Novel tRNA aminoacylation mechanisms

Novel tRNA aminoacylation mechanismsTerry Cathopoulis,a Pitak Chuawongab and Tamara L. Hendrickson*a

DOI: 10.1039/b618899k

In nature, ribosomally synthesized proteins can contain at least 22 different aminoacids: the 20 common amino acids as well as selenocysteine and pyrrolysine. Eachof these amino acids is inserted into proteins codon-specifically via an aminoacyl-transfer RNA (aa-tRNA). In most cases, these aa-tRNAs are biosynthesized directlyby a set of highly specific and accurate aminoacyl-tRNA synthetases (aaRSs).However, in some cases aaRSs with relaxed or novel substrate specificitiescooperate with other enzymes to generate specific canonical and non-canonicalaminoacyl-tRNAs.

Introduction

The aminoacyl-tRNAs (aa-tRNAs) are

at the heart of protein biosynthesis.

These effector molecules are responsible

for the codon-defined insertion of amino

acids into specific positions in nascent

proteins—a process that proceeds with

high efficiency and accuracy.1 The text-

book pathway for aa-tRNA biosynthesis

is through the action of twenty conserved

aminoacyl-tRNA synthetases (aaRSs),

with each enzyme specific for pairing

one of the 20 standard encoded amino

acids to the correct tRNA (or tRNA

isoacceptor set). The 20 aaRSs all

catalyze the same series of reactions to

generate their respective aa-tRNAs.

First, each enzyme condenses its cognate

amino acid (aa) with ATP to generate an

enzyme-bound aminoacyl-adenylate (aa-

AMP, eqn (1)); this step is sometimes

tRNA-dependent.2–7 Next, either the 29

or 39 OH on the 39 end of the cognate

tRNAaa reacts with this aa-AMP to

generate the correct aa-tRNAaa product

(eqn (2)). Each aaRS is specific for only a

given amino acid and a given isoacceptor

set of tRNAs—this exquisite pairing

guarantees accurate protein translation.1

aa + ATP + aaRS Aaa-AMP*aaRS + PPi

(1)

aa-AMP*aaRS + tRNAaa Aaa-tRNAaa + aaRS + AMP

(2)

The rule of 20 aaRSs for 20 encoded

amino acids largely holds true in

eukaryotes (including Saccharomyces

cerevisiae8 and humans9) and some

microorganisms like Escherichia coli.10

The purpose of this Highlight article,

however, is to describe the known

exceptions to this rule, where organisms

have either a limited (,20) or a non-

standard set of aaRSs or tRNA amino-

acylation reactions.11,12 Five different

cases will be discussed. The first three

will focus on how organisms circumvent

the need for three different aaRSs: The

glutaminyl-, asparaginyl-, and cysteinyl-

tRNA synthetases (GlnRS, AsnRS, and

CysRS, respectively). The last two will

examine how the non-standard amino

acids selenocysteine and pyrrolysine are

introduced site-specifically into proteins

via the biosynthesis and utilization of

selenocysteinyl-tRNASec (Sec-tRNASec)

and pyrrolysyl-tRNAPyl (Pyl-tRNAPyl),

respectively, and read-through of in-

frame stop codons. All five cases require

additional enzymes, beyond the common

aDepartment of Chemistry, Johns HopkinsUniversity, 3400 N. Charles St., Baltimore,MD 21218E-mail: [email protected];Fax: +1 410-516-8420; Tel: +1 410-516-6706bDepartment of Chemistry, KasetsartUniversity, Pahonyothin Rd., Chatuchak,Bangkok 10900, Thailand

Terry Cathopoulis earned a BAdegree in Chemistry fromHaverford College in 2004.He is currently a graduates t u d e n t w o r k i n g w i t hP r o f e s s o r T a m a r aHendrickson in the ChemistryDepartment at Johns HopkinsUniversity, where he is investi-gating indirect mechanisms fortRNA aminoacylation used byt h e h u m a n p a t h o g e nHelicobacter pylori.

Pitak Chuawong earned anundergraduate degree in

Chemistry at Kasetsart University in Bangkok, Thailand. In1999, Pitak received a DPST Fellowship from the Royal Thai

Government to study abroad.He chose to come to theUnited States to attend OregonState University, where heearned an MS degree based onhis research in organometallicchemistry with Professor KevinP. Gable. He then transferred toJohns Hopkins Universitywhere he earned his PhD work-ing in the lab of ProfessorTamara Hendrickson. His dis-sertation focused on the non-discriminating aspartyl-tRNAsynthetase from Helicobacterpylori. He is now a lecturer

and research scientist in the Department of Chemistry atKasetsart University.

Terry Cathopoulis Pitak Chuawong

HIGHLIGHT www.rsc.org/molecularbiosystems | Molecular BioSystems

408 | Mol. BioSyst., 2007, 3, 408–418 This journal is � The Royal Society of Chemistry 2007

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http://dx.doi.org/10.1039/b618899k

http://pubs.rsc.org/en/journals/journal/MB

http://pubs.rsc.org/en/journals/journal/MB?issueid=MB003006

aaRSs, for the biosynthesis of the rele-

vant aa-tRNAs.

