Efficient ribosomal peptidyl transfer critically relies on the presence of the ribose 2′-OH at A2451 of 23S rRNA

Article abstract of DOI:10.1021/ja0588454

The ribosomal peptidyl transferase center is a ribozyme catalyzing peptide bond synthesis in all organisms. We applied a novel modified nucleoside interference approach to identify functional groups at 9 universally conserved active site residues. Owing t

Full text of DOI:10.1021/ja0588454

Published on Web 03/14/2006
Efficient Ribosomal Peptidyl Transfer Critically Relies on the
Presence of the Ribose 2′-OH at A2451 of 23S rRNA
Matthias D. Erlacher,† Kathrin Lang,‡ Brigitte Wotzel,† Renate Rieder,‡
Ronald Micura,*,‡ and Norbert Polacek*,†
Contribution from the Innsbruck Biocenter, DiVision of Genomics and RNomics-Innsbruck
Medical UniVersity, Fritz-Pregl Strasse 3, 6020 Innsbruck, Austria, and Center for Molecular
Biosciences Innsbruck (CMBI), Institute of Organic Chemistry, Leopold Franzens UniVersity,
Innrain 52a, 6020 Innsbruck, Austria
Received December 31, 2005; E-mail: ronald.micura@uibk.ac.at; norbert.polacek@i-med.ac.at
Abstract: The ribosomal peptidyl transferase center is a ribozyme catalyzing peptide bond synthesis in all
organisms. We applied a novel modified nucleoside interference approach to identify functional groups at
9 universally conserved active site residues. Owing to their immediate proximity to the chemical center,
the 23S rRNA nucleosides A2451, U2506 and U2585 were of particular interest. Our study ruled out U2506
and U2585 as contributors of vital chemical groups for transpeptidation. In contrast the ribose 2′-OH of
A2451 was identified as the prime ribosomal group with potential functional importance. This 2′-OH renders
almost full catalytic power to the ribosome even when embedded into an active site of six neighboring
2′-deoxyribose nucleosides. These data highlight the unique functional role of the A2451 2′-OH for peptide
bond synthesis among all other functional groups at the ribosomal peptidyl transferase active site.
Introduction
have been proposed to be of functional relevance for translation
(ref 9 and reviewed in ref 2). While the complete deletion of
Peptidyl transfer is the fundamental process of protein
synthesis with amide bond formation as the crucial chemical
reaction and is catalyzed within the ribosomal peptidyl trans-
ferase center (PTC) of the large (50S) ribosomal subunit (Figure
1A). Biochemical and recent crystallographic studies revealed
that the PTC is composed entirely of 23S rRNA (ref 1 and
reviewed in ref 2), and consequently, catalysis of peptide bond
formation is based on a ribozyme mechanism, yet to be revealed
in its molecular details. In the crystallographic structure of the
50S subunit two ribosomal groups were seen within hydrogen-
bonding distance to the attacking R-amino group of aminoacyl-
tRNA, namely the adenine N3 and the ribose 2′-OH of A2451
(Figure 1B).1,3 A prominent hypothesis proposes that the N3 of
the universally conserved A2451 (E. coli numbering is used
here and throughout the manuscript) is involved in acid-base
catalysis and functions as a proton acceptor.1 Although many
aspects of the initial model did not survive the scrutiny of
subsequent experimental testing (refs 4-8 and reviewed in ref
2), A2451 remained the prime suspect for a catalytic nucleotide
even though other highly conserved residues, such as U2506,
U2585, or A2602, are also in close proximity to the active site
of the PTC. Among those residues U2585 and A2602 received
special attention since these were the structurally most flexible
nucleotides within the PTC in response to substrate binding and
A2602 had only marginal effects on transpeptidation and hence
excludes this residue for providing crucial functional groups
for amide bond formation,10 the role of U2585 and U2506
remained unclear. One way to experimentally test the functional
roles of these 23S rRNA nucleotides involves the generation
of site-specific chemically modified ribosomes with retained
functionality. Owing to the huge size and complexity of rRNAs
such an undertaking seemed impossible thus far. This problem
was overcome here by applying a novel approach (the gapped-
cp-reconstitution) that allows the site-specific incorporation of
non-natural nucleoside analogues into 23S rRNA of Thermus
aquaticus 50S subunits (Figure 1A). This approach is used in
the following to target the mechanistic role of A2451, U2506,
and U2585 in peptide bond formation and to clearly identify
the functional groups involved.
