Interstrand Aminoacyl Transfer in a tRNA Acceptor Stem-Overhang Mimic

Article abstract of DOI:10.1021/jacs.1c05746

Protein-catalyzed aminoacylation of the 3′-overhang of tRNA by an aminoacyl-adenylate could not have taken place prior to the advent of genetically coded peptide synthesis, and yet the latter process has an absolute requirement for aminoacyl-tRNA. There must therefore have been an earlier nonprotein-catalyzed means of generating aminoacyl-tRNA. Here, we demonstrate efficient interstrand aminoacyl transfer from an aminoacyl phosphate mixed anhydride at the 5′-terminus of a tRNA acceptor stem mimic to the 2′,3′-diol terminus of a short 3′-overhang. With certain five-base 3′-overhangs, the transfer of an alanyl residue is highly stereoselective with the l-enantiomer being favored to the extent of ～10:1 over the d-enantiomer and is much more efficient than the transfer of a glycyl residue. N-Acyl-aminoacyl residues are similarly transferred from a mixed anhydride with the 5′-phosphate to the 2′,3′-diol but with a different dependence of efficiency and stereoselectivity on the 3′-overhang length and sequence. Given a prebiotically plausible and compatible synthesis of aminoacyl phosphate mixed anhydrides, these results suggest that RNA molecules with acceptor stem termini resembling modern tRNAs could have been spontaneously aminoacylated, in a stereoselective and chemoselective manner, at their 2′,3′-diol termini prior to the onset of protein-catalyzed aminoacylation.

Full text of DOI:10.1021/jacs.1c05746

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Article
Interstrand Aminoacyl Transfer in a tRNA Acceptor Stem-Overhang
Mimic
Long-Fei Wu,‡ Meng Su,‡ Ziwei Liu, Samuel J. Bjork, and John D. Sutherland*
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ABSTRACT: Protein-catalyzed aminoacylation of the 3′-over-
hang of tRNA by an aminoacyl-adenylate could not have taken
place prior to the advent of genetically coded peptide synthesis,
and yet the latter process has an absolute requirement for
aminoacyl-tRNA. There must therefore have been an earlier
nonprotein-catalyzed means of generating aminoacyl-tRNA. Here,
we demonstrate efficient interstrand aminoacyl transfer from an
aminoacyl phosphate mixed anhydride at the 5′-terminus of a
tRNA acceptor stem mimic to the 2′,3′-diol terminus of a short 3′-
overhang. With certain five-base 3′-overhangs, the transfer of an
alanyl residue is highly stereoselective with the L-enantiomer being
favored to the extent of ∼10:1 over the D-enantiomer and is much
more efficient than the transfer of a glycyl residue. N-Acyl-aminoacyl residues are similarly transferred from a mixed anhydride with
the 5′-phosphate to the 2′,3′-diol but with a different dependence of efficiency and stereoselectivity on the 3′-overhang length and
sequence. Given a prebiotically plausible and compatible synthesis of aminoacyl phosphate mixed anhydrides, these results suggest
that RNA molecules with acceptor stem termini resembling modern tRNAs could have been spontaneously aminoacylated, in a
stereoselective and chemoselective manner, at their 2′,3′-diol termini prior to the onset of protein-catalyzed aminoacylation.
1d). The proximity between the 3′- and the 5′-termini of a
tRNA acceptor stem microhelix with a variant 3′-overhang has
been demonstrated by NMR spectroscopy,12 supporting this
idea.
INTRODUCTION
■
tRNA is the key adaptor molecule in the translation of a
nucleic acid message into a protein sequence.1 Although the L-
shaped 3D structure of tRNA and the way in which it functions
in translation are now known in atomic detail, the reasons for
tRNA being the size and shape it is are still unknown. Also
unknown is how the two-step mechanism of tRNA amino-
acylation via an aminoacyl-adenylate came to be. Although the
idea that primitive tRNA might have been “its own activating
enzyme” is very attractive as regards the emergence of
translation,2 it has not yet received any experimental support.