1. Indirect biosynthesis of Gln-tRNAGln in the absence ofGlnRS

In the late 1960’s, around the same

time that the nature of the different

aaRSs were defined, it was reported

that Bacillus subtilis lacks the ability

to directly generate Gln-tRNAGln

according to the pathway defined in

eqn (1) and (2), above.13 Instead, it was

demonstrated that Gln-tRNAGln is bio-

synthesized indirectly in two enzymatic

steps (Fig. 1), with the formation of Glu-

tRNAGln, followed by phosphorylation

and transamidation to generate Gln-

tRNAGln.14 These observations sug-

gested that B. subtilis does not have a

functional GlnRS, a fact that was con-

firmed when the B. subtilis genome

sequence was reported in 1997.15 It is

now clear that GlnRS is rare, being

found only in eukaryotes and amongst

a subset of bacteria, including E. coli.

Most bacteria and all archaea (at least

those sequenced to date) lack a glnS gene

and consequently generate Gln-tRNAGln

indirectly via the pathway shown in

Fig. 1.1,11

Two enzymes are required to complete

the reactions shown in Fig. 1: A non-

discriminating or a misacylating gluta-

myl-tRNA synthetase (ND-GluRS or

GluRS2, respectively) capable of

generating Glu-tRNAGln,16–19 and a

glutamine-dependent Glu-tRNAGln ami-

dotransferase (Glu-Adt or Asp/Glu-Adt,

see below).20 The details of this pathway

have been reviewed recently,21,22 so only

select highlights from the past few years

will be discussed in this article.

Misacylating glutamyl-tRNA

synthetases

Two different types of GluRSs are able

to generate the requisite Glu-tRNAGln

intermediate: A non-discriminating

GluRS (ND-GluRS, e.g. B. subtilis16

and Lactobacillus bulgaricus17) and

Tamara Hendrickson received her PhD inChemistry from the California Institute ofTechnology in 1996. She then conductedpost-doctoral research in molecular biol-ogy at the Massachusetts Institute ofTechnology and The Scripps ResearchInstitute. She joined the ChemistryDepartment at Johns Hopkins Universityas an Assistant Professor in 2000, whereshe also has a joint appointment in Biologyand is a member of the Program inMolecular and Computational Biophysicsand the Chemical Biology InitiativeProgram. Her research interests focus onprotein translation and post-translationalmodification reactions.

Tamara L. Hendrickson

Fig. 1 Gln-tRNAGln biosynthesis via the indirect transamidation pathway. (A) In organisms that lack GlnRS, Gln-tRNAGln is biosynthesized

indirectly. In the first step, a misacylating GluRS (either an ND-GluRS or GluRS2) generates Glu-tRNAGln. In the second step, Glu-Adt (GatDE)

or Asp/Glu-Adt (GatCAB) converts the glutamate side chain to glutamine by delivering ammonia from a molecule of glutamine; asparagine may

also be a source of ammonia. The two tRNAGln anticodons are given in parentheses. (B) Glu-Adt (GatDE) and Asp/Glu-Adt (GatCAB) each

catalyze the same three reactions to generate Gln-tRNAGln.

This journal is � The Royal Society of Chemistry 2007 Mol. BioSyst., 2007, 3, 408–418 | 409

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GluRS2 (e.g. Helicobacter pylori18,19 and