The gapped-cp-reconstitution system is based on the use of
circularly permuted (cp) 23S rRNA for 50S assembly. The cp-
(4) Polacek, N.; Gaynor, M.; Yassin, A.; Mankin, A. S. Nature 2001, 411,
498-501.
(5) Thompson, J.; Kim, D. F.; O’Connor, M.; Lieberman, K. R.; Bayfield, M.
A.; Gregory, S. T.; Green, R.; Noller, H. F.; Dahlberg, A. E. Proc. Natl.
Acad. Sci. U.S.A. 2001, 98, 9002-9007.
(6) Katunin, V. I.; Muth, G. W.; Strobel, S. A.; Wintermeyer, W.; Rodnina,
M. V. Mol. Cell 2002, 10, 339-346.
(7) Youngman, E. M.; Brunelle, J. L.; Kochaniak, A. B.; Green, R. Cell 2004,
117, 589-599.
(8) Beringer, M.; Bruell, C.; Xiong, L.; Pfister, P.; Katunin, V. I.; Mankin, A.
S.; Bo¨ttger, E. C.; Rodnina, M. V. J. Biol. Chem. 2005, 280, 36065-36072.
† Division of Genomics and RNomics-Innsbruck Medical University.
‡ Leopold Franzens University.
(1) Nissen, P.; Hansen, J.; Ban, N.; Moore, P. B.; Steitz, T. A. Science 2000,
289, 920-930.
(2) Polacek, N.; Mankin, A. S. Crit. ReV. Biochem. Mol. 2005, 40, 285-311.
(3) Hansen, J. L.; Schmeing, T. M.; Moore, P. B.; Steitz, T. A. Proc. Natl.
Acad. Sci. U.S.A. 2002, 99, 11670-11675.
(9) Bashan, A.; Agmon, I.; Zarivach, R.; Schlu¨nzen, F.; Harms, J.; Berisio,
R.; Bartels, H.; Franceschi, F.; Auerbach, T.; Hansen, H. A.; Kossoy, E.;
Kessler, M.; Yonath, A. Mol. Cell 2003, 11, 91-102.
(10) Polacek, N.; Gomez, M. G.; Ito, K.; Nakamura, Y.; Mankin, A. S. Mol.
Cell 2003, 11, 103-112.
9
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J. AM. CHEM. SOC. 2006, 128, 4453-4459
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A R T I C L E S
Erlacher et al.
Figure 1. The ribosomal peptidyl transferase center (PTC). (A) The crystallographic structure of the H. marismortui large ribosomal subunit viewed from
the interface side (left). The 23S rRNA and 5S rRNA are shown as green ribbons, and ribosomal proteins are in gray. The PTC is highlighted by nucleotides
of the 23S rRNA peptidyl transferase loop shown in red. Applying the “gapped-cp-reconstitution” approach allows us to site-specifically place a chemically
modified nucleoside inside the PTC (right). (B) View of the active site of the PTC structure. The distance between the nitrogen atom of the attacking amino
group of an aminoacyl-tRNA analogue (orange sphere) and the N3 or the ribose 2′-OH of A2451 (red) are indicated by black arrows. Tertiary interactions
are depicted by red dotted lines. (A) and (B) were generated from pdb files 1FFK and 1FG0,1 respectively. (C) The standard puromycin reaction.32 The
R-amino group (orange) of A-site bound puromycin attacks the ester bond of P-site bound formyl-[3H]-Methionine-tRNAfMet. For the P-site bound tRNA
only the 3′ CCA-aminoacyl fragment is depicted.
