GENE EXPRESSION

GSND 5113Q

Transfer RNA
Aminoacyl-tRNA synthetases

Emanuel Goldman, Ph.D.

New Jersey Medical School
Rutgers Biomedical Health Sciences (RBHS)
Rutgers, The State University of New Jersey
Department of Microbiology, Biochemistry & Molecular Genetics
International Center for Public Health (ICPH)
225 Warren Street, room E450T
Newark, NJ 07103 USA
Telephone: 973-972-4367
Fax: 973-972-3644 or -8982
Email: egoldman@njms.rutgers.edu
 tRNA
a. Generalized structure: cloverleaf in 2-D; "L-shaped" in 3-D (from X-ray crystallography).

b. Extensive use of modified bases; some function in decoding.

c. Antiparallel pairing with codon; wobble with 3rd codon position; "extended anticodon."

d. Aminoacyl-tRNA synthetases activate amino acids, charge CCA ends of cognate tRNAs, edit incorrect
amino acids (aa).

e. tRNA "identity" elements and families of isoacceptors.

f. Identity is critical because once the aa is attached to tRNA, it is committed to protein synthesis in response
to cognate codon to the tRNA, as shown experimentally with Raney nickel (described in lecture 5).

g. tRNA transcription: prokaryotic, from separate genes, as part of rRNA operons, and as "dual-function"
transcripts (with mRNA); eukaryotic, described in lecture 10)

h. tRNA processing: RNase P (which contains an RNA component responsible for catalysis, as well as
protein components).

NOMENCLATURE: prefix indicates amino acid the tRNA species is aminoacylated with (no prefix
means the tRNA is uncharged). Superscript indicates which family of tRNA isoaccepters this particular
tRNA belongs to, one of 20 families, one family per amino acid. Subscript indicates which specific
isoacceptor species this tRNA molecule is. Subscript is sometimes numerical, sometimes the anticodon.
EXAMPLE: Leu-tRNALeu
3
means the aminoacylated form of the 3rd leucyl-tRNA isoacceptor.
 tRNA Structure
A. B.

Rich A & Kim SH (1978)

The three-dimensional
structure of transfer RNA.
Scientific American 238:5262

A. Cloverleaf diagram is the 2-dimensional folding pattern of the transfer RNA (tRNA) molecule. Nucleotide bases found almost universally in
the same position in all tRNA sequences are indicated. The ladder-like stems are made up of complementary bases in different parts of the
polynucleotide chain that pair up and form hydrogen bonds, causing the chain to fold back on itself. The number of nucleotides in the various
stems and loops is generally constant except for two parts of the D loop designated and (which consist of from one to three nucleotides in
different tRNAs) and the variable loop (which usually has four or five nucleotides but may have as many as 21). Abbreviations: A, adenosine; G,
guanosine; C, cytidine; U, uridine; R, adenosine or guanosine; Y, cytidine or uridine; T, ribothymidine; and , pseudouridine.

B. Folding pattern of the polynucleotide chain in yeast phenylalanine tRNA is diagrammed. The sugar-phosphate backbone of the molecule is
represented as a coiled tube, with the cross rungs standing for the nucleotide base pairs in the stem regions. The short rungs indicate bases that are
not involved in basebase hydrogen bonding.

The 3' end of all tRNAs have the sequence CCA, with the amino acid attached by the tRNA synthetase to the
terminal adenosine residue. In eukaryotic cells, the 3 terminal CCA is not encoded but is enzymatically added post-transcriptionally.
 Selected modified nucleosides of transfer RNA

Adapted from p. 404 of "Modified nucleosides in tRNA, McCloskey JA, Nishimura S (1977) Accounts of Chemical Research 10:403
Example of tRNA modification:

is formed at the polynucleotide level by switching ribose from N1 to C5.

is found in tRNA, rRNA, and snRNA, but not in mRNA or viral RNA.

