Protein biosynthesis: aminoacyl-tRNA synthetases and aminoacylation

Protein biosynthesis:
aminoacyl-tRNA synthetases
and aminoacylation
Rya Ero
07.09.09

Aminoacyl-tRNA synthetases
(AaRS)
• charge tRNAs with amino acids (establish the
link between nucleic and amino acids)
• divided into two unrelated classes by based on
structural and biochemical properties
• highly specific - join 1 type of amino acid to all its
corresponding (cognate) tRNAs
• nomenclature:
Nomenclature of tRNA synthetases and charged tRNAs.
amino
acid
serine
leucine
cognate
tRNA
tRNA Ser
tRNA Leu
tRNA Leu
UUA
cognate aminoacyl tRNA
synthetase
seryl-tRNA synthetase
leucyl-tRNA synthetase
aminoacyl tRNA
(charged tRNA)
seryl- tRNA Ser
leucyl- tRNA Leu
leucyl- tRNA Leu
UUA

Comparison of class I and class II
AaRSs
Class I
Class II
Structure of the enzyme
active site
Interaction with the tRNA
Orientation of the bound
tRNA
Amino acid attachment
Enzymes for ...
Parallel β-sheet
(Rossman fold)
Minor groove of the
acceptor stem
variable loop faces away
from the enzyme
To the 2′-OH of the
terminal nucleotide of
tRNA
Arg, Cys, Gln, Glu, Ile,
Leu, Lys1, Met, Trp, Tyr,
Val
Antiparallel β-sheet
Major groove of the
acceptor stem
variable loop faces the
enzyme
To the 3′-OH of the terminal
nucleotide of the tRNA
Ala, Asn, Asp, Gly, His,
Lys2, Phe, Pro, Thr, Ser

Classification and subunit structure of AaRSs in
E. coli
Class I
Class II
Arg (α) Ala (α 4
)
Cys (α) Asn (α 2
)
Gln (α) Asp (α 2
)
Glu (α) Gly (α 2
β 2
)
IIe (α) His (α 2
)
Leu (α) Lys (α 2
)
Met (α) Phe (α 2
β 2
)
Trp (α 2
) Ser (α 2
)
Tyr (α 2
) Pro (α 2
)
Val (α) Thr(α 2
)

Evolution and sub-groups of AaRS

Class I active site (Rossman fold - 5 parallel β
strands connected by α helices), HIGH and
KMSKS signature sequences, extended
conformation of bound ATP

Class II active site, 3 conserved motifs:
• motif 1 - binding ATP (bent conformation) (α-helix)
• motif 2 - amino acid binding (β-sheet)
• motif 3 - dimerization

Comparison of class I and class II active sites

Recognition of tRNAs by AspRS (class II) and ArgRS
(class I), shown as outlines. The tRNAAsp shown will
bind to either. Sites on the opposite sides of tRNA
make contacts with the two AaRSs (blue balls -
contacts with AspRS, yellow balls - with ArgRS).

Class I aaRSs interact with the 5’ nucleotides of the
acceptor stem and class II aaRSs with the 3’
nucleotides.
The CCA arm of tRNA adopts different conformations
in complexes with the class I and class II AaRSs.

The structure of the complex between threonyl-tRNA
synthetase (class II) and tRNAThr reveals that the
synthetase binds to both the acceptor stem and the
anticodon loop of tRNA.

Glutaminyl-tRNA synthetase (class I) tRNAGln
complex reveals that the synthetase interacts with
base pair G10:C25 in addition to the acceptor step
and anticodon loop.

LysRS of most eukaryotes and bacteria is a class II
AaRS, but in archaea class I AaRS (blue – highly
conserved region, red – less conserved region).
Recognice same regions of tRNA, aminoacylation
mechanism differs.

Other functions of AaRS
• nuclear proofreading of tRNAs prior to their
export to cytoplasm (tRNA aminoacylation
takes place in cucleus, AaRSs must be
imported from cytoplasma)
• involved in transcriptional and translational
control of protein biosynthesis
• aminoacylation of tRNA-like structures (tmRNA
a.k.a. 10SaRNA )

• inhibiors of AaRSs can be used as antibiotics
• mupirocin inhibits IleRS of gram-positive (S.
aureus) and gram-negative bacteria (H.
influenzae)

In eukaryotes some AaRSs form complexes with
other proteins, function not known

tRNA

tRNA identity elements
• signals in tRNA required by AaRSs for correct
aminoacylation, “second genetic code”
• each AaRS must be able to distinguish the correct
(cognate) tRNA from all the others
• isoacceptor tRNAs have mostly the same set of
identity determinants
• swapping identity elements between tRNAs can
cause the AaRS to add amino acid to the wrong
tRNA
• identity of tRNA is determined by its primary,
secondary and tertiary structure