23S rRNAs were generated by covalently connecting the natural
ends of 23S rRNA and introducing new endpoints at appropriate
positions in the PTC, thereby placing gaps in the 23S rRNA
sequence encompassing the nucleoside under investigation. This
missing rRNA segment was chemically synthesized and used
in consort with the cp-23S rRNA to reconstitute 50S subunits
(Figure 2A). Even though the applied technique has the
advantage of performing modified nucleoside interference
studies in the ribosome, its intrinsic limitations have to be
considered.10,11 Importantly, transpeptidation in reconstituted
large ribosomal subunits proceeds clearly slower than the known
in vivo rate of ∼15 s-1 (see Materials and Methods). Therefore
this experimental system is not applicable to measure subtle
changes in transpeptidation but turned out to be valuable to
pinpoint crucial groups in the PTC whose chemical modification
significantly hampered the overall rate of the reaction. In
addition, the gapped-cp-reconstitution is not very efficient, and
compared to full length 23S rRNA transcripts only 10-20% of
cp-23S rRNA assembled into active particles (ref 11 and data
not shown). Within the limits of our experimental system, we
recently showed that base modifications at A2451, including
those which eliminate the proton acceptor capabilities at N3,
only marginally affected the transpeptidation rates in the
puromycin reaction (Figure 1C).11 Even the deletion of the entire
nucleobase, by placing a ribose-abasic site analogue at position
2451, was largely tolerated. However, the additional removal
of the ribose 2′-OH, thereby generating a deoxy-abasic site,
markedly inhibited amide bond synthesis. Comparing the
activities of these two abasic variants suggested that the 2′-OH
at A2451 plays an important role in catalytic activities of the
PTC.11
Results and Discussion
Elimination of the entire nucleobase at A2451 is a rather
invasive modification destroying multiple interactions with other
highly conserved residues in the PTC (Figure 1B). It has been
shown by others that results of active site mutants that disrupt
tertiary interactions are difficult to interpret.12 We therefore
intended to further substantiate our hypothesis of the A2451
2′-OH being involved in peptide bond formation by synthesis
and incorporation of 3-deaza-2′-deoxyadenosine at position 2451
(Figure 2B; see also Materials and Methods). The advantage
of 3-deaza-2′-deoxyadenosine is that it eliminates both potential
proton acceptors at A2451 simultaneously and yet maintains
(11) Erlacher, M. D.; Lang, K.; Shankaran, N.; Wotzel, B.; Hu¨ttenhofer, A.;
Micura, R.; Mankin, A. S.; Polacek, N. Nucleic Acids Res. 2005, 33, 1618-
1627.
(12) Hesslein, A. E.; Katunin, V. I.; Beringer, M.; Kosek, A. B.; Rodnina, M.
V.; Strobel, S. A. Nucleic Acids Res. 2004, 32, 3760-7370.
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Nucleoside Analogue Interference in the Ribosome
A R T I C L E S
Figure 2. Modified nucleoside interference probing of the PTC. (A) Secondary structure of the PTC of gapped-cp-reconstituted 50S subunits showing the
new endpoints of the cp-23S rRNA at positions 2468 and 2440 (5′ and 3′, respectively). The chemically synthesized 26-nucleotide RNA, which compensates
for the missing rRNA segment, is shown in red. The region where modified nucleosides have been introduced is underlined in blue. (B) Chemical synthesis
of 3-deaza-2′-deoxyadenosine (c3dA) and 3-deazaadenosine (c3A) containing RNA (Supporting Information; see also Schemes 1 and 2).26 (C) Relative
initial peptidyl transferase rates (krel) in the puromycin reaction (ref 11) of ribosomes carrying the synthetic RNA fragment with the wild-type sequence (wt),
single 2′-deoxyribose nucleosides at the indicated positions (d), or the c3dA analogue at 2451 (Supplemental Table 1). (D) Relative initial rates of ribosomes
containing seven 2′-deoxyribose nucleosides from positions G2447 to A2453 (all DNA) or the same 26-mer with a single 2′-OH reintroduced at position
A2451 (all DNA + 2′-OH (2451)) in the puromycin reaction. The krel values are given above the respective bars. The rate of ribosomes containing the “all
DNA” oligo could not be measured at the early time points and was therefore estimated to be <0.007 compared to the wt. The initial rates in (C) and (D)
were determined from points in the linear range of the reaction within the first 10 min of incubation. Values shown represent the mean and standard
deviation of at least two time-course experiments.
all the other functional groups of adenosine. This allowed us
for the first time to investigate the catalytic performance of a
PTC where the two prime suspects for crucial functional
ribosomal groups were modified concurrently without decisively
destroying the active site architecture. If the N3 of A2451
participates in transpeptidation, introducing 3-deaza-2′-deoxy-
adenosine would add to the harmful effect of the 2′-deoxyad-
enosine modification. However, the removal of the second
potential proton acceptor did not additionally inhibit peptide
bond formation in the standard puromycin assay (Table 1 and
Figure 2C). This finding, combined with N3 removal alone
having little effect,11 underlines that the main inhibition stems
from the elimination of the ribose 2′-OH at A2451 and indicates
that the contribution of the nucleobase to catalysis is only minor.
