Specificity of translation for N-alkyl amino acids

Article abstract of DOI:10.1021/ja073487l

We examine the specificity of translation for various primary and secondary amino acid analogues. A synthetase-free, pure, E. coli translation system is used to prevent competing reactions, and three different tRNA adaptor:codon pairs are used to control

Full text of DOI:10.1021/ja073487l

Published on Web 08/25/2007
Specificity of Translation for N-Alkyl Amino Acids
†
‡
‡
§
Baolin Zhang, Zhongping Tan, Lucas Gartenmann Dickson, Madhavi N. L. Nalam,
‡
,†
Virginia W. Cornish, and Anthony C. Forster*
Department of Pharmacology and Vanderbilt Institute of Chemical Biology, Vanderbilt UniVersity Medical Center,
2
222 Pierce AVenue, NashVille, Tennessee 37232, Department of Chemistry, Columbia UniVersity,
3
000 Broadway, New York, New York 10027, and Department of Pathology, Brigham and Women’s Hospital,
HarVard Medical School, 77 AVenue Louis Pasteur, Boston, Massachusetts 02115
Received May 16, 2007; E-mail: a.forster@vanderbilt.edu
The universal genetic code encodes 19 amino acids and just one
N-alkyl amino acid (“imino acid”), proline (Pro). This raises some
interesting questions. Given that several N-alkyl amino acids are
major products of prebiotic synthetic experiments and meteorite
1
analyses, why did Nature select Pro? Is Pro incorporated into
protein less efficiently than an amino acid by the translation
apparatus? Are other N-alkyl amino acids excluded from incorpora-
tion, for example, by proofreading? Can translation be engineered
to incorporate other N-alkyl amino acids efficiently? Answers bear
on plans to translate mRNA templates into libraries of N-alkyl
amino acid polymers for genetic selection of drug leads.2 N-
Alkylation is desirable because it can confer the important pharma-
cological properties of protease resistance, membrane permeability,
and even oral availability (e.g., cyclosporin A).
Figure 1. The 24 translation assays. (A) L-amino acids and N-alkyl-L-
amino acids synthesized in NVOC-amino-protected form on the dinucleotide
minusCA
The above questions have been investigated experimentally using
chemoenzymatically synthesized N-alkyl aminoacyl-transfer RNAs
pdCpA for ligation onto tRNA
transcripts (see Supporting Informa-
tion). A, Ala; F, Phe; P, Pro; hP, 4-hydroxy-Pro; NMA, N-methyl-Ala; NMF,
N-methyl-Phe; NBA, N-butyl-Ala; NBF, N-butyl-Phe. (B) Coding sequences
of mRNAs: f, formyl; X, amino/N-alkyl amino acid from (A).
(tRNAs) and in vitro translation systems. Efficiencies are typically
reported as the yield of product with an unnatural aminoacyl-tRNA
in comparison with the control yield of 100% from all wild-type
aminoacyl-tRNAs. Results can be summarized as follows. Pro was
incorporated at 46% efficiency from an unnatural tRNA that could
aminoacyl-tRNAs were assayed in a full translation cycle by
3
programming incorporation of H-Val immediately downstream
(Figure 1B).
3
As a positive control, we translated mRNA MTFV into fMTFV
incorporate Ala at 28%. Pro analogues ranged in incorporation
3
-5
using all-natural tRNAs precharged in vitro with all-natural amino
acids by aminoacyl-tRNA synthetases. The yield of this peptide
(Figure 2, legend) was essentially the same as the yields when
efficiencies from 43% to undetectable.
N-Methyl amino acids
3,4,6,7
ranged from 72% to undetectable.
N-Ethyl alanine incorporation
3
was undetectable. It is difficult to draw general conclusions from
these results because of the wide range of reported efficiencies and
the paucity of experiments. Interpretation may be complicated by
the large number of variables between the experimental systems:
prokaryotic versus eukaryotic, presence of competitors and editing
by aminoacyl-tRNA synthetases in crude systems versus absence
in synthetase-free pure systems, tRNA adaptors that are natural ver-
sus methylated natural versus unnatural, tRNA anticodons that are
natural versus unnatural, different messenger RNA (mRNA) codons
for incorporation, different amino acids preceding incorporation,
and requirement for amino acid incorporation downstream or not.
Of the various translation systems available for comparison of
amino acid analogues, a purified E. coli system free of aminoacyl-
Phe
natural Phe-tRNA was substituted with our chemoenzymatically
PheB
PheB
synthesized Phe-tRNA
or Ala-tRNA
(Figure 2A, A and F
on the x-axis). This validated the ability of certain synthetic
substrates to saturate the assay.
Ala, Phe, Pro, and 4-Hydroxy-Pro. The 12 incorporation
efficiencies (Figure 2, right) ranged from quantitative to very
efficient, with the cyclic secondary amino acids being comparable
with the primary amino acids. All incorporation efficiencies were
substantially greater than those reported for amino acids from
suppressor tRNAs in crude translation systems,10 presumably due
to a lack of competitors in our system.