Features of :
(a) N1-H group, extra H-bond donor
(b) histidine-like structure - catalysis?
(c) transacylation: N1-acyl in high energy state
Positions of some modified nucleosides in tRNA

Eubacteria Eukaryotes
 WOBBLE

U opposite the third codon position I opposite the third codon position can pair with U, C, or A
can pair with A or G
WOBBLE PAIRING
(as originally proposed)
Base in Anticodon Base in Codon
Wobble enables inosine G U or C
to form hydrogen bonds C G
with bases other than A U
those in standard base pairs.
U A or G
I A, U, or C

Antiparallel codon-anticodon pairing

tRNAs
3-UACGACACC-5 anticodons
5-AUGCUGUGG-3 codons
Met Leu Trp amino acids
direction of reading of mRNA
 WOBBLE (updated)

Q, queuosine (#32 in slide 4); I, inosine (#17); L, lysidine (#33) From Osawa et al., 1992

"Extended anticodon"
Other nucleotides in the anticodon loop (i.e., in addition to the anticodon) can influence the efficiency of anticodon interaction with the codon

Role of modifications in coding specificity

Examples:
Nucleotide 34 in tRNA (first anticodon position, wobble base):
Q (#32 in slide 4) in all E. coli tRNAs which read XAU or XAC codons (where X = G, A, C, or U)
(difference from Table above is because Table designation is also for eukaryotes)
mcm5s2U (eukaryotes) & mam5s2U (E. coli) (#9, #10 in slide 4) in tRNAs which read XAA or XAG codons
(where X = G, A, or C [U leads to stop codon])
Nucleotide 37 in tRNA (adjacent base 3 to anticodon, i.e., next to base which pairs with 1st codon position):
ms2i6A (#21 in slide 4) (i = isopentenyl) in tRNAs which read UXX codons
t6A (#22 in slide 4) (t = threonine) in tRNAs which read AXX codons
Some modification mutants have diminished decoding activity, leading to altered regulation in attenuation:
hisT mutation in E. coli fails to make modification at positions 38 & 39 in anticodon loop of tRNAHis.
trpX mutation in E. coli fails to make ms2i6A modification at position 37 in anticodon loop of tRNATrp.
 tRNA identity elements

Schematic representation of the three-dimensional structure of tRNA. Circles indicate the position of nucleotides, the size of which is
proportional to the documented frequency with which they are involved in recognition by AARSs. The acceptor stem refers to the
helical structure formed by base pairing of bases 1 to 7 with bases 72 to 66 respectively. The discriminator base is the unpaired
nucleotide at position 73. (Figure modified by WH McClain from McClain WH, Nicholas HB [1987] J Mol Biol 194:63542)
 Recognition elements of some tRNAs

from Pallanck, Pak, Schulman 1995 Courtesy of Hieronim Jakubowski

 Unique features of initiator tRNAs

formylation

P site binding/initiation P site binding/initiation

The three G-C base pairs in the stem portion of the anticodon arm are in all initiator tRNAs. This feature is necessary
and sufficient to identify a tRNA as an initiator, i.e., for entry at the P site instead of the A site of the ribosome.
 tRNA Genes in the Human Genome
With the publication of the complete sequence of the human genome (Nature 409: 860, 2001), there is now a wealth of data on
the numbers of tRNA genes that are cognate to each codon. In the published draft of the human genome, there are 497 identified
tRNA genes and 324 putative tRNA pseudogenes; this analysis allows for the possibility that a few more tRNA genes will be
identified as the sequence is refined, but the overall number is not expected to change by very much.
Several of these genes represent gene duplications that encode identical tRNA isoacceptors. The figure below (Fig. 34 from
Nature 409:860) delineates the number of tRNA genes in the human genome with anticodons corresponding to each codon in the
universal genetic code. Apparently omitted from this figure is the specialized seryl-tRNA that is used to decode the UGA stop
codon under specialized circumstances for incorporating selenocysteine (described in following slides). The absence of any other
tRNAs to decode the stop codons settles the long-standing nagging issue of whether human cells have the capacity to suppress
nonsense codons; we now know that if there is any such suppression it is not tRNA-based. The vast database is only beginning to
be mined for information, and this is an exciting area for future bioinformatics research.