It has recently been demonstrated that the minimal A-site
substrate puromycin is hypersensitive to active site changes.7
To test the performance of chemically modified ribosomes with
substrates that better mimic the in vivo situation, we employed
here a peptidyl transferase assay using full-length tRNAs. Even
though the reduced activities of 50S subunits containing 2′-
deoxyribose modifications at 2451 in general could be margin-
ally rescued using genuine aminoacyl-tRNA, the activities were
still reproducibly weaker compared to the corresponding ribose
variants (Table 1 and Supplemental Figure 3). These data
emphasize the critical role of the ribose 2′-OH at A2451 for
transpeptidation independent of the A-site substrate used.
To investigate possible functional implications of U2585 or
U2506 in transpeptidation, we constructed cp-23S rRNAs that
allow placing 2′-deoxyuridine or a ribose-abasic site at these
inner shell residues (Figure 3). Our data showed that none of
those modifications had an inhibitory effect on peptide bond
synthesis in both the puromycin and the full-length tRNA assay
(Table 1). In fact, the abasic 2585 and 2506 variants reproduc-
ibly showed slightly accelerated reaction rates in the puromycin
assay. This contrasts results employing 50S subunits carrying
natural base mutations at U2585 or U2506 which showed
moderate to strong reductions in transpeptidation using in vitro
assembled (ref 10 and data not shown) or in vivo derived
particles.7 Removal of the entire nucleobase at these positions
obviously eliminates the structural distortions that are likely
present in the A, C, or G mutants which in turn allows a more
unrestricted accommodation of puromycin in the A-site. This
conclusion is supported by recent crystallographic structures
suggesting that for efficient peptide bond formation the nucleo-
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A R T I C L E S
Erlacher et al.
Table 1. Activities of Chemically Modified Ribosomes in the
Puromycin (Pmn) Reaction and in a Two tRNA Peptidyl
Transferase Assay
Pmn reaction
two tRNA assay
a
a
23S rRNA position
modification
krel
krel
wt
A2451
none
1.00
1.00
0.81
0.22
0.20
0.05
0.50
1.17
0.91
0.91
0.97
c3A
0.77b
0.15
c3dA
dA
d-abasic
r-abasic
dU
r-abasic
dU
r-abasic
0.11b
<0.01b
0.53b
1.03
1.49
0.99
U2585
U2506
2.21
a Initial transpeptidation rate (krel) of ribosomes carrying the wild-type
(wt) synthetic RNA-oligomer was taken as 1.00 and compared to ribosomes
containing modified nucleoside analogues at positions A2451, U2585, or
U2506. krel were determined from experimental points in the linear range
of the reactions (Supplemental Figure 3). b Data taken from ref 11. Tested
nucleoside analogues: 3-deazaadenosine (c3A), 3-deaza-2′-deoxyadenosine
(c3dA), 2′-deoxyadenosine (dA), 2′-deoxyuridine (dU), 2′-deoxyabasic (d-
abasic), or the ribose-abasic site analogue (r-abasic).
bases at U2506 and U2585 have to move away in the activated
PTC to create space for an optimal positioning of the attacking
R-amino group of the A-site substrate relative to the peptidyl-
tRNA ester.13 In this way, the abasic U2585 and U2506
ribosomes seem to mimic the activated state of the PTC and
therefore accelerate the puromycin reaction.
Introduction of a single DNA nucleoside into the catalytic
center can theoretically inhibit the activity of ribozymes by
impeding the fine-tuning of the productive conformational state
or by changing the overall hydrogen bonding potential.14 To
investigate the requirement of ribose 2′-hydroxyl groups in the
PTC in a thorough manner, we individually introduced DNA
residues at every position of the synthetic RNA neighboring
A2451, which is not involved in Watson-Crick interactions
(Figure 2A). The placement of 2′-deoxyribose nucleosides at
all tested positions did not compromise peptide bond formation,
with the single exception of A2451 (Figure 2C). This clearly
demonstrates that the inhibitory effect of the 2′-OH removal is
specific for A2451. Simultaneous removal of all 2′-hydroxyls
from positions G2447 to A2453 reduced the activity below the
detection limit (Figure 2D and Supplementary Figure 4).
Significantly, reimplantation of a single 2′-OH at A2451 almost
completely restored full transpeptidation activity (Figure 2D).