tRNA has a quasi-symmetric cloverleaf 2D structure (Figure
1a), and this along with the hint of symmetry derived from
sequence alignment of the 5′-half and 3′-half3−5 and the
phenomenon of split tRNA genes6 has led to suggestions that
tRNA might have arisen by duplication.3−5 Such a duplication
could occur in a number of ways,5,7,8 but we were intrigued by
the suggestion that two copies of an RNA half the length of
tRNA became joined end to end as this places the join in the
anticodon loop (Figure 1b).5,6 The synthesis of aminoacyl
phosphate mixed anhydrides has been demonstrated under
prebiotically plausible conditions,9−11 and we now wondered if
the proximity of the 3′- and 5′-termini that would be required
to seal the anticodon loop by ligation might, at the symmetry-
related acceptor termini of a proto-tRNA, allow transfer of an
aminoacyl group from the 5′-phosphate to the 2′,3′-diol of a
3′-overhang with a folded-back conformation (Figure 1c and
In seminal work, Tamura and Schimmel demonstrated
transfer of an aminoacyl group from the 5′-phosphate of a
separate helper oligonucleotide to the 2′,3′-diol of a tRNA
acceptor stem-overhang mimic using an additional bridging
oligonucleotide that brought the reactive termini together in
the form of a nicked duplex (Figure S1a).13,14 Although this
work demonstrated that aminoacylation of a tRNA acceptor
stem-overhang mimic is possible, we felt that it suffers from a
number of shortcomings from an evolutionary perspective.
First, it provides no explanation as to why the 3′-overhang of a
tRNA should be the length and sequence it is, as any sequence
of overhang longer than a few nucleotides could allow nicked
duplex transfer using appropriate helper and bridging
oligonucleotides. Second, there is no obvious way apparent
for the sequence of the duplex to influence the side chain
Received: June 7, 2021
Published: July 20, 2021
© 2021 The Authors. Published by
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https://doi.org/10.1021/jacs.1c05746
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Figure 1. Model for the origin of tRNA by duplication suggests the possibility of interstrand aminoacyl transfer at the acceptor stem overhang. (a)
Cloverleaf 2D structure of extant tRNA with the anticodon loop and extended conformation 3′-overhang highlighted (dark blue). (b) Two copies
of RNA annealed to each other to generate a cloverleaf proto-tRNA with two folded-back conformation 3′-overhangs (dark blue). (c) Close up of
the stem 3′-overhang at the bottom of the structure depicted in b showing the proximity that would be required for attack of the 3′-hydroxyl group
on the activated 5′-phosphate (black) to generate a loop. (d) Close up of the stem 3′-overhang at the top of the structure depicted in b showing
how the same proximity shown in c might allow transfer of an aminoacyl group from a mixed anhydride with the 5′-phosphate (black) to the 3′-
hydroxyl group of the folded-back conformation 3′-overhang.
selectivity of the aminoacyl transferindeed, the aminoacyl
transfer that was demonstrated for alanyl, leucyl, and
phenylalanyl residues showed no chemoselectivity.13 Finally,
it does not explain why, in extant biology, tRNA has a 5′-
phosphate introduced by pre-tRNA cleavage in a reaction that
has all the hallmarks of being the vestige of an ancient
process.15 In the case of the aminoacyl transfer that we
envisaged from a 5′-phosphate to the 2′,3′-diol of a folded-
back 3′-overhanghereinafter nicked loop transfer (Figure
S1b)the proximity required to facilitate aminoacyl transfer
would derive from the structure of the acceptor stem-overhang
mimic itself12 rather than requiring helper and bridging
oligonucleotides. We felt that nicked loop transfer might
better explain aspects of tRNA structure from an evolutionary
perspective for several reasons. First, not all lengths and
sequences of overhang are likely to adopt any of the range of
conformations that one could imagine would be conducive to
aminoacyl transfer. Finding a correlation between those that do
and the nature of the tRNA 3′-overhang in extant biology
would offer a hint as to why tRNA has the 3′-overhang length
and sequence it does, though other functions could have
influenced this, witness, for example, the binding of 3′-CCA
termini to the 23S rRNA in extant biology.16,17 Second, the
range of overhang conformations potentially allowing amino-
acyl transfer would afford opportunities for both stereo-
selectivty and chemoselectivity resulting from interactions
between the aminoacyl group and the RNA during transfer.