Acidithiobacillus ferrooxidans19). In most

prokaryotes, the GluRS is non-discrimi-

nating; these enzymes have relaxed

tRNA specificities and generate the

misacylated product Glu-tRNAGln, in

addition to their cognate product, Glu-

tRNAGlu.21 In contrast, eukaryotes

and some bacteria (e.g. E. coli),

particularly those with a functional

GlnRS, have a canonical discriminating

GluRS (D-GluRS) that only generates

Glu-tRNAGlu.21

The first crystal structure of an ND-

GluRS, from the thermophilic bacterium

Thermosynechococcus elongatus, was

recently reported.23 A comparison of this

structure to the Thermus thermophilus

D-GluRS24 revealed important differ-

ences behind the divergent patterns of

tRNA recognition used by each of

these enzymes. Variations in the

anticodon-binding domain are particu-

larly important. The anticodons of

tRNAGlu and tRNAGln share two

of three nucleotides—C34 or U34

(the U is post-transcriptionally modified

to 5-methylaminomethyl-2-thiouridine

(mnm5s2U)),25,26 and U35. Position 36

is the codon-defining position for the two

tRNAs: it is a C36 in tRNAGlu, but a

G36 in tRNAGln. Thus, an ND-GluRS

accommodates both C and G in this

position whereas a D-GluRS is specific

for C36. In fact, known D-GluRSs

contain a critical arginine (Arg358 in

the T. thermophilus D-GluRS) that forms

two hydrogen bonds with C36 in

tRNAGlu.24 In contrast, ND-GluRSs

have smaller amino acids in this position

(Gly366 in T. elongatus ND-GluRS),

creating a larger, less specific cavity that

can accommodate the two different

nucleotides.23

Instead of utilizing a single ND-

GluRS, some bacteria utilize two

GluRSs (GluRS1 and GluRS2) for the

biosynthesis of a complete set of Glu-

tRNAGlu and Glu-tRNAGln isoaccep-

tors.18,19 In Helicobacter pylori, for

example, GluRS1 is discriminating and

aminoacylates only the two tRNAGlu

isoacceptors and not tRNAGln. In order

to complete the set of required aa-

tRNAs, H. pylori GluRS2 produces

Glu-tRNAGln. The activities of GluRS1

and GluRS2 are complementary and

together they ensure the availability of a

complete set of aa-tRNAs.18,19

Interestingly, GluRS2 has lost the ability

to generate Glu-tRNAGlu, its ‘‘cognate’’

product, suggesting that this enzyme

might represent an intermediate in the

evolution of an as of yet unknown or

future bacterial GlnRS.18 (Known

bacterial GlnRSs are eukaryotic in

origin.27,28) The importance of the

anticodon-binding domain in directing

the unique tRNA recognition pattern

of H. pylori GluRS2 has also been

demonstrated.29

The GluRS1/GluRS2 duplication in

Acidithiobacillus ferrooxidans paints a

slightly different picture. In this case,

GluRS1 is non-discriminating and ami-

noacylates tRNAGlu and one tRNAGln

isoacceptor (tRNAGlnCUG); the GluRS2 is

still specific only for tRNAGlnUUG.19 This

tRNA specificity has been correlated to

the length of the D-stem in the different

tRNAs, with GluRS2 recognizing

tRNAGlnUUG, which has a shorter D-stem,

and GluRS1 recognizing the three

tRNAs with longer D-stems.19 The exact

mechanism of D-stem recognition

remains unknown.

Glu-Adt (GatDE)—conversion of Glu-

tRNAGln to Gln-tRNAGln in archaea

The second step in indirect Gln-tRNAGln

biosynthesis is the glutamine- and ATP-

dependent conversion of Glu-tRNAGln

into Gln-tRNAGln (Fig. 1A and B).20

This conversion is accomplished in a

three-step process. (1) Glutamine (and/or

possibly asparagine) is hydrolyzed to

produce ammonia and glutamate (or

aspartate) (Fig. 1B, rxn (1)).30 The

resultant ammonia remains sequestered

within the enzyme. (2) The amino

acid carboxylate in Glu-tRNAGln is

phosphorylated to c-phosphoryl-Glu-

tRNAGln (Fig. 1B, rxn (2)).14 3) The

ammonia is delivered to the activated

c-carbonyl to generate Gln-tRNAGln, the

final product (Fig. 1B, rxn (3)).

In archaea, these reactions are cata-

lyzed by the Glu-tRNAGln amidotrans-

ferase (Glu-Adt), a heterodimer

composed of the GatD and GatE

subunits.31,32 Two crystal structures of

archaeal Glu-Adt orthologs have been

reported: the apo-enzyme from

Pyrococcus horikoshii33 and a complex

of GatDE:tRNAGln from Methano-

thermobacter thermoautotrophicus.34

These structures show that GatDE is an

a2b2 heterodimer. The GatD subunit

shares sequence and structure homology

with Asparaginase A and contains the

glutaminase active site (Fig. 1, rxn (1)).

Its glutaminase activity is tightly coupled

to the binding of Glu-tRNAGln and the

integrity of the GatDE heterodimer.

Four conserved amino acids, Thr101,

Thr177, Asp178, and Lys254, are critical

for glutaminase activity, with one of the

two threonines likely serving as the active

site nucleophile within a Thr-Lys-Asp

catalytic triad, analogous to that found

in L-asparaginases.32

GatE contains the kinase and trans-

amidase active sites (Fig. 1, rxn (2) and

(3)) and is also solely responsible for

tRNA recognition. Several conserved

residues in GatE (e.g. His15, Glu157,

and Glu184) are critical for both kinase

and transamidase activity, but not for

glutaminase activity. In contrast, a few

mutations have been identified (e.g.