The fact that this sole 2′-OH renders essentially full activity
even when surrounded by six 2′-deoxyribonucleosides makes
the scenario of a misfolded PTC upon DNA residue insertions
less plausible. In a previous in vivo study using snoRNA-guided
methylation, the modification of the A2451 2′-OH resulted in
a lethal growth phenotype in yeast.15 One possible explanation
for this in vivo result could be ribosome assembly defects upon
ribose methylation at this site. To investigate whether the A2451
2′-OH modification compromised the 50S assembly in our in
vitro system we have previously compared the sucrose gradient
profiles of “wild type” and 2′-deoxy-A2451 50S subunits, which
turned out to be indistinguishable.11 More importantly, “wild
type” and 2′-deoxy-A2451 ribosomes showed full activities in
Figure 3. Chemical mutagenesis at U2585 and U2506. (A) Structure of
the inner shell residues A2451 (red), U2506 (green), U2585 (magenta), and
A2602 (blue). Distances between functional groups of the 23S rRNA
residues to the A-site-bound puromycin (Pmn) are indicated by black arrows.
The position of the nitrogen atom of the attacking amino group is encircled
in orange. The figure was generated from pdb file 1FG0.1 (B, C) Secondary
structures of the PTC of gapped-cp-reconstituted 50S subunits used to
chemically modify U2585 (B) or U2506 (C). The chemically synthesized
RNA fragments that are used to fill the gap of the cp-23S rRNA during
reconstitution are shown in red.
the second fundamental chemical reaction of the PTC, namely
in release factor mediated peptide release.11 Both, peptide bond
formation and peptide release, were shown to be single turnover
reactions under our assay conditions, and therefore the peptide
release activities suggested that comparable fractions of catalyti-
cally active 50S subunits were assembled with the wild-type
RNA oligomer or the 2′-deoxy-A2451 variant.11 These data
combined with the new findings presented in Figures 2 and 3
(13) Schmeing, T. M.; Huang, K. S.; Strobel, S. A.; Steitz, T. A. Nature 2005,
438, 520-524.
(14) Gordon, P. M.; Fong, R.; Deb, S. K.; Li, N.; Schwans, J. P.; Ye, J.; Piccirilli,
J. A. Chem. Biol. 2004, 11, 237-246.
(15) Liu, B.; Fournier, J. RNA 2004, 10, 1130-1141.
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Nucleoside Analogue Interference in the Ribosome
A R T I C L E S
Scheme 1. Synthesis of 3-Deazaadenosine 8a
highlight the exceptional functional role of the A2451 2′-OH
for peptide bond formation.
Previous studies employing standard mutagenesis failed to
unequivocally identify the crucial functional group(s) of 23S
rRNA for peptide bond formation.4-8,12,16 Here we applied a
novel approach that allows the chemical modification of single
23S rRNA groups inside the large ribosomal subunit. Within
the limits of the experimental system, our chemical mutagenesis
study discloses the ribose 2′-OH at A2451 as the prime candidate
in the PTC with vital importance to forge an amide bond. The
unperturbed pKa of a ribose 2′-OH is far from neutral and
therefore unfavorable to directly participate in acid/base chem-
istry. We favor the view that the universally conserved A2451
plays a role as a molecular trigger that senses the substrates in
the active site and properly aligns its ribose 2′-OH group for
coordinating the attacking R-amino group. This interpretation
is in line with recent crystallographic data showing the attacking
amine to be in hydrogen bonding distance to the A2451 2′-OH
in the ground state.13,17 Alternatively, and even though not seen
in the currently available crystal structures from the Steitz and
Strobel laboratories,13,17 the 2′-OH of A2451 could be involved
in transition state stabilization. In support of this possibility, a
molecular dynamics simulation study was presented that predicts
the A2451 2′-OH to be part of a H-bond network that stabilizes
the transient tetrahedral intermediate (TI) of peptide bond
formation.18 Removal of the ribose 2′-OH at A2451 was
calculated to significantly destabilize the relative free energy
of the TI by ∼3 kcal/mol. Irrespective of the specific role of
the A2451 2′-OH, it is likely that the evolutionary pressure that
conserved the nucleobase identity at this residue ensures correct
positioning of its 2′-OH group for efficient peptide bond
catalysis. A similarly important role for another universally
conserved adenosine has recently been proposed for the 2′-OH
of the terminal nucleoside of peptidyl-tRNA.19,20 The currently
available data suggest that effective peptide bond catalysis relies
on the presence of two ribose 2′-OH groups; one located in the
ribosome (at A2451 of 23S rRNA) and the other on the P-site
substrate (at A76 of P-tRNA), rather than on any nucleobase
functional group.