Finally, were we to demonstrate nicked loop transfer from an
aminoacyl mixed anhydride of the 5′-phosphate, it would
suggest why tRNA ancestors might have had to have 5′-
phosphate termini (Figure 1a and 1d), though again other
functions could also have contributed to selection, for example,
the role of the 5′-phosphate in peptidyl-tRNA hydrolysis.18
the basis of the idea that tRNA is the result of duplication, we
designed an initial tRNA acceptor stem-overhang mimic by
convergence from anticodon loop and acceptor stem-overhang
lengths and sequences in extant biology (Figure S2). Our plan
was then to modify the length and sequence of this initial
mimic. Alanine was chosen as the first amino acid to
investigate because it is one of the simplest of the chiral
proteinogenic amino acids.
Conventional synthesis was used to aminoacylate a 5′-
phosphorylated oligonucleotide (5′-pAGCGA-3′). The L-
alanyl-phosphate mixed anhydride donor strand (5′-L-Ala-
pAGCGA-3′) sample thereby prepared also contained the
starting oligonucleotide either because the aminoacylation
failed to proceed to completion or because of product
hydrolysis. The mixed anhydride donor strand was then
annealed to an acceptor strand (5′-UCGCUUGCCA-3′,
underlining indicates overhang sequence) in a near neutral
pH solution, optionally containing added salts (Supporting
Information and Table S1). Aliquots of the reaction mixture
were then taken over a period of time and analyzed by HPLC
whereupon it became apparent that the peak for the mixed
anhydride donor strand decreased over time (Figure S3). As
this peak decreased, the peak for the 5′-phosphorylated donor
strand (5′-pAGCGA-3′) increased, the peak for the acceptor
strand decreased, and a new peak appeared. This new peak
reached a maximum and then decreased, albeit more slowly
than the peak for the mixed anhydride. This slower hydrolysis
behavior was consistent with the peak being due to the
aminoacyl-transfer product as aminoacyl esters are more stable
than aminoacyl phosphate mixed anhydrides.19 A MALDI-
TOF mass spectrum of the reaction mixture had peaks with
masses consistent with both the unreacted acceptor strand and
an alanyl derivative thereof (Figure 2a). Proof that the
derivative was an L-alanyl ester of the 2′,3′-diol of the acceptor
strand (and not an amide of a nucleobase amino group, or an
ester of an internal 2′-hydroxyl group) was obtained by
digesting the RNA in the reaction mixture with RNase A19 and
comparing the HPLC elution profile of the degradation
products with similar profiles for authentic standards of the
2′,3′-diol esters of adenosine with L- and D-alanine (Figure 2b
and Figures S4−S6). Taken with the other data, the
RESULTS AND DISCUSSION
■
Experimentally, we envisaged using HPLC to analyze amino-
acyl transfer from a mixed anhydride donor strand to a
complementary acceptor strand with a 3′-overhang. The large
number of permutations of amino acid and 3′-overhang lengths
and sequences coupled to a relatively low-throughput analytical
method meant that we needed to establish starting points. On
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Figure 2. Characterization of nicked loop L-alanyl-transfer. (a) MALDI-TOF mass spectrum of products of aminoacyl transfer from 5′-L-Ala-
pAGCGA-3′ to an acceptor strand with sequence 5′-UCGCUUGCCA-3′. Mass clusters for the acceptor strand (found 3096.885, calcd 3096.44)
and its L-alanyl diol ester product (5′-UCGCUUGCCA-L-Ala, found 3167.935, calcd 3167.44) are indicated. Measured mass difference, 71.050,
C3H5NO [Ala-H2O], calcd 71.037. (b) Enzyme digestion confirming that the diol of the acceptor strand’s 3′-terminal adenosine is aminoacylated
by aminoacyl transfer. (c) L-Ala transfer in a tRNA acceptor arm mimic: 5′-UCGCUUUCCA-3′; 5′-L-Ala-pAGCGA-3′. Peaks for the donor, donor
mixed anhydride, and acceptor strands are indicated. Peak presumed to be due to the diol ester transfer product is highlighted by the dashed box.