N-Methyl Amino Acids. For N-Me-Ala, incorporation was very
AsnB
AlaB
2
efficient from tRNA
tRNA
and tRNA
but only intermediate from
tRNA synthetases is highly suitable. It is well-defined, flexible,
PheB
(Figure 2, right). For N-Me-Phe, incorporation was
and incorporates unnatural amino acids selectively and efficiently.
We thus used this system to compare the incorporation of amino/
N-alkyl amino acids (Figure 1A). To examine the effects of different
codons, anticodons, and tRNA bodies, we constructed two new
tRNA adaptors with wild-type anticodons to compare with our origi-
intermediate from all three tRNAs. This demonstrates that es-
sentially quantitative incorporation of an N-Me amino acid is
possible, provided that it is matched with a suitable tRNA adaptor
and used in a purified system. It has been speculated that the natural
pairings of amino acids and their cognate tRNAs may be optimal
for delivery by the aminoacyl-tRNA carrier protein, elongation
factor Tu, and for incorporation by the ribosome.11 Our data with
unmodified tRNAs, together with our earlier intermediate efficiency
Asn
AsnB 8
nal E. coli tRNA -based unmodified tRNA (termed tRNA
).
9
They are based on two other unmodified E. coli tRNAs, termed
PheB
AlaB
tRNA
and tRNA
(Figure 2, left). Synthetic amino/N-alkyl
†
Vanderbilt University Medical Center.
Columbia University.
Harvard Medical School.
‡
§
incorporations of these same N-Me amino acids from two other
AsnB
adaptors,7 indicate that the pairing is particularly
tRNA
11316
9
J. AM. CHEM. SOC. 2007, 129, 11316-11317
10.1021/ja073487l CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
incorporations for N-Me and N-Bu amino acids. It is hard to explain
this preference by arguing that translation co-evolved with Pro or
Pro
Pro-tRNA because the structures of the 20 natural amino/N-alkyl
amino acids differ greatly and we used three different tRNA bodies.
An alternative explanation is that Pro is more chemically reactive
than N-Me amino acids and much more reactive than N-Bu amino
acids. The five-membered Pro ring is less hindered than the freely
rotating N-Me, which is less hindered than the larger N-Bu. Indeed,
solid-phase peptide synthesis couples Pro more efficiently than
1
3
N-Me amino acids and other bulky amino acid analogues, and
Pro analogues and N-Me amino acids are the most common N-alkyl
amino acids incorporated by cellular nonribosomal peptide syn-
thetases.14 If the incorporation preferences for our purified E. coli
system are in fact governed by the innate chemical reactivities of
various N-alkyl amino acids, analogous preferences would be
predicted for the translation machinery of every organism. Our data
suggest that many N-alkyl amino acids in the cell may be excluded
from incorporation into proteins by the translation apparatus, even
though some can be charged onto tRNAs by aminoacyl-tRNA
synthetases.15 Finally, the data support exploration of Pro analogues
and N-Me amino acids, each charged on several tRNAs, as
substrates for engineering ribosomal synthesis of genetically
selectable libraries of protease-resistant, N-alkyl, peptide ligands.
Acknowledgment. A.C.F.’s work began in the Department of
Pathology, Brigham and Women’s Hospital, Harvard Medical
School, under the exceptional mentorship of Dr. Stephen Blacklow.
We thank Drs. Michael Pavlov and M a˚ ns Ehrenberg for advice
and materials. This work was supported by NIH grants (to A.C.F.
and V.W.C.), American Cancer Society grants (to A.C.F.), and the
Swiss National Foundation (to L.G.D.).
Figure 2. Efficiencies of incorporation of amino/N-alkyl amino acids from
synthetic tRNAs into peptide tetramers in the 24 assays of Figure 1. Left:
natural E. coli tRNAs (black with purple anticodons) and their synthetic
unmodified counterparts (changes in blue). Right: yields of peptide tetramers
in pure, synthetase-free translations incubated at 37 °C for 40 min. (-)
Background counts defined by omitting cognate aminoacyl-tRNAs for the
Supporting Information Available: Figure S1 and additional
experimental details. This material is available free of charge via the
Internet at http://pubs.acs.org.
Phe
third codons. Addition of wild-type Phe-tRNA in (A) gave the positive
PheB
control (95 ( 5%; not plotted). Ala-tRNA
gave 100 ( 2%, and the
References
other two Ala-charged unmodified tRNAs also saturated tetrapeptide
synthesis (normalized to 100%). Yields with all other substrates were
calculated relative to their respective Ala-tRNAs. Standard deviations of
quadruplicate experiments are shown. See Supporting Information.
(1) Weber, A. L.; Miller, S. L. J. Mol. EVol. 1981, 17, 273-84.