For each of the 64 codons, we show: the

corresponding amino acid; the observed frequency of
the codon per 10,000 codons; the codon; predicted
wobble pairing to a tRNA anticodon (black lines); an
unmodified tRNA anticodon sequence; and the number
of tRNA genes found with this anticodon. For example,
phenylalanine is encoded by UUU or UUC; UUC is
seen more frequently, 203 to 171 occurrences per
10,000 total codons; both codons are expected to be
decoded by a single tRNA anticodon type, GAA, using
a G/U wobble; and there are 14 tRNA genes found
with this anticodon. The modified anticodon sequence
in the mature tRNA is not shown, even where post-
transcriptional modifications can be confidently
predicted (for example, when an A is used to decode a
U/C third position, the A is almost certainly an inosine
in the mature tRNA). The Figure also does not show
the number of distinct tRNA species (such as distinct
sequence families) for each anticodon; often there is
more than one species for each anticodon.
tRNA Transcription
Prokaryotic
as separate genes
as part of rRNA operons (slide in lecture 3)
as "dual-function" transcripts (with mRNA)

Eukaryotic
discussed in lecture 10
 tRNA Processing
RNase III (a double-stranded ribonuclease);
for tRNA genes co-transcribed with rRNA

RNase D (a 3-5 exonuclease)

RNase P (contains an RNA component responsible for catalysis,

as well as one [in eubacteria] or more protein components).

Eukaryotic RNase P processing

Modified from an image at

http://www.science.ca/scientists/scientistprofile.php?pID=3&pg=1
 tRNA-Dependent Amino Acid Transformations

Courtesy of Hieronim Jakubowski from Ibba, Curnow, & Soll 1997

Courtesy of Hieronim Jakubowski from Ibba, Curnow, & Soll 1997
 Selenocysteine Incorporation During Translation Elongation

The unusual amino acid selenocysteine (a derivative of cysteine in which the sulfur atom is replaced by a selenium atom)
is an essential component in a small number of proteins. These proteins occur in prokaryotes and eukaryotes ranging from E.
coli to humans. In all cases, selenocysteine is incorporated into protein during translation in response to the codon UGA.
This codon usually serves as a termination codon but occasionally, in a particular context in the message, is used to specify
selenocysteine instead, which has thus been called "the 21st amino acid."

In E. coli, the products of four genes (selA, B, C, and D) are required for the incorporation of selenocysteine. The product
of the selC gene is an isoacceptor of tRNASer whose anti- codon is UCA (complementary to the UGA codon). The first step
in the incorporation of selenocysteine is catalyzed by seryl-tRNA synthetase. The products of selA and D function in the
subsequent conversion of Ser-tRNASer to selenocysteinyl-tRNASer. Proposed pathway:

tRNASer seryl-tRNASer phosphoseryl-tRNASer selenocysteinyl-tRNASer

Incorporation of selenocysteinyl-tRNASer into protein in response to the UGA codon requires SelB (the protein product of
the selB gene in E. coli). SelB is homologous in sequence to EF-Tu and probably replaces it in translation by specifically
recognizing selenocysteinyl-tRNA and possibly UGA in the appropriate context. Selenocysteinyl-tRNASer, in combination
with SelB, therefore competes with termination factors for recognition of this termination codon. For this to occur, a stem-
loop structure in the mRNA is required just downstream of the UGA codon specifying selenocysteine. This structure is
similar to other "stimulators" involved in recoding events, such as those used in some programmed translational frameshifts.

Other proteins of unknown function that are homologous to EF-Tu are known to exist. Conceivably, these proteins might
participate in the incorporation of other rare amino acids into unique positions in proteins. Mutants of EF-Tu have also been
found which stimulate ribosomes to read through stop codons, perhaps in analogy to the role of SelB in allowing UGA to be
read as selenocysteine.

Some archaebacteria contain the unusual amino acid "pyrrolysine", which is incorporated translationally in response to
UAG codons by a unique tRNA charged by a dedicated tRNA synthetase.
 Selenocysteine incorporation continued

Baron & Bock 1995

B

A serine-accepting tRNA (tRNASec,

selC) mediates incorporation of
selenocysteine into fdhF protein in
E. coli. The A5a-U67a base pair is
crucial for selenium incorporation:
deletion mutant is inactive.
Courtesy of Hieronim Jakubowski
 Incorporation of selenocysteine in protein

Baron & Bock 1995

Biosynthesis and incorporation of selenocysteine into proteins of Escherichia coli compared with that of the 20 standard
amino acids. The selenocysteine pathway involves the action of seryl-tRNA synthetase and the sel gene products. The
discrimination of the UGA (selenocysteine) from UGA (stop) codons is mediated by a stem-loop structure immediately
3' to the UGA codons in the fdhF and fdnG mRNAs. Selenium is also incorporated in the anticodon of tRNAGlu and
tRNALys isoacceptors; the modification at U34 is 5-methylaminomethyl-2-selenouridine (mnm5se2U).