(d) Time courses showing the stereoselectivity for L-Ala over D-Ala nicked loop transfer with acceptor strands UCGCUUGCCA and
UCGCUUUCCA. Conditions: each oligoribonucleotide 100 μM, NaCl 100 mM, MgCl2 5 mM, HEPES 50 mM, pH 6.8, 10 °C.
observation of the 2′,3′-diol esters of adenosine with L-alanine
among the digestion products confirms that aminoacyl transfer
takes place from the donor strand to the 2′,3′-diol terminus of
the acceptor strand. We then screened the effects of added
salts, pH, and temperature on the reaction (Table S1). It was
found that under near optimal conditions (100 μM of each
aminoacyl donor and acceptor strands, 100 mM NaCl, 5 mM
MgCl2, 50 mM HEPES at pH 6.8, 10 °C), the observed yield
of L-alanyl transfer was 30%. Taking into account the impurity
of the mixed anhydride donor strand and its hydrolysis, the
corrected yield was 55% (Table 1, Figure S3, Method).
We then proceeded to investigate the effect of overhang
length and sequence and probe potential stereoselectivity and
chemoselectivity of the transfer with D-alanine and other amino
acids, but we first addressed a slight concern. Our random
choice of G as the additional residue to insert in the overhang
to make it five nucleotides long gave a sequenceUGCCA
that was partially self-complementary. Binding of two stem-
overhang complexes to each other could therefore potentially
generate a doubly nicked duplex where aminoacyl transfer in
the configuration described by Tamura and Schimmel might
be possible,13 although the C:C mismatch would be expected
to significantly destabilize a duplex comprising two such
overhangs arranged in an antiparallel fashion. Accordingly, we
synthesized additional oligonucleotides in which the G of the
overhang was changed to the other three canonical
nucleotides, thereby removing self-complementarity. No trans-
fer was detectable with the overhang with C in place of the G,
but in the other two cases L-alanyl-transfer was efficient
(corrected yields with the UACCA and UUCCA overhangs
were 30% and 57%, respectively. Table 1, Figure 2c, Table S2,
and Figures S7−S9). As the transfer with the UACCA or
UUCCA overhangs must go through the folded-back
conformation, these results suggest that the transfer with the
UGCCA overhang similarly proceeds via the folded-back
conformation as we envisaged.
We next addressed the stereochemistry question. Incubation
of a conventionally synthesized D-alanyl-phosphate mixed
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a
Table 1. Nicked Loop Aminoacyl Transfer of tRNA Acceptor Arm Mimics
aminoacyl donor RNA strand
5′-D-Ala-pAGCGA
5′-L-Ala-pAGCGA
5′-Gly-pAGCGA
acceptor RNA strand
observed yield
corrected yield
observed yield
corrected yield
observed yield
corrected yield
5′-UCGCUUGCCA-3′
5′-UCGCUUACCA-3′
5′-UCGCUUCCCA-3′
5′-UCGCUUUCCA-3′
5′-UCGCUUGCCC-3′
5′-UCGCUUGCCG-3′
5′-UCGCUUGCCU-3′
5′-UCGCUUGCCA(2′d)-3′
5′-UCGCUUGCCA(3′d)-3′
5′-UCGCUUGCCCA-3′
5′-UCGCUACCA-3′
30%
16%
N.D.
34%
N.D.
14%
N.D.
4%
N.D.
22%
N.D.
1%
55%
30%
N.D.
57%
N.D.
25%
N.D.
6%
N.D.
38%
N.D.
2%
10%
N.D.
N.D.
4%
N.D.
8%
N.D.
−
−
15%
N.D.
N.D.
6%
N.D.
12%
N.D.
−
9%
N.D.
N.D.
2%
N.D.
N.D.
N.D.
−
11%
N.D.
N.D.
3%
N.D.
N.D.
N.D.
−
−
−
−
15%
N.D.