Arg221Ala) that disrupt all three

enzyme activities, highlighting the tight

coupling between the GatD-catalyzed

glutaminase activity and the GatE

active site.34 The GatD and GatE

active sites are connected by a 40 A

channel, lined with hydrophilic residues,

which is positioned to promote ammonia

delivery from the GatD glutaminase

active site to the transamidation site in

GatE.34

The co-crystal structure of M. thermo-

autotrophicus GatDE complexed with

tRNAGln revealed that GatE, and not

GatD, binds tRNAGln, and this binding

is independent of the anticodon.34 GatE

binds to the top of the L-shaped tRNA,

forming contacts with the TYC helix and

the D-loop, and positioning the tRNA

acceptor stem in the transamidase active

site. Site-directed mutagenesis demon-

strated that the contacts between

tRNAGln and Gln240 are important for

amidotransferase activity; Arg503 and

Asn463 also form key contacts with the

tRNA D-stem. Additionally, analysis of

mutations in tRNAGln demonstrated that

U19 and A20 are specifically critical for

amidotransferase activity and that the

G1?A72 base pair in tRNAAsn is a key

antideterminant.

Finally, GatE contains an AspRS-like

insertion domain, which has been

proposed to play a role in prohibiting

complex formation between AspRS,

Asp-tRNAAsn, and GatDE, thus


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preventing GatDE from utilizing Asp-

tRNAAsn as a substrate.34

Asp/Glu-Adt (GatCAB)—conversion

of Glu-tRNAGln to Gln-tRNAGln in

bacteria and archaea

In bacteria, some organelles, and some

archaea, Glu-tRNAGln is converted into

Gln-tRNAGln by the Asp-tRNAAsn/Glu-

tRNAGln amidotransferase (Asp/Glu-

Adt, so named because this enzyme also

converts Asp-tRNAAsn into Asn-

tRNAAsn, see below). Asp/Glu-Adt is

heterotrimeric and is composed of the

GatC, GatA, and GatB subunits.

Interestingly, some archaea have both

GatDE and GatCAB.22,31 Organisms

containing both amidotransferases lack

GlnRS and AsnRS, consequently, Asp/

Glu-Adt is present to ensure Asn-

tRNAAsn biosynthesis. In these cases, it

is likely that both GatDE and GatCAB

are contributing to Gln-tRNAGln bio-

synthesis in vivo.

The first set of crystal structures of

GatCAB (from Staphylococcus aureus)

was recently reported35 and, when com-

bined with biochemical analyses, gives

rise to a detailed picture of Asp/Glu-

Adt.20,30,35–38 The GatA subunit shares

homology with certain amidases and

contains the glutaminase active site;

GatA is not structurally related to

GatD. Glutamine hydrolysis proceeds

via the formation of an acyl-enzyme

intermediate at the glutamine c-carbonyl

with Ser178 (S. aureus numbering). The

importance of this serine was first

demonstrated experimentally30 and the

actual acyl-enzyme intermediate was

directly observed in a crystal structure

of GatCAB with glutamine.35

GatB contains the kinase and amido-

transferase active sites and is closely

related to GatE, but GatB lacks an

AspRS-like domain. A 30 A hydrophilic

tunnel connects the active sites of GatA

and GatB.35 The fact that both GatDE

and GatCAB contain hydrophilic tunnels

separates these enzymes from other

ammonia-generating enzymes that typi-

cally contain long hydrophobic tunnels

in order to maintain the ammonia in a

state of deprotonation.39 Thus, it has

been proposed that GatCAB transports

an ammonium cation through its tunnel,

possibly by a series of protonation and

deprotonation events; consequently, a

mechanism for deprotonation prior

to or concomitant with transamidation

is required.35,39 It is likely that a

protonation–deprotonation mechanism

is used by GatE as well.34 A co-crystal

structure with tRNAGln bound to

GatCAB has not been reported, however

the similarities between GatB and GatE

suggest that Glu-tRNAGln will bind in a

manner similar to that observed in

the GatDE-tRNAGln complex structure.

Mutagenesis experiments have pointed to

the U1?A72 base pair in the tRNAGln

acceptor stem as a positive identity

determinant and the insertion of an extra

U into the D-loop as an antideterminant

to prevent Glu-tRNAGlu from binding.

Finally, GatC is a small protein of less

than 100 amino acids. In the GatCAB

crystal structure, GatC is wrapped

around the GatA/GatB interface and

perhaps plays a role in stabilizing or

assembling the protein complex.35

2. Indirect biosynthesis of Asn-tRNAAsn in the absence ofAsnRS

In organisms that lack AsnRS (e.g. H.

pylori), Asp-tRNAAsn is generated by a

non-discriminating AspRS (ND-AspRS)