a (a) 5 equiv of tbs-Cl, 10 equiv of imidazole in DMF, rt, 20 h, 98%; (b)
2.5 equiv of 2,4-dinitrochlorobenzene, 2.5 equiv of K2CO3 in DMF, 80 °C,
2.5 h, 93%; (c) ethylenediamine, 50 °C, 6 h, 70%; (d) 1.4 equiv of
p-toluenesulfonyl chloride in pyridine, rt, 20 h, 95%; (e) 4.1 equiv of isoamyl
nitrite in CH2I2, 100 °C, 45 min, 60%; (f) 4 equiv of ethinyl trimethylsilane,
1.2 equiv of NEt3, 5 mol % (PhCN)2PdCl2 in CH3CN, 110 °C, 20 h; (g) 7
N NH3/MeOH, 120 °C, 14 h, 50% over two steps; (h) 9 equiv of polymer
supported fluoride (Amberlite IRA 900 fluoride-form; macroporous, 20-
50 mesh, 3.0 mmol/g loading) in toluene, 110 °C, 6 h, 79%. tbs-Cl ) tert-
butylchlorodimethylsilane; DMF ) N,N-dimethylformamide.
functional residues that build the inner shell of the PTC, such
as A2602, U2585, or U2506, are not critically required to
promote transpeptidation. The data presented here explain why
the importance of the A2451 2′-OH escaped the detection by
regular mutagenesis studies, since the ribose 2′-OH group
remained untouched in all the previous mutants. Our study
suggests that the A2451 ribose 2′-OH represents a key functional
ribosomal group for effective peptide bond synthesis.
Conclusion
Even though the crystal structures of the ribosome and its
individual subunits provide a wealth of detailed insights, the
molecular mechanism of peptide bond formation remains
controversial and far from being fully understood (reviewed in
ref 2). Here we have applied a novel experimental device which
allows us to chemically alter functional groups on specific 23S
rRNA nucleosides inside the PTC. This modified nucleoside
interference approach clearly identified the ribose 2′-OH of
A2451 to be of crucial functional importance. The single 2′-
OH was necessary and sufficient to provide almost full catalytic
power to the PTC, even when introduced into an active site of
neighboring 2′-deoxyribose nucleosides. The other potentially
Materials and Methods
Syntheses of Phosphoramidite Building Blocks of c3A and c3dA.
The chemical syntheses of 2′-O-tris(triisopropylsilyl)-3′-O-[(N,N-di-
isopropylamino)-(â-cyanoethoxy)phosphino]-5′-O-(4,4′-dimethoxy-
trityl)-N6-benzoyl-3-deazaadenosine 12 (c3A) and of 2′-deoxy-3′-O-
[(N,N-diisopropylamino)-(â-cyanoethoxy)phosphino]-5′-O-(4,4′-dimeth-
oxytrityl)-N6-benzoyl-3-deazaadenosine 19 (c3dA) started from inosine
and proceeded in 12 steps (for c3A) and 15 steps (for c3dA) via the
5-amino-4-imidazolecarboxamide (AICA) riboside derivative 3 (Schemes
1 and 2). Our synthesis strategy took previous reports by Matsuda,
Piccialli, McLaughlin, Watanabe, Robins, and their respective co-
workers into account.21-25 Detailed procedures and characterization data
are provided in the Supporting Information.
(16) Bayfield, M. A.; Thompson, J.; Dahlberg, A. E. Nucleic Acids Res. 2004,
32, 5512-5518.
(17) Schmeing, T. M.; Huang, K. S.; Kitchen, D. E.; Strobel, S. A.; Steitz, T.
A. Mol. Cell 2005, 20, 437-448.
(21) Minakawa, N.; Sasabuchi, Y.; Kiyosue, A.; Kojima, N.; Matsuda, A. Chem.
Pharm. Bull. 1996, 44, 288-295.
(22) DeNapoli, L.; Messere, A.; Montesarchio, D.; Piccialli, G.; Varra, M. J.
Chem. Soc., Perkin Trans. 1 1997, 2079-2082.
(23) Bevers, S.; Xiang, G.; McLaughlin, L. W. Biochemistry 1996, 35, 6483-
6490.
(18) Trobro, S.; Aqvist, J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 12395-
12400.
