Probing the Role of an Active Site Aspartic Acid in Dihydrofolate Reductase

Karginov, Vladimir A.; Mamaev, Sergey; An, Hongyu; Cleve, M D Van; Hecht, Sidney M.; Komatsoulis, George A.; Abelson, John

doi:10.1021/ja971099l

Cited by 71 publications

(95 citation statements)

References 68 publications

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“…Based on several reports, we expect that the ␤-amino acids will not be translated (56)(57)(58). Conversely, at least some of the N-Me amino acid AARS substrates should be efficient translation substrates (8,(58)(59)(60). Several ␣,␣-disubstituted analogs have been tested in translation after chemoenzymatic charging onto either a suppressor tRNA (61) or a modified tRNA Asn (58).…”

Section: Discussionmentioning

confidence: 99%

Enzymatic aminoacylation of tRNA with unnatural amino acids

Hartman

Josephson²,

Szostak³

2006

Proc. Natl. Acad. Sci. U.S.A.

138

172

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The biochemical flexibility of the cellular translation apparatus offers, in principle, a simple route to the synthesis of drug-like modified peptides and novel biopolymers. However, only Ϸ75 unnatural building blocks are known to be fully compatible with enzymatic tRNA acylation and subsequent ribosomal synthesis of modified peptides. Although the translation system can reject substrate analogs at several steps along the pathway to peptide synthesis, much of the specificity resides at the level of the aminoacyl-tRNA synthetase (AARS) enzymes that are responsible for charging tRNAs with amino acids. We have developed an AARS assay based on mass spectrometry that can be used to rapidly identify unnatural monomers that can be enzymatically charged onto tRNA. By using this assay, we have found 59 previously unknown AARS substrates. These include numerous side-chain analogs with useful functional properties. Remarkably, many ␤-amino acids, N-methyl amino acids, and ␣,␣-disubstituted amino acids are also AARS substrates. These previously unidentified AARS substrates will be useful in studies of the specificity of subsequent steps in translation and may significantly expand the number of analogs that can be used for the ribosomal synthesis of modified peptides.aminoacyl-tRNA synthetases ͉ enzyme specificity ͉ MALDI-TOF mass spectrometry ͉ translation

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Section: Discussionmentioning

confidence: 99%

Enzymatic aminoacylation of tRNA with unnatural amino acids

Hartman

Josephson²,

Szostak³

2006

Proc. Natl. Acad. Sci. U.S.A.

138

172

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“…N-alkyl groups in polypeptides and proteins kink 3-dimensional structures, result in slow isomerization between cis and trans conformations in the case of Pro, and can improve the pharmacological properties of peptides by decreasing their degradation by proteases and increasing their membrane permeability. To investigate these special properties or endow them on translation products, there have been many studies incorporating N-alkylamino acids (''imino acids'') in translation (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). Small linear N-alkylamino acids ( Fig.…”

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confidence: 99%

“…Small linear N-alkylamino acids ( Fig. 1 Upper Right), because of their similarity to Pro, were expected to incorporate well in translation, but the efficiencies were variable (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). Incomplete incorporations in purified translation systems (8)(9)(10)(11)(12)(13) are particularly puzzling because of the lack of competing reactions, incubation times of tens of minutes, and an average codon translation time comparable with that in Escherichia coli (45 ms) (14).…”

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confidence: 99%

Slow peptide bond formation by proline and other N -alkylamino acids in translation

Pavlov

Watts²,

Tan³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

295

350

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Proteins are made from 19 aa and, curiously, one N-alkylamino acid (''imino acid''), proline (Pro). Pro is thought to be incorporated by the translation apparatus at the same rate as the 19 aa, even though the alkyl group in Pro resides directly on the nitrogen nucleophile involved in peptide bond formation. Here, by combining quench-flow kinetics and charging of tRNAs with cognate and noncognate amino acids, we find that Pro incorporates in translation significantly more slowly than Phe or Ala and that other N-alkylamino acids incorporate much more slowly. Our results show that the slowest step in incorporation of N-alkylamino acids is accommodation/peptidyl transfer after GTP hydrolysis on EF-Tu. The relative incorporation rates correlate with expectations from organic chemistry, suggesting that amino acid sterics and basicities affect translation rates at the peptidyl transfer step. Cognate isoacceptor tRNAs speed Pro incorporation to rates compatible with in vivo, although still 3-6 times slower than Phe incorporation from Phe-tRNA Phe depending on the Pro codon. Results suggest that Pro is the only N-alkylamino acid in the genetic code because it has a privileged cyclic structure that is more reactive than other N-alkylamino acids. Our data on the variation of the rate of incorporation of Pro from native Pro-tRNA Pro isoacceptors at 4 different Pro codons help explain codon bias not accounted for by the ''tRNA abundance'' hypothesis.non-natural amino acids ͉ ribosomes ͉ tRNA ͉ EF-Tu ͉ GTPase