that, like ND-GluRS, has dual tRNA

substrate specificity and aminoacylates

both tRNAAsp and tRNAAsn.22,40

Indirect biosynthesis of Asn-tRNAAsn

(Fig. 2) parallels that of Gln-tRNAGln

biosynthesis, with some notable excep-

tions. First, AsnRS is more widespread

than GlnRS, consequently this indirect

pathway is less common in bacteria but is

still prevalent in archaea.41 Second,

transamidation is catalyzed by Asp/Glu-

Adt (GatCAB) only, as Asp-tRNAAsn is

not a substrate for Glu-Adt (GatDE).31

Third, some organisms lack asparagine

synthetase and consequently rely on this

indirect pathway as the sole route for

asparagine production.42

Crystal structures of several different

canonical discriminating AspRSs (D-

AspRS) have been reported and reviewed

elsewhere.40 There are two divergent

types of ND-AspRSs – one of archaeal

origin and the other of bacterial origin.41

The structure of an archaeal-type ND-

AspRS from T. thermophilus has been

solved.43 Not surprisingly, given the

importance of anticodon recognition by

most aaRSs, key differences in the

structures of the anticodon-binding

domains of D-AspRSs and ND-AspRSs

were revealed upon comparison of repre-

sentative crystal structures. Similar to

ND-GluRSs, recognition of position 36

in the anticodons of tRNAAsp (C36) and

tRNAAsn (U36) was shown to be critical

for the divergent tRNA specificities of

D-AspRSs and ND-AspRSs; insertion of

an AsnRS-like loop from an ND-AspRS

into a D-AspRS was sufficient to convert

the D-AspRS into an ND-AspRS.43

Furthermore, mutation of a single pro-

line (P77) in the anticodon-binding

domain of the Deinococcus radiodurans

archaeal-type ND-AspRS was sufficient

to convert this enzyme into a

D-AspRS.44

A crystal structure of a bacterial-type

ND-AspRS has not yet been reported,

however the anticodon-binding domains

of two different ND-AspRS orthologs

have been analyzed by site-directed

Fig. 2 Asn-tRNAAsn biosynthesis via the indirect transamidation pathway. Asn-tRNAAsn can be biosynthesized indirectly via a pathway

analogous to that shown in Fig. 1. In the first step, an ND-AspRS generates Asp-tRNAAsn. Next, Asp/Glu-Adt (GatCAB) converts the glutamate

side chain to glutamine by delivering ammonia from glutamine or asparagine. The two tRNAAsn anticodons are given in parentheses.


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mutagenesis. The pathogenic bacterium

Pseudomonas aeruginosa PAO1 (P. aeru-

ginosa PAO1) utilizes a bacterial type

ND-AspRS for Asp-tRNAAsn synth-

esis.45 Mutations in this ND-AspRS

(H31L and G83K, and the double

mutant H31L/G83K) were designed

based on sequence conservation and

analyzed for variations in tRNA specifi-

city. Each of these mutant proteins

exhibited greater tRNAAsp specificity

when tested against total tRNA from

E. coli.46 However, the specificity gain

for tRNAAsp was very small when these

mutants were tested against P. aerugi-

nosa PAO1 total tRNA.46 Anticodon-

binding domain mutations were also

introduced into the H. pylori ND-

AspRS and were shown to increase

tRNAAsp aminoacylation over

tRNAAsn.47 It is interesting that muta-

tions haven’t yet been identified that

increase recognition of tRNAAsn over

tRNAAsp.

Asp-tRNAAsn is converted into Asn-

tRNAAsn by Asp/Glu-Adt (Fig. 2), via

the same mechanism as described for the

indirect biosynthesis of Gln-tRNAGln

(see previous section). Although a co-

crystal structure of GatCAB with bound

tRNAAsn has not yet been reported,

identity determinants for GatCAB recog-

nition of this tRNA have been evaluated

using the Neisseria meningitidis GatCAB.

As was shown for tRNAGln and

described above, tRNAAsn contains a

U1?A72 base pair that is essential for

GatCAB activity with this tRNA.