2%
25%
N.D.
4%
17%
N.D.
N.D.
23%
N.D.
N.D.
5′-UCGCUUCCA-3′
a
N.D.: product not detected. −:, experiments not performed.
anhydride donor strand (5′-D-Ala-pAGCGA-3′, along with 5′-
pAGCGA-3′) with the acceptor strand (5′-UCGCUUGCCA-
3′) resulted in only low amounts (≤15% corrected yield) of
the aminoacyl-transfer product according to HPLC analysis
(Table 1, Figure 2d, and Figure S10), giving an L:D
stereoselectivity of ∼4:1. However, a considerably higher L:D
stereoselectivity of ∼10:1 was observed when using an
acceptor strand with the UUCCA overhang (Figure 2d and
Figure S11), and the stereoselectivity with the UACCA
overhang is likely similar (though we could not quantitate it
because in this case the L-alanyl-transfer yield is lower and the
D-alanyl-transfer product could not be detected) (Table 1).
Tamura and Schimmel reported a stereoselectivity of ∼4:1
with the preference also being for transfer of L-configured
aminoacyl residues, but in their case, for synthetic expediency,
the donor strand was composed of DNA (Figure S1a).13 We
measured the L:D stereoselectivity for nicked duplex transfer in
an all-RNA system and found it to be ∼2:1 (Table S3 and
Figures S12−S14).
Moving on to the question of whether there is any
aminoacyl side chain chemoselectivity in the transfer, we
opted to investigate glycyl-transfer next. This decision was
predicated on the expectation that as glycine is likely to have
been one of the most abundant prebiotic amino acids,20,21
transfer of other aminoacyl residues would have had to
contend with significant competing glycyl transfer. We studied
glycyl transfer using the same four acceptor strands that we had
used before (Table 1 and Table S4). With the UCCCA
acceptor strand there was again no detectable transfer, and
while there was transfer to the UGCCA (11% corrected,
Figures S15 and S16) and UUCCA (3% corrected) acceptor
strands with glycyl donor, both were lower than in the case of
L-alanyl transfer. Intriguingly, we observed no transfer of a
glycyl residue with the UACCA acceptor strand, in stark
contrast to the good transfer yield of an L-alanyl residue (30%
corrected). Transfer to the UUCCA and UACCA acceptor
strands is thus both highly stereoselective for an L-alanyl
residue over a D-alanyl residue and highly chemoselective for
transfer of an L-alanyl residue over a glycyl residue (Table 1).
In addition, we found that nicked duplex transfer of a glycyl
residue in our all-RNA system still took place in reasonable
yield (17% corrected, Figure S17), thus strengthening the case
for nicked duplex transfer being significantly less chemo-
selective than nicked loop transfer.
We then changed our focus to the 3′-terminal nucleotide of
the overhang. We synthesized three more acceptor strands
based on our original UGCCA acceptor strand but in which
the 3′-terminal A was changed to the other canonical
nucleotides. We found that L-alanyl transfer to the acceptor
strand with a UGCCG overhang was still reasonably efficient
(25% corrected, Figure S18), and D-alanyl transfer was less so,
but glycyl transfer was not detected. No alanyl or glycyl
transfer with pyrimidine-terminated acceptor strands was
detected. The preference for a purine at the 3′-terminus of
the acceptor strand is thus apparent, and we suggest that it
might be explained by the stabilization of the folded-back
conformation by stacking of the nucleobase of the last
overhang residue on top of the last base pair of the stem. In
an attempt to get an indication of the regioselectivity of
transfer to the 2′,3′-diol, we employed deoxynucleotide-
terminated acceptor strands, but no significant aminoacyl
transfer was observed with either a UGCC-2′-dA overhang or a
UGCC-3′-dA overhang (less than 6%, Figures S19 and S20).