Differences in the structure of tRNASec compared to other tRNAs prevents tRNASec from reading ordinary UGA codons that
do not have the downstream stimulator stem-loop. [Li WQ, Yarus M (1992) Bar to normal UGA translation by the
selenocysteine tRNA. J Mol Biol 223:9-15.]
 Selenocysteine incorporation in archaea & eukaryotes
Sec-tRNASec

eEFSec

SBP2
ribosome SBP2 SECIS
3 UTR
mRNA UGA

RFs
adapted from fig. 4 of Copeland (2003) Gene 312:17-25

A model of Sec incorporation. We propose that SBP2 binds as at least a dimer simultaneously to the ribosome and the SECIS element and thus
may prevent release factor access. In addition, SBP2 may play an active role in delivering the eEFSec/Sec-tRNA complex to the ribosomal A site.

The SECIS hairpin is localized in the 3' untranslated region of mRNA

eEFSec is the mammalian homolog of SelB

SBP2 is a SECIS RNA binding protein

 AMINOACYLATION

Aminoacylation of tRNA is a two-step reaction.
In the first step (equation 1), which is generally
referred to as amino acid activation, an amino
acid (AA) is activated to form enzyme (E)-bound
aminoacyl adenylate.
E + AA + ATP EAA-AMP + PPi (1)
In the second step (equation 2), the amino acid is
transferred from the adenylate to tRNA.
EAA-AMP + tRNAAA E + AA-tRNAAA + AMP (2)
Note that the aminoacyl-tRNA link is a high energy
bond, which is the source of energy for peptide bond
formation on the ribosome.
Two classes of aminoacyl-tRNA synthetases
Class I Class II
C (Cys) A (Ala)
E (Glu) D (Asp)
I (Ile) F (Phe)
L (Leu) G (Gly)
M (Met) H (His)
Q (Gln) K (Lys)
R (Arg) N (Asn)
V (Val) P (Pro)
W (Trp) S (Ser)
Y (Tyr) T (Thr)

Class I:
Two conserved regions of amino acids: HIGH and KMSKS: "Rossmann fold"
Amino acid attached to the 2'OH on the ribose of the 3'-terminal adenosine
Generally, for the bulkier amino acids

Class II:
Three conserved regions in active site; includes GLER motif
Amino acid attached to the 3'OH on the ribose of the 3'-terminal adenosine
Generally, for the smaller amino acids

NOTE: once amino acid is attached to ribose at either the 2 or 3 positions, it

isomerizes to the other position, and can be at either for protein synthesis.
Amino acid MetRS IleRS LeuRS ValRS Accuracy of the
Methionine 1000 0.8 Adenylate Formation
Homocysteine 5.0 2.5 8.3 0.2 Reaction Is Low
Cysteine 0.3 1.0
Jakubowski & Goldman, 1992
Isoleucine 1000 1.5 0.2
Leucine 1.0 1000
Valine 7.0 1000
Threonine 0.2 4.0

Amino acid MetRS IleRS LeuRS ValRS LysRS

Accuracy of
Methionine 106 <1
Aminoacyl-tRNA
Homocysteine <1 <1 <1 <1 <1
Formation Is High
Cysteine <1 <1 1.7
Isoleucine 106 <1 200 Jakubowski 1999, 2001

Leucine <1 106 7.6

Valine 2.9 106
Threonine <1 2.9 63
Alanine <1 <1 3.4
Lysine 106
Arginine 630
Ornithine <1
Courtesy of Hieronim Jakubowski
 Aminoacyl-tRNA synthetases edit mistakes

E + AA + ATP EAA-AMP EAA-tRNA E + AA-tRNA

(a) (b)
E + AA + AMP EtRNA + AA
Pre-transfer (a) and post-transfer (b) editing removes errors in amino acid
selection
Jakubowski & Fersht 1981