Furthermore, in tRNAAsp, the G1?C72

base pair and the supernumerary U20A

D-loop base serve as anti-determinants

for GatCAB recognition.48

3. Indirect biosynthesis of Cys-tRNACys in the absence ofCysRS

Most organisms contain a functional

copy of the cysS gene and biosynthesize

Cys-tRNACys directly using a cognate

CysRS.41 However, a few archaea lack a

copy of the cysS gene and instead

biosynthesize Cys-tRNACys indirectly.11

Two possible solutions to this problem

were first put forth in the literature: One,

that either ProRS49,50 or, two, that a

protein with weak similarity to CysRS,51

were responsible for the direct generation

of Cys-tRNACys biosynthesis. However,

as a greater understanding of this system

was obtained, both of these hypotheses

lost support.52

Bioinformatic analyses pointed to the

possibility that an open reading frame

(ORF) of unknown function in

M. jannaschii (MJ1660) might function

as a class II CysRS (The canonical

CysRS is class I).53 Shortly thereafter,

MJ1660 was shown to be a novel

O-phosphorylseryl-tRNA synthetase

(SepRS),54 which catalyzes the specific

attachment of phosphoserine (Sep) to

tRNACys (Fig. 3A). MJ1660 is accom-

panied by MJ1678, which encodes

for a Sep-tRNA:Cys-tRNA synthase

(SepCysS) – this enzyme converts Sep-

tRNACys into Cys-tRNACys, using

pyridoxal phosphate (PLP) as a

cofactor.54 In an interesting parallel to

the indirect biosynthesis of Asn-

tRNAAsn, this SepRS/SepCysS pathway

to Cys-tRNACys serves as the only

route for cysteine biosynthesis in

Methanococcus maripaludis.54

The archaeal SepRS is a standard

aaRS based on the reactions it catalyzes

(Rxns 1 and 2 above); its uniqueness

stems from the fact that phosphoserine is

its amino acid substrate and misacylated

phosphoseryl-tRNACys (Sep-tRNACys) is

its direct product (Fig. 3A). This protein

has been designated a class II aaRS54 and

it recognizes a set of identity nucleotides

in tRNACys that is closely related to the

identity set used by canonical CysRSs.55

The archaeal SepCysS is an interesting

protein that catalyzes the conversion of

Sep-tRNACys to Cys-tRNACys in a PLP-

dependent fashion (Fig. 3A).54 In the

biochemical characterization of SepCysS,

Na2S was used as the sulfur donor, and

the identity of the natural sulfur donor

remains uncharacterized. SepCysS is a

cysteine desulfurase that shares simila-

rities with NifS and IscS, two other

enzymes that are involved in the incor-

poration of sulfur into different metabo-

lites.56 In general, cysteine desulfurases

like NifS, and IscS all generate and

utilize cysteine persulfides as their reac-

tive sulfur-donating species.57 Thus, it

seems likely that SepCysS uses a similar

persulfide mechanism to donate sulfur to

Sep-tRNACys. In addition, the use of

PLP as a cofactor is reminiscent of

selenocysteinyl-tRNASec (Sec-tRNASec)

biosynthesis, where PLP promotes

the conversion of either Ser-tRNASec or

Sep-tRNASec to dehydroalanyl-tRNASec,

prior to selenium incorporation (see

Section 4, below).58 By combining these

similarities, a hypothetical mechanism

for SepCysS can be proposed (Fig. 3B):

One can imagine that the phosphate

group in Sep-tRNACys would be elimi-

nated to generate dehydroalanyl-

tRNACys via formation of a PLP adduct

with the phosphoseryl amino group. The

electron sink provided by the PLP would

drive deprotonation and elimination of

the phosphate group (delocalization into

PLP is not shown). Next, sulfur from a

SepCysS persulfide group could react

with this adduct to generate PLP-

modified Cys-tRNACys; the resultant

enzyme-bound disulfide would be

reduced by reaction with another

SepCysS cysteine residue and the PLP

group would be transferred to an active

site lysine. Proof (or disproof) of this

proposed mechanism awaits further

characterization of SepCysS.

4. Indirect biosynthesis ofselenocysteinyl-tRNASec

In 1976, it was reported that a subunit of

Clostridium stricklandii glycine reductase

contains a selenocysteine amino acid.59

Ten years later, it was demonstrated that

selenocysteine is directly incorporated

into some proteins via read-through of

an in-frame UGA stop codon during

ribosomal protein biosynthesis.60,61

These discoveries led to selenocysteine

being labeled the 21st amino acid. It is

now well established that selenocysteine

is incorporated into proteins via seleno-

cysteinyl-tRNASec (Sec-tRNASec) and

that this intermediate is biosynthesized

and utilized in all three domains of life.

(For a recent review on this topic, see

Bock et al.62)

In bacteria, Sec-tRNASec is generated

in two steps: The first is aminoacylation

by seryl-tRNA synthetase (SerRS) to

generate Ser-tRNASec (Fig. 4).63 The

second step is catalyzed by the enzyme

selenocysteine synthase, a PLP-depen-

dent enzyme (encoded by selA), that uses

monoselenium phosphate as the selenium

donor (Fig. 4A).58 The SelA-catalyzed

conversion of Ser-tRNASec into Sec-

tRNASec is a multi-step process

(Fig. 4B). The first step is the formation

of a Schiff’s base between the serine

amino group in seryl-tRNASec (R = H,


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Fig. 4B) and PLP. This adduct undergoes