Sequence elements:
Mostly non-modified nucleosides (except E. coli
tRNAIle, tRNAGlu and tRNALys)
• N73 discriminator position (except E. coli
tRNAThr and tRNAGlu)
• anticodon nucleosides (except E. coli tRNAAla,
tRNASer and tRNALeu)
• first base pairs in acceptor stem (mostly N1:N72
and N2:N71)
Nucleosides in tRNA core are infrequently used
as identity determinants

G3
tRNA Ala
U70
G20
A73
G34 A36
A35
tRNA Phe
G1
G2
AU3
G73
C72
C71
UA70
C11
G24
G35 A36
U37
tRNA
fMet
tRNA Ser

Structural elements:
identity elements may also act indirectly through
changing the conformation of tRNA recognized by
AaRS
• lengths of variable and D loops are important for E.
coli tRNAGln, tRNAAla and yeast tRNAPhe.
• long extra arm in tRNASer is important for
recognition
• major recognition element for E. coli AlaRS is
G3:U70 wobble base pair in tRNAAla. Not certain
whether AlaRS recognices the specific chamical
groups or the acceptor stem conformation induced
by this base pair.

In case of tRNAAla a
single base pair (G3-
U70) in the right
context of the
acceptor stem is
necessary and
sufficient for
recognition by AlaRS
Microhelix recognized by AlaRS. A stem-loop
containing just 24 nt corresponding to the tRNA
acceptor stem is aminoacylated by AlaRS.

• Anti-determinants: negative signals that keep
tRNAs from aminoacylation by non-cognate
AaRSs. Include modified nucleosides and
structural elements. May be located in different
regions of tRNA
• Permissive elements: not directly involved in
determining the identity of tRNA, form a context
for optimal recognition of tRNA identity elements
by AaRSs
Different identity elements may have additive,
cooperative, anti-cooperative, or independent
effects

Conserved nucleosides (left) and identity
elements (right) in E. coli tRNAs

• identity elements mostly clustered in acceptor
stem and anticodon region
• these two regions come into close contact with
AaRSs
• aaRSs mostly contact with tRNA hydrogen
bonds are formed beween identity nucleosides
in tRNA and amino acids in AaRSs
• stronger and weaker identity elements exist

Aminoacylation reaction
• there is a different AaRS for each amino acid (20
in all) in most cells
• recognition of amino acid and tRNA varies
among AaRSs
• all AaRSs catalyze the same 2-step ATPdependent
reaction
(1) aaRS + ATP:Mg2+ + aa ↔ aaRS:aa:AMP + PPi :Mg2+
(2) aaRS:aa:AMP + tRNA ↔ aaRS + aa-tRNA + AMP

Step 1 – activation of amino acid
aa + ATP + AaRS ↔ AaRS·aa-AMP + PPi
carboxyl oxygen of amino acid reacts
with α-phosphate of ATP,
aminoacyladenylate (high energy
intermediate) is formed and PPi
released
Step 2 – A76 of uncharged tRNA reacts
with aminoacyladenylate
AaRS·aa-AMP + tRNA ↔ AaRS + aatRNA
+AMP
nucleophilic attac of 2’ or 3’ hydroxyl
group of tRNA 3’-terminal adenosine to
carboxyl grooup of aminoacyladenylate,
ester bond is formed between tRNA
and amino acid

The result of aminoacylation by a class II AaRS, the
amino acid being attached via its -COOH group to
the 3′-OH of the terminal nucleotide of the tRNA. A
class I AaRS attaches the amino acid to the 2′-OH
group.

adenosine P P P +
ATP
Step 1
O
C
O
P
P
CH
R
+
NH3
amino acid
O
C C A
+
O C CH NH
P P 3
OH
R
2-aminoacyl tRNA
adenosine P
O
+
C CH NH3
~ O R aminoacyl adenylate
or
Step 2
C C A
P
P
OH
OH
C C A
P
P
O C CH
+
NH3
R
OH
O
3-aminoacyl tRNA

• some class I AaRS (GlnRS, GluRS, ArgRS and class
I LysRS) require prior binding of tRNA for step 1
• binding of tRNA occurs in presence of polyamine or
divalent cation (Mg2+)
• hydrolysis of the pyrophosphate to inorganic
phosphate (by pyrophosphatase) drives the reaction
forward
• class I AaRSs join the aa to the 2'-OH, subsequent
transesterification reaction shifts the amino acid to
the 3' position
• the amino acid-tRNA bond is of ‘higher energy’ than
the peptide bond which helps the ribosome
transpeptidate

Structure of the large fragment of ThrRS reveals
that the amino acid-binding site includes a zinc ion
that coordinates threonine through its amino and
hydroxyl groups.