Aminoacyl-tRNA synthetases with editing function:

MetRS, IleRS, LeuRS, ValRS, LysRS, AlaRS, PheRS, ProRS, ThrRS

Some aminoacyl-tRNA synthetases do not need to edit:

ArgRS, CysRS, TyrRS, AspRS, SerRS

Courtesy of Hieronim Jakubowski

 Example of editing by aa-tRNA synthetases: homocysteine
Leu
O O O
H2 N CH C OH H2 N CH C OH H2 N CH C OH
CH2 CH CH3 CH CH3
Ile CH CH3 CH2 CH3 Val
CH3 CH3

O O O
H2 N CH C OH H2 N CH C OH H2 N CH C OH
CH2 CH2 CH2
Met CH2 CH2 CH2 Lys
S SH CH2
CH3 Homocysteine CH2
NH2

The non-protein amino acid homocysteine, an intermediate in methionine biosynthesis, is activated to the
aminoacyl adenylate by several aa-tRNA synthetases, then edited with formation of homocysteine thiolactone.
AARS + Hcy + ATP AARSHcyAMP + PPi

Hcy thiolactone
NH2
NH2
O

O
AM P - AM P
Jakubowski & Fersht 1981 SH S Courtesy of Hieronim Jakubowski
 Synthetic/Editing Active Site of Methionyl-tRNA Synthetase
Synthesis of Met-tRNAMet
Specificity subsite Specificity subsite

CH3 CH3 CH3

S S S

+ ATP + tRNA

Thiol NH2 Thiol NH2 NH2

subsite C subsite C C
O O O
HO AMP tRNA

Editing of Homocysteine
Specificity subsite Specificity subsite

H
S

- AMP NH2
Thiol NH2 Thiol NH2 S C
HS O
subsite C subsite C
O O
AMP AMP

Courtesy of Hieronim Jakubowski

Kim et al. 1993, Jakubowski 1996, 2000, 2001
 Mischarging of tRNA and human disease
The specificity sub-site of MetRS (previous slide) can be fooled by S-nitroso-homocysteine, which has an NO group
attached to the sulfur atom instead of 'H'. This can be attached to tRNA-Met and inserted into protein.

The nitroso group is labile, and when it is removed, the protein is left with homocysteine in the protein chain instead of
methionine. This may be a factor in why homocysteine is a risk factor for atherosclerosis, since proteins bearing
homocysteine are damaged and foreign, and could facilitate development of plaque on artery walls.

There aren't many known diseases based on this kind of event, probably because if mischarging occurred at a
significant level, that would be a lethal mutation in the embryo.

However, there do appear to be some neurodegenerative diseases based on this kind of mechanism. See the abstract
below (from Dunlop RA, Cox PA, Banack SA, Rodgers KJ (2013) The non-protein amino acid BMAA is misin-
corporated into human proteins in place of L-serine causing protein misfolding and aggregation. (PLoS ONE 8: e75376).

"Mechanisms of protein misfolding are of increasing interest in the aetiology of neurodegenerative diseases characterized
by protein aggregation and tangles including Amyotrophic Lateral Sclerosis (ALS), Alzheimers disease (AD), Parkinsons
disease (PD), Lewy Body Dementia (LBD), and Progressive Supranuclear Palsy (PSP). Some forms of
neurodegenerative illness are associated with mutations in genes which control assembly of disease related proteins. For
example, the mouse sticky mutation sti, which results in undetected mischarging of tRNA-Ala with serine resulting in the
substitution of serine for alanine in proteins causes cerebellar Purkinje cell loss and ataxia in laboratory animals.
Replacement of serine 422 with glutamic acid in tau increases the propensity of tau aggregation associated with
neurodegeneration. However, the possibility that environmental factors can trigger abnormal folding in proteins remains
relatively unexplored. We here report that a non-protein amino acid, -N-methylamino-L-alanine (BMAA), can be
misincorporated in place of l-serine into human proteins. We also report that this misincorporation can be inhibited by l-
serine. Misincorporation of BMAA into human neuroproteins may shed light on putative associations between human
exposure to BMAA produced by cyanobacteria and an increased incidence of ALS."