SelA-mediated dehydration to generate

dehydroalanyl-tRNASec, which is subse-

quently converted to Sec-tRNASec via the

addition of monoselenium phosphate,

followed by hydrolysis to release PLP

from the correctly charged tRNA.58,64,65

Interestingly, SelA will also convert

phosphoseryl-tRNASec into Sec-

tRNASec in vitro, however a bacterial

Ser-tRNASec kinase has not been identi-

fied and Ser-tRNASec is the presumed

in vivo substrate.66

The combined action of SerRS and

selenocysteine synthase make the bio-

synthesis of Sec-tRNASec an indirect

process, analogous to the mechanisms

described above for the biosynthesis of

Gln-tRNAGln, Asn-tRNAAsn, and Cys-

tRNACys. In contrast to incorporation of

these coded amino acids, however, sele-

nocysteine is incorporated into a small

subset of proteins at positions noted by

the opal stop codon UGA. This process

is guided by SelB, a Sec-tRNASec-specific

elongation factor,64 and by recognition

of a hairpin loop structural element

called the selenocysteine insertion

sequence (SECIS) in the encoding

mRNA.67

In bacteria, the SECIS hairpin is

located within the encoded gene ORF,

immediately downstream from the in-

frame UGA destined for selenocysteine

incorporation.67 SelB recognizes this

SECIS and forms a complex between it,

GTP, and Sec-tRNASec in order to load

the aa-tRNA onto the ribosome at the

right time.68 The crystal structure of SelB

shows that this protein shares structural

features with elongation and initiation

factors.69

In eukaryotes and archaea, Sec-

tRNASec biosynthesis is accomplished

via a similar pathway to that used by

bacteria (Fig. 4A). The most notable

difference is that Ser-tRNASec is phos-

phorylated by an O-phosphorylseryl-

tRNASec kinase (PstK), prior to

modification by a SelA analog named

SecS or SepSecS;70–72 this use of Sep-

tRNACys as an intermediate is similar to

the indirect biosynthesis of Cys-tRNACys

(see Fig. 3B).54 Presumably this phos-

phorylation event improves elimination

to generate the PLP-dehydroalanyl-

tRNASec intermediate (Fig. 4B, R =

OPO322). Expression of SepSecS/SecS

or PstK alone was insufficient to restore

selenocysteine incorporation into pro-

teins in an E. coli selA deletion strain;

however, co-expression of PstK and

SepSecS/SecS led to successful incorpora-

tion of selenocysteine into formate dehy-

drogenase H in this same strain, clearly

demonstrating that phosphorylseryl-

tRNASec is the substrate for mammalian

and archaeal SepSecS/SecS.72 Homologs

of PstK have also been identified in

Fig. 3 Indirect biosynthesis of Cys-tRNACys. (A) In a small set of microorganisms, Cys-tRNACys is biosynthesized indirectly. In the first step,

tRNACys is aminoacylated with phosphorylserine; this reaction is catalyzed by SepRS. Next, the phosphorylseryl-tRNACys is converted into Cys-

tRNACys, in a PLP-dependent reaction catalyzed by SepCysS; the origin of the sulfur is unknown. (B) Proposed mechanism for SepCysS, based on

its homology with other sulfur-donating enzymes and the mechanism for Sec-tRNASec biosynthesis (See Fig. 4); delocalization of electrons into

PLP have been omitted for simplicity. In the final steps (denoted by two arrows), Cys-tRNACys is released by reduction of its disulfide bond to

SepCysS and release of PLP.


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mammals, Caenorhabditis elegans,

Methanopyrus kandleri, and M. jan-

naschii, but not in yeast, bacteria or

plants.70

In eukaryotes, the SECIS is located in

the 39 untranslated region (UTR), distal

to the in-frame UGA codon and past the

ORF stop codon.73 The eukaryotic SelB

still forms a complex with Sec-tRNASec

and GTP but it does not bind the

eukaryotic SECIS.74 Instead, a eukaryo-

tic SECIS-Binding Protein (eSBP2) binds

these elements, completing the complex

formation required for Sec-tRNASec

insertion into the ribosome.75 Evidence

also suggests that ribosomal protein L30

participates in the formation of this Sec-

tRNASec elongation complex.76

The archaeal system for selenocysteine

incorporates features of both the eukary-

otic and bacterial systems. Like eukary-

otes, Sec-tRNASec is biosynthesized by

SerRS, PstK, and SepSecS/SecS (Fig. 4B,

R = OPO322).72 There is an archaeal

ortholog of SelB and inactivation of this

selB gene in Methanococcus maripaludis

ablated selenoprotein biosynthesis,

demonstrating that this protein is the

archaeal Sec-tRNASec elongation

factor.77 As in eukaryotes, the archaeal

SECIS is removed from the in-frame

UGA and is in the 39 UTR (with one

example of the SECIS in the 59 UTR in

M. jannaschii78), however archaea do not

have an apparent analog of SBP2,

suggesting that this SECIS is recognized

by SelB alone or in conjunction with one

or more other as of yet unidentified

proteins.79

5. Direct biosynthesis ofpyrrolysyl-tRNAPyl

The first observation of pyrrolysine as

a proteinogenic amino acid arose

through research on the ability of the

archaeal family, Methanosarcinacea, to

use methylamines in the production

of methane. This process involves a

number of methyltransferases, including

monomethylamine methyltransferase

(MMAMT).80

In 1998 it was reported that MMAMT

contained a traditional stop codon within

its open reading frame81 and by 2002 it

was confirmed by crystal structure that

the amino acid at the corresponding

position was a lysine joined to a (4R,

5R)-4-substituted pyrroline-5-carboxy-

late via an amide linkage.82 Further

analysis and the use of synthetic stan-

dards have identified a methyl group

substituent at position 4 (Fig. 5).83,84

Pyrrolysine is unique amongst the

tRNA aminoacylation pathways dis-

cussed herein because it is directly

charged onto its cognate tRNAPyl

(encoded by pylT) by a novel aaRS,

pyrrolysyl-tRNA synthetase (PylRS,

encoded by pylS), without further

enzyme modification of the aminoacyl-

tRNA.85–87 Five genes are essential

Fig. 4 Indirect biosynthesis of Sec-tRNASec. A) Sec-tRNASec biosynthesis begins with serylation of tRNASec, a reaction catalyzed by SerRS.