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A R T I C L E S
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Scheme 2. Synthesis of 3-Deazaadenosine Phosphoramidite 12
and of 2′-Deoxy-3-deazaadenosine Phosphoramidite 19a
Peptidyl Transferase Assays. Gapped-cp-reconstituted 50S subunits
for investigating A2451 were assembled, reassociated with native T.
aquaticus 30S subunits and subsequently used in the puromycin reaction
as described previously.11 Under these conditions the puromycin reaction
with reconstituted 50S containing full-length 23S rRNA transcripts
proceeds with a rate of ∼0.6 min-1, while ribosomes composed of
gapped-cp-reconstituted 50S carrying the wt-oligo sequence promote
transpeptidation with a rate of ∼0.012 min-1 (data not shown). To
investigate U2585 or U2506, cp-23S rRNAs were constructed in
analogy to the published procedure.11 To generate the DNA templates
for cp-23S rRNA transcription, the forward and reverse PCR primer
pairs TAATACGACTCACTATAG(2523)GGGCTGAAGAAGGTCCC and
G
(2483)CCGCTGTGGACGCTCGG as well as TAATACGACTCA-
CTATAG(2623)GGCGCAGGAGGCTTGAG and C(2576)GCGTGCCG-
CTTTAATG were used for the cp-23S rRNA constructs that placed
gaps around positions U2506 and U2585, respectively. The T7 promoter
sequences in the forward primers are italic, and the positions defining
the new 5′ and 3′ ends of the cp-23S rRNAs are bold and numbered
according to E. coli nomenclature.
To assess the peptidyl transferase activity of reconstituted ribosomes
using full-length tRNA substrates, 50S subunits were assembled from
20 pmol cp-23S rRNA and 40 pmol synthetic RNA oligonucleotide as
described in ref 11 with the only difference that 8 pmol native T.
aquaticus 30S subunits were present during the entire reconstitution
procedure. Subsequently, the 70S ribosomes were programmed with
60 µg poly(U) and incubated with 3 pmol unlabeled N-acetyl-Phe-
tRNAPhe for 15 min at 37 °C for P-site binding in a buffer containing
12.1 mM Tris/HCl pH 7.5, 8.4 mM Hepes/KOH pH 7.5, 18.1 mM
MgCl2, 5 mM MnCl2, 519 mM NH4Cl, 3.6 mM spermidine, 0.05 mM
spermine, 4.1 mM â-mercaptoethanol, and 0.12 mM EDTA. The
peptidyl transferase reaction was initiated by the addition of 3 pmol
[3H]Phe-tRNAPhe (20 000 cpm/pmol) and performed at 37 °C in a
final volume of 46 µL. The reaction was stopped by the addition of
6.1 µL of 10 M KOH and incubation at 37 °C for 15 min. Subsequently
121 µL of 1 M KH2PO4 and 87 µL of 1 M HCl were added. At this
pH value (pH 2.5) the reaction product, N-acetyl-Phe-[3H]Phe, can be
extracted into ethyl acetate (extraction efficiency was essentially
quantitative) and was analyzed by liquid scintillation counting. At this
pH unreacted [3H]Phe carries a positive charge and is not coextracted
with the dipeptidyl product to a significant extent. In control experiments
with 3 pmol completely hydrolyzed [3H]Phe-tRNAPhe (20 000 cpm/
pmol), only 400 cpm could be extracted into the organic phase. In
peptidyl transferase reactions the background activity (usually ∼500
cpm) that results from possible minor 50S contaminations of the T.
aquaticus 30S subunit preparation was subtracted from all experimental
time points. Compared to the standard puromycin reaction employing
gapped-cp-reconstituted 50S, the transpeptidation rate in this full-length
tRNA assay was accelerated to ∼0.05 min-1. tRNAPhe (Sigma) was
aminoacylated using PheRS as described in ref 30, acetylated using
acetic anhydride (see ref 31), and HPLC purified according to ref 11.