(2) (a) Forster, A. C.; Weissbach, H.; Blacklow, S. C. Anal. Biochem. 2001,
297, 60-70. (b) Tan, Z.; Blacklow, S. C.; Cornish, V. W.; Forster, A. C.
Methods 2005, 36, 279-90.
(
(
3) Ellman, J. A.; Mendel, D.; Schultz, P. G. Science 1992, 255, 197-200.
4) (a) Chung, H. H.; Benson, D. R.; Schultz, P. G. Science 1993, 259, 806-
important for N-Me amino acids. Results are difficult to predict:
PheB
our expectations that tRNA
would be the best adaptor for Phe
9
. (b) Karginov, V. A.; Mamaev, S. V.; An, H.; Van Cleve, M. D.; Hecht,
AlaB
and N-Me-Phe and that tRNA
would be the best adaptor for
S. M.; Komatsoulis, G. A.; Abelson, J. N. J. Am. Chem. Soc. 1997, 119,
166-76.
5) (a) Josephson, K.; Hartman, M. C. T.; Szostak, J. W. J. Am. Chem. Soc.
2005, 127, 11727-35. (b) Lummis, S. C. R.; Beene, D. L.; Lee, L. W.;
Lester, H. A.; Broadhurst, R. W.; Dougherty, D. A. Nature 2005, 438,
8
Ala and N-Me-Ala were not fully borne out. Data are consistent
(
with the rather varied incorporation efficiencies from different tRNA
bodies reported for N-Me amino acids3
,4,6,7
12
and other amino acids.
2
48-52. (c) Choudhury, A. K.; Golovine, S. Y.; Dedkova, L. M.; Hecht,
N-Butyl Amino Acids. Incorporations were undetectable (limit
of sensitivity ) 5%; Figure 2, right). This was surprising given
the lack of competing cognate incorporations but is consistent with
S. M. Biochemistry 2007, 46, 4066-76.
(
6) (a) Bain, J. D.; Wacker, D. A.; Kuo, E. E.; Chamberlin, A. R. Tetrahedron
1
991, 47, 2389-400. (b) Frankel, A.; Millward, S. W.; Roberts, R. W.
Chem. Biol. 2003, 10, 1043-50. (c) Merryman, C.; Green, R. Chem. Biol.
2004, 11, 575-82.
3
the failure to incorporate N-ethyl alanine in a crude system. Note
(
7) Tan, Z.; Forster, A. C.; Blacklow, S. C.; Cornish, V. W. J. Am. Chem.
Soc. 2004, 126, 12752-3.
that with the mRNA MTAV, measured dpm was paradoxically less
3
than the minus cognate tRNA control. This is because the H-Val
(8) Forster, A. C.; Tan, Z.; Nalam, M. N. L.; Lin, H.; Qu, H.; Cornish, V.
W.; Blacklow, S. C. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 6353-7.
incorporation for the minus cognate tRNA control was unexpectedly
higher than for the minus mRNA control due to missense transla-
tion, and the N-Bu aminoacyl-tRNAs inhibited missense translation
by 4-fold. Translation inhibition was not nonspecific because 14C-
Thr incorporation was unaffected, indicating that N-Bu aminoacyl-
tRNAs can bind to the ribosomal A site. Direct evidence for codon-
specific binding of an N-Bu aminoacyl-tRNA was obtained by 3
min ribosome binding assays (Figure S1 in Supporting Information).
Returning to our original questions, is Pro a privileged N-alkyl
amino acid for incorporation by the translation apparatus? In support
of this, three different tRNA bodies gave efficient incorporations
for 12/12 amino acid/Pro/hydroxy-Pro assays but only for 4/12
(
9) (a) Harrington, K. M.; Nazarenko, I. A.; Dix, D. B.; Thompson, R. C.;
Uhlenbeck, O. C. Biochemistry 1993, 32, 7617-22. (b) Ma, C.; Kudlicki,
W.; Odom, O. W.; Kramer, G.; Hardesty, B. Biochemistry 1993, 32,
7939-45.
(
10) Cornish, V. W.; Mendel, D.; Schultz, P. G. Angew. Chem., Int. Ed. Engl.
1995, 34, 621-33.
(
11) LaRiviere, F. J.; Wolfson, A. D.; Uhlenbeck, O. C. Science 2001, 294,
165-8.
(12) Cload, S. T.; Liu, D. R.; Froland, W. A.; Schultz, P. G. Chem. Biol. 1996,
3, 1033-8.
(
(
13) Humphrey, J. M.; Chamberlin, A. R. Chem. ReV. 1997, 97, 2243-66.
14) Nonribosomal peptide amino acids are listed at http://bioinfo.lifl.fr/norine/.
(15) Hartman, M. C. T.; Josephson, K.; Szostak, J. W. Proc. Natl. Acad. Sci.
U.S.A. 2006, 103, 4356-61.
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