Next, the Ser-tRNASec is converted to Sec-tRNASec in a PLP-dependent reaction that is catalyzed by SelA in bacteria (top pathway). In eukaryotes

and archaea, Ser-tRNASec is phosphorylated by PstK and the resultant c-phosphoseryl-tRNASec is converted to Sec-tRNASec by SepSecS/SecS

(bottom pathway). B) The apparent mechanism catalyzed by SelA in the generation of Sec-tRNASec; delocalization of electrons into PLP have been

omitted for simplicity.


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for pyrrolysine biosynthesis and

translational incorporation (pylT, pylS,

pylB, pylC, and pylD), and these genes

are in a pylTSBCD operon in

Methanosarcina barkeri. This operon

has been identified in all sequenced

Methanosarcina and Methanococcoide

genomes and, interestingly, in the

unrelated bacterium Desulfitobacterium

hafniense.85

The five genes in the pylTSBCD

operon (from M. acetivorans) are neces-

sary and sufficient to introduce pyrroly-

sine into the genetic code of E. coli.88

Consequently, it has been concluded that

the pylBCD genes represent the pyrroly-

sine biosynthetic machinery and a

mechanism for pyrrolysine biosynthesis

has been proposed.88

Transfer RNAPyl is robustly amino-

acylated with pyrrolysine by PylRS85–87

and can be weakly aminoacylated with

lysine when incubated with both LysRS1

and LysRS2 from M. barkeri.89 It

remains to be seen whether or not the

pyrrolysine biosynthetic machinery will

utilize this Lys-tRNAPyl as a substrate to

convert it to Pyl-tRNAPyl, but this

observation raises the intriguing possibi-

lity that M. barkeri may biosynthesize

Pyl-tRNAPyl both directly and indirectly.

PylRS is specific for tRNAPyl and

recognizes this tRNA’s G73 discrimina-

tor base and the G1?C72 acceptor stem

base pair as major identity elements.90

PylRS shows some amino acid promis-

cuity and can charge tRNAPyl with

N-e-D-prolyl-lysine and with N-e-cyclo-

pentyloxycarbonyl-L-lysine in vitro and

in vivo in E. coli.91

Like selenocysteine, pyrrolysine is

encoded by a stop codon (The amber

stop codon, UAG, in this case).80,92 In

analogy to the SECIS element in seleno-

cysteine incorporation, a pyrrolysine

insertion sequence (PYLIS) has been

suggested and a structural element

proposed.12,93,94 Alternatively, genomic

studies on the frequency of UAG codons,

both internally and as stop codons, in

pyrrolysine-containing species have

shown that the frequency of this stop

codon is extremely low compared to

species that do not express pyrroly-

sine.95,96 This statistical observation has

led to the suggestion that pyrrolysine

insertion at the amber codon may merely

be competitive to termination, or even

completely reassigned in some species. In

fact, recent reports have demonstrated

that Pyl can be inserted into proteins in

the absence of a PYLIS;91,97 this read-

through is analogous to classical amber

suppression and is consistent with the

fact that elongation factor TU binds Pyl-

tRNAPyl.98 However, the presence of the

PYLIS mRNA motif enhances pyrroly-

sine incorporation instead of premature

termination.97

6. Conclusion

As highlighted in this review, it is now

well established that aa-tRNAs can be

biosynthesized via unexpected, indirect

mechanisms and through the use of non-

standard amino acids. Still, the aaRSs

are always involved in one way or

another and the critical role played by

these enzymes in maintaining transla-

tional accuracy cannot be overstated.

At the present time, the number of

encoded amino acids stands at 22 and it

remains to be seen if, and how many,

other non-standard amino acids may be

directly incorporated into proteins via

aa-tRNAs. A genomic search for tRNA

genes of unknown function suggests that

non-standard amino acid incorporation

is rare and may be limited only to

selenocysteine and pyrrolysine.99 With

the widespread use of some of the path-

ways discussed herein, it seems unlikely

that life would have limited itself to

22 amino acids. In time, particularly as

genomic data from more obscure organ-

isms become available, it is enticing to

consider the possibility that more novel

tRNA aminoacylation mechanisms will

be discovered.

Acknowledgements

The authors thank Professor Joseph

Krzycki for providing a copy of a

manuscript prior to publication,

Professor Zan Luthey-Schulten and

Anurag Sethi for helpful discussions,

and the reviewers for thoughtful

comments.

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Novel tRNA aminoacylation mechanisms