limiting, TRAP does not bind to

the trp leader RNA transcript and formation of the A:B

antiterminator is favored. This situation allows tran-

scription to proceed into the trp operon structural genes,

which can now be expressed to produce the enzymes to

synthesize tryptophan.

THE TRAP PROTEIN

In this system the ability of TRAP to sense the levels of

intracellular tryptophan and to then inﬂuence the

structure of the nascent trp leader mRNA transcript

is the basis for attenuation control of the trp operon.

TRAP is a ring-shaped protein composed of 11

identical subunits (Figure 3). TRAP is activated to

bind its RNA targets by binding 11 molecules of

L-tryptophan in clefts between adjacent subunits.

The detailed mechanism by which tryptophan binding

activates TRAP to bind RNA is not known, however,

recent studies suggest that tryptophan binding induces

a conformational change in TRAP as well as reduces

the ﬂexibility of the protein. Upon activation by

tryptophan, TRAP binds to its RNA target consisting

of the 11 (G/U)AG repeats by wrapping the single-

stranded RNA around the outer perimeter of the

protein ring (Figure 3). The phosphodiester backbone

is on the outside of the RNA ring and the bases point

in toward the protein. The speciﬁcity of this interaction

derives mainly from hydrogen bonds between several

amino acids of each TRAP subunit including Glu36,

Lys37, Lys56, and Arg58, and the bases on the

(G/U)AG repeats. The spacer nucleotides do not

contact the protein, and their identity is not crucial

for TRAP recognition and binding. This mechanism of

wrapping the RNA around the protein ring explains

both the speciﬁcity of the TRAP–RNA interaction as

well as how TRAP binding alters the structure of the

trp leader RNA to regulate attenuation.

THE ROLE OF PAUSING IN TRAP

EDIATED ATTENUATION

In addition to the terminator and antiterminator

structures, there is an RNA hairpin at the 5

end of

the trp leader mRNA (Figure 2;5

stem-loop).

Although this structure has been shown to be import-

ant for proper attenuation control of the trp operon, it

is not a transcriptional pause signal analogous to the 1:2

structure of the E. coli trp attenuation system. The role

of this 5

stem-loop may be to enhance the rate of

TRAP binding to the trp leader RNA. This is an

important consideration because TRAP must bind to its

target before RNA polymerase transcribes beyond

the terminator structure, otherwise TRAP binding

would not be able to inﬂuence attenuation. Recent

studies published by Yakhnin and Babitzke indicate

that RNA polymerase pauses at U107 (Figure 2)

during transcription of the trp leader region, and that

this pausing is dependent on the NusA transcription

factor. This pause would provide additional time for

TRAP to bind to the nascent trp leader RNA and

promoter termination.

FIGURE 3 Ribbon diagram of TRAP complexed with an RNA

containing 11 GAG repeats separated by AU spacers. The TRAP

protein is shown in ribbon diagrams with each subunit depicted

in a different color. The RNA is shown in stick models and

the 11 molecules of bound L-tryptophan are shown in space-

ﬁlling models.

270 trp OPERON AND ATTENUATION

ANTI-TRAP AND THE ROLE

OF T

RNA

IN REGULATING

THE

B. SUBTILIS TRP GENES

In this attenuation system the regulatory signal is the

level of free tryptophan available to activate TRAP to

bind RNA. In the previously described E. coli trp

attenuation system, free tryptophan is sensed by the

DNA binding trp repressor protein and the regulatory

signal for attenuation is the availability of charged

tRNA

Trp

. The level of charged tRNA

Trp

also inﬂuences

attenuation control of the B. subtilis trp operon,

although by a very different mechanism than in the

E. coli system. In B. subtilis the rtpA gene encodes a

protein called anti-TRAP (AT). AT inﬂuences expression

of the trp genes by binding to tryptophan-activated

TRAP and preventing it from binding RNA, thus

elevating expression of trp genes. Expression of rtpA,

which is in a two-gene operon together with ycbK

(unknown function), is regulated in response to changes

in the level of uncharged tRNA

Trp

by a mechanism

known as T-box antitermination. High levels of

uncharged tRNA

Trp

induce expression of AT, which

prevents TRAP from down-regulating expression of the

trp genes. Hence even if free tryptophan levels are high

enough to activate TRAP, if the level of charging of

tRNA

Trp

is low, then the trp genes will be expressed.

Thus while both E. coli and B. subtilis regulate

expression of the trp genes in response to changes in

levels of both free tryptophan and aminoacylated

tRNA

Trp

, they have evolved very different mechanisms

to do so.

OCCURRENCES OF TRAP MEDIATED

ATTENUATION IN BACTERIA

TRAP mediated attenuation control of the trp operon

has till date been only observed in B. subtilis and several

related bacilli including B. pumilus, B. stearothermo-

philus, B. caldotenax,andB. halodurans, as well as in

Clostridium thermocellum. In contrast to the leader-

peptide-dependent mechanism described previously,

the RNA-binding protein-dependent attenuation mecha-

nism has never been characterized for any other amino

acid biosynthetic operons beside tryptophan even

though it would seem to be easily adaptable by simply

changing the amino acid that activates the protein to

bind its RNA target. Perhaps with the explosion of

bacterial genomic information currently becoming

available we might soon discover such a system.