Lastly, we tried to interrogate the effect of overhang length
on the nicked loop aminoacyl transfer. With an acceptor strand
having a UGCCCA overhang, we observed transfer of L-alanyl,
D-alanyl, and glycyl residues in corrected yields of 38%, 25%,
and 23%, respectively (Table 1, Figures S21−S23). When we
shortened the overhang to ACCA or UCCA, there was no
significant transfer of either L- or D-alanyl residues or glycyl
residues (no more than 4% corrected yield, Table 1 and
Figures S24 and S25). We therefore conclude from a sparse
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a
Table 2. Nicked Loop N-Acetylaminoacyl Transfer of tRNA Acceptor Arm Mimics
N-acetylaminoacyl donor RNA strand
5′-Ac-L/D-Ala-pAGCGA
5′-Ac-Gly-pAGCGA
observed yield corrected yield
acceptor RNA strand
observed yield
corrected yield
5′-UCGCUUGCCA-3′
5′-UCGCUUACCA-3′
5′-UCGCUUCCCA-3′
5′-UCGCUUUCCA-3′
5′-UCGCUUGCCC-3′
5′-UCGCUUGCCG-3′
5′-UCGCUUGCCU-3′
5′-UCGCUUGCCA(2′d)-3′
5′-UCGCUUGCCA(3′d)-3′
5′-UCGCUUGCCCA-3′
5′-UCGCUACCA-3′
20%
9%
8%
14%
N.D.
12%
N.D.
3%
25%
10%
9%
16%
N.D.
13%
N.D.
4%
32%
27%
36%
21%
5%
46%
36%
46%
28%
7%
25%
5%
36%
7%
17%
14%
20%
23%
4%
23%
19%
32%
37%
6%
2%
2%
11%
10%
2%
14%
13%
3%
5′-UCGCUUCCA-3′
a
N.D.: product not detected.
sampling of different aminoacyl groups and overhang sequence
space that efficient chemo- and stereoselective nicked loop
transfer in a tRNA acceptor stem-overhang mimic is possible
with acceptor strands having overhangs of five or six
nucleotides. The stereo- and chemoselectivity of aminoacyl
transfer is dependent on the nature of the overhang sequence.
However, ribosomal peptide synthesis uses not only aminoacyl
tRNA but also N-acylaminoacyl tRNA, so we now switched
our attention to investigate whether such N-acylaminoacyl
species could also be produced by interstrand transfer in a
tRNA acceptor stem-overhang mimic.
We started our investigation of N-acylaminoacyl transfer
using a conventionally synthesized N-acetylglycyl phosphate
mixed anhydride (5′-Ac-Gly-pAGCGA-3′) as the donor
strand. Incubation of an acceptor strand (5′-UCGCUUGC-
CA-3′) with the donor strand was again monitored by HPLC
time course analysis (Methods and Supporting Information).
Under optimal conditions, the transfer to the UGCCA
overhang proceeded in good yield (32% observed, 46%
corrected, Table 2, Tables S5 and S6, and Figures S26−
S28). Changing the second base of the overhang had little
effect on yield in complete contrast to the case with the
transfer of an unprotected glycyl residue (Table 2 and Figures
S29−S31). In particular, for the acceptor strand with a
UCCCA overhang, very efficient N-acetylglycyl transfer was
observed (36% observed, 46% corrected, Figure S31).
Changing the last base of the overhang showed that a purine
is again important for efficient transfer (Table 2 and Figures
S32−S34). Increasing the length was tolerated, the UGCCCA
overhang undergoing N-glycyl transfer in good yield (32%
corrected, Table 2 and Figure S35). Decreasing the length was
now also tolerated, the UCCA overhang undergoing transfer of
an N-acetylglycyl residue in 37% corrected yield (Figure S36),
although the ACCA overhang was a poor substrate (6%
corrected, Figure S37). The better aminoacyl transfer yield
with a UCCA overhang relative to an ACCA overhang
correlates with the dominant folded-back conformation of the
former relative to the latter.12 Efficient transfer was also
observed to deoxy-terminated acceptor strands. On this
occasion, both UGCC-2′-dA and UGCC-3′-dA overhangs
underwent efficient reaction (23% and 19% corrected,
respectively, Figures S38 and S39), suggesting that transfer
to the diol-terminated overhang of the same sequence might
not be regioselective. The significant difference between the
transfer of N-acetylglycyl residues and of unprotected glycyl
residues indicates that the protonated amino group of the latter
might play an important role in this process, possibly through
intramolecular salt-bridge formation with attendant conforma-
tional effects.