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“…By replacing the codon corresponding to Ser 284 in luciferase with a TAG stop codon, plasmid pTrcLuc-Wt was modified to give pTrcLucSt284. The linearized plasmids were transcribed in vitro to afford the requisite mRNAs.Preparation of Mono-and Bisaminoacyl-tRNA CUA s-Mono-and bisaminoacyl-tRNAs were prepared by a T4 RNA ligase-mediated ligation of the chemically synthesized pdCpA derivatives (12,13) 3,4 with the abbreviated suppressor tRNA-C OH , the latter of which was prepared as described previously (8,14). Ligation reactions were carried out as described previously (8, 9).…”

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confidence: 99%

Tandemly Activated tRNAs as Participants in Protein Synthesis

Wang

Zhou

Lodder

et al. 2006

Journal of Biological Chemistry

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While all studies of protein synthesis to date have employed monoaminoacylated transfer RNAs, there have been reports that bisphenylalanyltRNA is formed by Thermus thermophilus phenylalanyl-tRNA synthetase. Such tandemly activated tRNAs have now been prepared by chemicoenzymatic techniques and are shown to function in both prokaryotic and mammalian protein synthesizing systems. They exhibit characteristics consistent with their possible utility under extreme conditions in natural systems and have important potential advantages for protein elaboration in cell free systems. Mechanistically, the bisaminoacylated tRNAs bind to the ribosomal A-site and utilize the aminoacyl moiety attached to the 3 -position of the terminal adenosine for addition to the growing polypeptide chain. Following translocation to the P-site and transfer of the formed peptidyl moiety, the donor tRNA dissociates from the ribosome as a monoaminoacylated tRNA capable of functioning in a subsequent polypeptide elongation step.Aminoacyl-tRNAs are key intermediates in protein biosynthesis. These adaptor molecules (1) not only faithfully recognize the appropriate mRNA codon but also transfer aminoacyl residues onto the growing peptide chain, thus bridging the information gap between nucleic acids and proteins. The amino acid residue is attached to the 2Ј(3Ј)-hydroxyl group of the 3Ј-terminal adenosine residue of tRNA via an ester linkage. The esterification of a tRNA with an amino acid is mediated by an aminoacyl-tRNA synthetase. There are 20 different aminoacyl-tRNA synthetases, each having a cognate amino acid and substrate tRNA. While the attachment of the amino acid occurs specifically either on the 2Ј-or 3Ј-OH group of the 3Ј-terminal adenosine moiety in tRNA (2), the resulting aminoacyl ester is quite activated and equilibriates rapidly between the 2Ј-and 3Ј-positions (3-5).Stepanov et al. (6, 7) have described the formation of bis(2Ј,3Ј-O-phenylalanyl)-tRNAs by phenylalanyl-tRNA synthetase from Thermus thermophilus; the enzyme was shown to form a tandemly activated tRNA using T. thermophilus tRNA Phe as substrate, as well as Escherichia coli tRNA Phe and an RNA transcript identical in sequence with E. coli tRNA Phe . While the formation of bisphenylalanyl-tRNAs by T. thermophilus phenylalanyl-tRNA synthetase has been known for some time, possible functions of bisacylated tRNAs have apparently not been studied. This may reflect the difficulty in obtaining such species by conventional methods on a preparative scale.Presently, we have prepared several tRNAs having amino acids attached to both the 2Ј-and 3Ј-positions of the 3Ј-terminal adenosine moiety. The ability of these tandemly activated tRNAs to participate in protein synthesis is described, as is an analysis of the mechanistic details of that participation. EXPERIMENTAL PROCEDURESConstruction of Expression Plasmids-Plasmids pThis15 and pTHD8 were used to express wild-type DHFR 2 in different in vitro translation systems. Plasmid pThis15 was used in a rabbit reticulocyte lysate system (8),...

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Probing the Role of an Active Site Aspartic Acid in Dihydrofolate Reductase

Cited by 71 publications

References 68 publications

Enzymatic aminoacylation of tRNA with unnatural amino acids

Enzymatic aminoacylation of tRNA with unnatural amino acids

Slow peptide bond formation by proline and other N -alkylamino acids in translation

Tandemly Activated tRNAs as Participants in Protein Synthesis

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