Active site structure of ThrRS, recognices 3’
CCA sequence, which enters a “pocket”

Accuracy of aminoacylation
Accurate amino acid and tRNA recognition by
AaRSs is crucial for protein synthesis
Recognition of tRNAs by the synthetase is
controlled at two steps:
• AaRS has a greater affinity for its cognate tRNA
• aminoacylation of the incorrect tRNA is very slow
Selection based on the differences of the rates is
known as kinetic proofreading

AaRSs use chemical properties, size and shape
to distinguish between amino acids
• In case of HisRS, ProRS and LysRS only
binding of the correct amino acid induces
conformational change in AaRSs required for
aminoacylation reaction to occur
• moving parts are KMSKS sequence (class I) or
loop of motif 2 (class II)

• AaRS have difficulties discriminating some
amino acids (for example Ile and Val are similar)
• AaRS should make many mistakes(Linus
Pauling, 1958)
ValRS incorporates Ile instead of Val into
proteins only with the frequency of 1:3000

Absolute accuracy of aminoacylation cannot be
achieved
• tRNALys is aminoacylated by LysRS with:
Arg 1:1600
Thr 1:16000
Met 1:32000
Leu 1:132000
Cys 1:256000
Ser 1:750000
The frequency of errors in aminoacylation is less
than 10 -5

tRNA editing
Some AaRSs remove their own coupling errors
through hydrolytic editing
• Pre-transfer editing – non-cognate
aminoacyladenylate is hydrolyzed befor its
transfer to tRNA
• Post-transfer editing – ester bond between
amino acid and non-cognate tRNA is hydrolyzed

• some AaRS (IleRS, ValRS, LeuRS and ThrRS)
have editing sites in addition to acylation sites
• these two sites function as a a double sieve to
ensure a very high fidelity
• wrong amino acid cannot bind to active site and
correct amino acid is rejected by the editing site
• hydrolysis of correct product is slow and hydrolysis
of the “wrong” product is quick
• in case of IleRS 80% of editing is pre-transfer, in
case of ValRS and LeuRS mostly post-transfer
editing (double sieve)

The flexible CCA arm of an aminoacyl-tRNA can move
the amino acid between the activation site and the
editing site. If the amino acid fits well into the editing
site, the amino acid is removed by hydrolysis.

Pyrococcus horikoshii LeuRS-tRNALeu complex

• Val is rejected based on
chemical properties (Zn ion
in active site)
• Ser is rejected based on
size
Activation and editing sites in threonyl-tRNA
synthetase (class II)

Unusual aminoacylation
mechanisms
Exceptions to the one-to-one rule (Francis
Crick 1958) that there is a specific AaRS for
each amino acid (there should be 20 different
AaRS in every organism)
1. Two different AaRSs may be expressed for
single amino acid:
• two LysRS genes in E. coli – lysU and lysS
• two ThrRS and TyrRS in B. Subtilis

2. When GlnRS is missing (B. subtilis)
then Gln-tRNAGln is formed in 2 steps:
ND-
GluRS
Glu + tRNA Gln + ATP Glu-tRNA Gln + AMP + PPi
AdT
Gln + Glu-tRNA Gln + ATP Gln-tRNA Gln + ADP + Pi + Glu
D-GluRS Glu-tRNA Glu
ND-GluRS Glu-tRNA Glu and Glu-tRNA Gln
AdT is an amidotransferase
EF-Tu does not bind Glu-tRNAGln, avoiding
misincorporation of Glu instead of Gln into proteins

RS
D-GluRS: C36 – Arg358 discriminates tRNA Glu and tRNA Gln
ND-GluRS: Glu instead of Arg cannot discriminate tRNA Glu from tRNA Gln

3. When AsnRS is missing (in T. thermophilus
and D. radiodurans in addition to the lack of
GlnRS) then Asn-tRNAAsn is formed in 2 seps:
ND-
AspRS
tRNA Asn Asp-tRNA Asn Asn-tRNA Asn
AdT
D-AspRS Asp-tRNA Asp
ND-AspRS Asp-tRNA Asp
and Asp-tRNA Asn
Amidotransferase (AdT) is same for both GlntRNAGln
and Asp-tRNAAsp synthesis, amido
group donor is also glutamine

4. Some thermophilic archaea (Methanococcus
jannaschii) lack cysS gene encoding for
CysRS
Possess unique ProCysRS synthetase able to
generate both Pro-tRNAPro and Cys-tRNACys

5. Selenosysteine - the 21-st amino acid and
Sec-tRNASec formation
tRNA Sec SerRS
Ser-tRNA Sec selA
Sec-tRNA Sec
selD

•aminoacylation of tRNASec by SerRS (Ser-tRNASec)
•selenocysteine synthase SelA converts to aminoacryltRNASec
•SelD adds selenide to aminoacryl group and SectRNASec
is formed

6. Initiator fMettRNA
is
formylated MettRNA
Synthesized by
methionyl-tRNA
formyltransferase
(MTF)

MTF

Additional reading:
1. TiBS, July 2000, vol 25 “The adaptor
hypothesis revisited”
2. TiBS, June 1997, vol 22 “Structural and
functional considerations of the aminoacylation
reaction”
3. BioEssays, Oct. 1993 No 10 “Aminoacyl-tRNA
synthetase family: Modules at work”
4. Current opinion in structural biology, Feb. 2000
vol10 No1 “The many routes of bacterial tRNAs
after aminoacylation”