Intrigued, we next dealt with stereochemistry issues
associated with N-acetylalanyl transfer. Activation of the
carboxylate group of N-acyl amino acids can lead to the
formation of 5(4H)-oxazolones that are prone to racemization
via their enol tautomers.11,22 Accordingly, we first developed
coupling chemistry which largely circumvents this issue and
thereby prepared N-acetyl-L-alanyl-phosphate and N-acetyl-D-
alanyl-phosphate mixed anhydride donor strands (5′-Ac-L-Ala-
pAGCGA-3′ and 5′-Ac-D-Ala-pAGCGA-3′) starting from
either enantiomer of N-acetylalanine or a mixture of both
(5′-Ac-L/D-Ala-pAGCGA-3′) starting from the racemate11 (see
Supporting Information for details). We then incubated the
donor strand 5′-Ac-L/D-Ala-pAGCGA-3′ with a UGCCA
overhang acceptor strand and observed transfer products in
an overall 25% corrected yield (Table 2, Tables S7 and S8, and
Figure S40). Digestion of the transfer products with RNase A
followed by HPLC analysis revealed that the 2′,3′-diol esters of
adenosine with N-acetyl-L-alanine had approximately the same
abundance as the 2′,3′-diol esters of adenosine with N-acetyl-
D-alanine (Figures S41−S43). Digestion of the transfer
products from the 5′-Ac-L-Ala-pAGCGA-3′ donor strand
with RNase followed by HPLC analysis revealed that the
2′,3′-diol esters of adenosine with N-acetyl-L-alanine greatly
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predominated over those of N-acetyl-D-alanine, the latter
resulting from a slight loss of enantiomeric purity during
preparation of the mixed anhydride. Similarly, using the 5′-Ac-
D-Ala-pAGCGA-3′ donor strand gave predominantly the 2′,3′-
diol esters of adenosine with N-acetyl-D-alanine after RNase
digestion. Furthermore, using a 1:1 mixture of both donor
strands (made by mixing an equal amount of separately made
5′-Ac-L-Ala-pAGCGA-3′ and 5′-Ac-D-Ala-pAGCGA-3′) re-
sulted in approximately equal amounts of the 2′,3′-diol esters
of adenosine with N-acetyl-L-alanine and N-acetyl-D-alanine
after RNase A digestion (Figures S41−S43). These experi-
ments showed that there is no stereoselectivity in the transfer
of N-acetyl-alanyl residues to the acceptor stand with the
UGCCA overhang, which differs significantly from the high
stereoselectivity observed in the transfer of unacylated alanyl
residues. The transfer still took place when the G of the
UGCCA overhang was changed to the other three canonical
bases, and again, it was found that there was a big preference
for a purine at the last position (Table 2 and Figures S44−
S49). Finally, the effect of overhang length on the transfer of
N-acetylalanyl was similar to the case with N-acetylglycyl
transfer (Table 2 and Figures S50−S52).
precursors by RNase P to generate a 5′-phosphate is clearly
ancient, and yet the 5′-phosphate still plays a role in tRNA
function. We suggest that the 5′-phosphate had a crucial
functional role in early aminoacylation chemistry, and when
the transition to the modern mechanism of aminoacylation
took place, the 5′-phosphate adapted to novel functions in the
Central Dogma. We further suggest an ancestral function for a
3′-overhang of 4−7 nucleotides and a sequence with a U at the
beginning and a purine at the end, allowing adoption of a
folded-back conformation12 that stacked on the last base pair
of the stem and enabled nicked loop aminoacyl transfer. We
speculate that during the evolutionary transition from using an
aminoacyl mixed anhydride of the 5′-phosphate of the tRNA
acceptor arm to using an aminoacyl adenylate, an overhang was
retained at the 3′-terminus but its length and sequence
underwent a slight change to give the near-canonical ACCA of
extant biology.5
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.1c05746.