a (a) (1) 8.5 equiv of TMS-Cl in pyridine, 0 °C, 50 min; (2) 5.7 equiv
of Bz-Cl in pyridine, rt, 3.5 h, 90%; (b) (1) 1.2 equiv of DMF-DMA in
pyridine, rt, 1.5 h; (2) 2.7 equiv of DMT-Cl in pyridine, rt, 18 h, 62%; (c)
4 equiv of AgNO3, 3.6 equiv of TIPS-Cl in pyridine/THF, rt, 40 h, 60%;
(d) 3.3 equiv of CEP-Cl, 10 eq Me2NEt in CH2Cl2, rt, 4 h, 90%; (e) 1.3
equiv of TBDS-Cl2 in pyridine, rt, 5 h, 81%; (f) (1) 5.8 equiv of TMS-Cl
in pyridine, 0 °C, 50 min; (2) 6.6 equiv of Bz-Cl in pyridine, rt, 3.5 h,
93%; (g) 1.2 equiv of TCD in DMF, rt, 4 h, 99%; (h) 1.5 equiv of n-Bu3SnH,
0.2 equiv of AIBN in toluene, 75 °C, 3 h, 64%; (i) 6 equiv of polymer
supported fluoride (Amberlite IRA 900 fluoride-form; macroporous, 20-
50 mesh, 3.0 mmol/g loading) in toluene, 110 °C, 5 h, 93%; (j) 1.5 equiv
of DMT-Cl in pyridine, rt, 18 h, 49%; (k) 3 equiv of CEP-Cl, 10 equiv of
Me2NEt in CH2Cl2, rt, 4 h, 80%. TMS-Cl ) chlorotrimethylsilane; Bz-Cl
) benzoyl chloride; DMF-DMA ) N,N-dimethylformamide dimethylacetal;
DMT-Cl ) 4,4′-dimethoxytrityl chloride; TIPS-Cl ) triisopropylchlorosi-
lane; THF ) tetrahydrofuran; CEP-Cl ) 2-cyanoethyl-N,N-diisopropyl-
chlorophosphoramidite; TBDS-Cl2 ) 1,3-dichloro-1,1,3,3-tetraisopropyl-
disiloxane; TCD ) 1,1′-thiocarbonyldiimidazole; im ) imidazole; AIBN
) R,R′-azo-isobutyronitrile.
Acknowledgment. We thank Alexander Mankin, Sean Con-
nell, Daniel Wilson, and Karl Grubmayr for valuable sugges-
tions. N.P. thanks Alexander Hu¨ttenhofer for generous support.
This work was supported by the Austrian Science Foundation
FWF (P16932 and P18709 to N.P.; P17864 to R.M.), the bm:
bwk (GEN-AU Project cluster ‘Non-coding RNAs’), and the
“Tiroler Wissenschaftsfonds” (UNI-404/109 to N.P. and UNI-
404/39 to R.M.).
Oligoribonucleotide Synthesis, Deprotection, and Purification.
Oligoribonucleotides containing c3A and c3dA nucleosides were
synthesized using the 2′-O-TOM-methodology.26-29 Details are provided
in the Supporting Information.
(25) Robins, M. J.; Wilson, J. S.; Hansske, F. J. Am. Chem. Soc. 1983, 105,
4059-4065.
(26) Micura, R. Angew. Chem., Int. Ed. 2002, 41, 2265-2269.
(27) Pitsch, S.; Weiss, P. A.; Jenny, L.; Stutz, A.; Wu, X. HelV. Chim. Acta
2001, 84, 3773-3795.
(28) Ho¨bartner, C.; Kreutz, C.; Flecker, E.; Ottenschla¨ger, E.; Pils, W.;
Grubmayr, K.; Micura, R. Monatshefte fu¨r Chemie - Chemical Monthly
2003, 134, 851-873.
(30) Polacek, N.; Swaney, S.; Shinabarger, S. D.; Mankin, A. S. Biochemistry
2002, 41, 11602-11610.
(31) Triana-Alonso, F. J.; Spahn, C. M.; Burkhardt, N.; Rohrdanz, B.; Nierhaus,
K. H., Methods Enzymol. 2000, 317, 261-276.
(29) Ho¨bartner, C.; Rieder, R.; Kreutz, C.; Puffer, B.; Lang, K.; Polonskaia,
A.; Serganov, A.; Micura, R. J. Am. Chem. Soc. 2005, 127, 12035-12045.
(32) Monro, R. E.; Marcker, K. A. J. Mol. Biol. 1967, 25,347-350.
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4458 J. AM. CHEM. SOC. VOL. 128, NO. 13, 2006

Nucleoside Analogue Interference in the Ribosome
A R T I C L E S
Supporting Information Available: Synthetic procedures and
characterization data of 3-deaza-adenosine 12 and 3-deaza-2′-
deoxyadenosine phosphoramidites 19 for RNA solid-phase
synthesis. Supplemental Figures 1-4 and Supplemental Table
1. This material is available free of charge via the Internet at
http://pubs.acs.org.
JA0588454
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