Materials and methods, supplementary data and figures
(PDF)
CONCLUSIONS
■
On the basis of a model for the origin of tRNA by
duplication3,5,6 and structural data on acceptor stem variants,12
a range of tRNA acceptor stem-overhang mimics was found to
undergo interstrand aminoacyl and N-acetylaminoacyl transfer
without the need for other oligonucleotides or auxiliaries.
Depending on the nature of the aminoacyl residue and the
overhang length and sequence, the transfer reactions can be
highly stereoselective and chemoselective as well as efficient.
The prebiotic formation of aminoacyl phosphate mixed
anhydrides is feasible,9,10 and taken together with the
aminoacyl-transfer process revealed herein, this suggests that
tRNA acceptor stems or their forerunners could have become
aminoacylated before the evolution of aminoacyl-tRNA
synthetase ribozymes or enzymes. This is consistent with
early speculation that primitive tRNA might have been “its
own activating enzyme”.2 Furthermore, the chemistry of the
transfer reaction closely resembles the second step of the
reaction catalyzed by aminoacyl-tRNA synthetase enzymes23
and could have foreshadowed it evolutionarily in keeping with
the principle of continuity.24
AUTHOR INFORMATION
Corresponding Author
■
John D. Sutherland − MRC Laboratory of Molecular Biology,
Cambridge CB2 0QH, United Kingdom; orcid.org/0000-
0001-7099-4731; Email: johns@mrc-lmb.cam.ac.uk
Authors
Long-Fei Wu − MRC Laboratory of Molecular Biology,
Cambridge CB2 0QH, United Kingdom; Present
Address: Howard Hughes Medical Institute, Department
of Molecular Biology, and Center for Computational and
Integrative Biology, Massachusetts General Hospital,
Boston, Massachusetts 02114, United States; Department
of Genetics, Harvard Medical School, Boston,
Massachusetts 02115, United States; orcid.org/0000-
0002-0635-1624
Meng Su − MRC Laboratory of Molecular Biology, Cambridge
CB2 0QH, United Kingdom; orcid.org/0000-0001-6558-
8712
Although the generation of selectively aminoacylated tRNA-
like molecules is a necessary prelude to coded peptide
synthesis, it is not sufficient as aminoacyl transfer from one
such species to the aminoacyl group of another would generate
a dipeptidyl-RNA that would be extremely prone to
diketopiperazine loss.25 Thus, the finding that N-acylaminoacyl
residues can similarly be transferred is also importantthe
prebiotic synthesis of N-acylaminoacyl phosphate mixed
anhydrides also is feasible11 (see also Supporting Information).
Development of a high-throughput assay will allow the
aminoacyl-transfer reaction to be systematically explored
across stem and overhang sequence space for all of the
canonical amino acids or a subset thereof. This should shed
further light on potential stereochemical relationships between
amino acid side chains and RNA sequences.2,7,24,26
Ziwei Liu − MRC Laboratory of Molecular Biology,
Cambridge CB2 0QH, United Kingdom; orcid.org/0000-
0002-1812-2538
Samuel J. Bjork − MRC Laboratory of Molecular Biology,
Cambridge CB2 0QH, United Kingdom
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.1c05746
Author Contributions
‡L.-F.W. and M.S.: These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Finally, we suggest that the chemistry we uncovered might
partly explain certain structural aspects of tRNA. The most
striking one is the overall shape of the tRNA acceptor arm,
especially the characteristic termini. The processing of tRNA
We thank Dr. Mark Skehel for help with MALDI-TOF Mass
Spectroscopy. We also thank J.D.S. group members for fruitful
discussions. This research was supported by the Medical
Research Council (MC_UP_A024_1009) and the Simons
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Journal of the American Chemical Society
pubs.acs.org/JACS
Article
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1968, 38, 381−393.
Foundation (290362 to J.D.S.). S.J.B. was supported by a
Cambridge Marshall Scholarship.
(25) Weber, A. L.; Orgel, L. E. Poly(U)-directed peptide-bond
formation from the 2′(3′)-glycyl esters of adenosine derivatives. J.
Mol. Evol. 1980, 16, 1−10.
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