Biochemistry 2004, 43, 6159-6166
6159
The Affinity of Elongation Factor Tu for an Aminoacyl-tRNA Is Modulated by the Esterified Amino Acid† Taraka Dale, Lee E. Sanderson, and Olke C. Uhlenbeck* Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern UniVersity, EVanston, Illinois 60208 ReceiVed December 19, 2003; ReVised Manuscript ReceiVed March 2, 2004
ABSTRACT: When different mutations were introduced into the anticodon loop and at position 73 of YFA2, a derivative of yeast tRNAPhe, a single tRNA body was misacylated with 13 different amino acids. The affinities of these misacylated tRNAs for Thermus thermophilus elongation factor Tu (EF-Tu)‚GTP were determined using a ribonuclease protection assay. A range of 2.5 kcal/mol in the binding energies was observed, clearly demonstrating that EF-Tu specifically recognizes the side chain of the esterified amino acid. Furthermore, this specificity can be altered by introducing a mutation in the amino acid binding pocket on the surface of EF-Tu. Also, when discussed in conjunction with the previously determined specificity of EF-Tu for the tRNA body, these experiments further demonstrate that EF-Tu uses thermodynamic compensation to bind cognate aminoacyl-tRNAs similarly.
Elongation factor Tu (EF-Tu)1 is the bacterial G protein responsible for delivering all elongator aminoacyl-tRNAs (aa-tRNAs) to the ribosomal A site during translation. When EF-Tu is complexed with GTP, EF-Tu binds cognate elongator aa-tRNAs with similar, high affinities, despite differences in the tRNA sequences and properties of the amino acid side chains (1, 2). Thus, while EF-Tu requires the presence of the esterified amino acid for tight tRNA binding, it has been considered nonspecific with respect to the identity of the aa-tRNA. However, recent experiments have demonstrated that EF-Tu shows high specificity for both the esterified amino acid and the tRNA body. LaRiviere et al. (3) found that tRNAs acylated with noncognate amino acids bind EF-Tu over a broad range of affinities, varying from 60-fold weaker to 120-fold tighter than tRNAs acylated with their cognate amino acid. Quantitative analysis of the binding of 4 cognate aa-tRNAs and 12 misacylated tRNAs to EF-Tu revealed that the thermodynamic contributions of the esterified amino acid and the tRNA body are independent of one another and that the affinities are arranged in such a way that cognate aa-tRNAs have a uniform affinity for EFTu. Thus, an amino acid that has a strong thermodynamic contribution to the overall affinity is esterified to a cognate tRNA that has a weak thermodynamic contribution, and vice versa. This phenomenon has been termed thermodynamic compensation (3). One implication of thermodynamic compensation is that there is a range of thermodynamic contributions made by the esterified amino acid to the overall affinity of an aatRNA for EF-Tu. The available X-ray cocrystal structures of the EF-Tu‚GDPNP‚Phe-tRNAPhe and EF-Tu‚GDPNP‚CystRNACys complexes show that the side chains of both phen† This work was funded by National Institutes of Health Grant GM37552 to O.C.U. * To whom correspondence should be addressed. Phone: (847) 4915139. Fax: (847) 491-5444. E-mail:
[email protected]. 1 Abbreviations: EF-Tu, elongation factor Tu; aa-tRNA, aminoacyltRNA; aaRS, aminoacyl-tRNA synthetase.
ylalanine and cysteine bind in the same pocket on the surface of EF-Tu (4, 5). This suggests that each esterified amino acid will interact with EF-Tu at this site by making slightly different contacts with the pocket, thus resulting in differences in binding affinities of the amino acid for EF-Tu. The range of amino acid affinities could be as large as the 700fold difference in tRNA affinities that was deduced using 19 different E. coli elongator tRNAs esterified with the same amino acid (6). The goal of the present paper was to directly test the specificity of Thermus thermophilus EF-Tu for the esterified amino acid. This aim was accomplished by misacylating a tRNAPhe variant, YFA2, with a number of different of amino acids and determining the affinities of these aa-tRNAs for EF-Tu. When a constant tRNA body was maintained, any changes in the aa-tRNA binding affinity could be attributed to changes in the amino acid identity. The ability of EF-Tu to interact differentially with the various esterified amino acids was further examined by making a mutation in the amino acid binding pocket of EF-Tu and determining the effect on the specificity of amino acid binding. EXPERIMENTAL PROCEDURES tRNA Mutagenesis and Preparation. Variants of the YFA2 gene (7) were created using site-directed mutagenesis (QuikChange Site-Directed Mutagenesis Kit, Stratagene). Transcription templates were prepared by PCR amplification of plasmid DNA. In vitro transcription (8) was performed in reactions containing 40 mM Tris-HCl (pH 8.0), 10 mM dithiothreitol (DTT), 20 mM Mg(OAc)2, 25 mM NaCl, 2 mM spermidine, 2.5 mM of each NTP, 20 mM GMP, 50 µg/mL bovine serum albumin, and 30 µg/mL T7 RNA polymerase. tRNAs were purified on 10% denaturing polyacrylamide gels. Aminoacylation Reactions. Aminoacylation reactions were performed with 30 mM Hepes (pH 7.0), 30 mM KCl, 15 mM MgCl2, 5 mM DTT, 4 mM ATP, 2-3 µM tRNA,
10.1021/bi036290o CCC: $27.50 © 2004 American Chemical Society Published on Web 04/27/2004
6160 Biochemistry, Vol. 43, No. 20, 2004 10-25 µM [3H]amino acid (10-100 Ci/mmol), 10 U/mL of inorganic pyrophosphatase (Sigma), 250 nM-2 µM of purified aminoacyl-tRNA synthetase (aaRS), and (in some cases) 20% DMSO. After a 15 min incubation at 37 °C, reactions were quenched with phenol/choloroform (pH 5.5) and subjected to phenol/chloroform and chloroform extractions. Reactions were precipitated with ethanol, the pellet was resuspended in 5 mM NaOAc (pH 5), and the samples were divided into aliquots and stored at -80 °C. Protein Purification. Histidine-tagged E. coli GluRS, ArgRS, and GlyRS were purified from plasmids provided by T. Yokogawa and K. Nishikawa (Gifu University, Japan). A total of 100 mL of BL21 pLysS cells was grown to A600 ) 0.7, followed by an incubation for 4 h in the presence of 0.5 mM IPTG. Harvested cells were resuspended in 10 mL of 20 mM Tris-HCl (pH 7.6), 1 mM MgCl2, 200 mM NaCl, 10 mM β-mercaptoethanol, 20 mM imidazole, 0.01% Triton X-100, and 5% glycerol (Buffer A), containing 50 µM 4-(2aminoethyl)-benzenesulfonyl fluoride (ABSF), and lysed by sonication. The lysate was cleared by centrifugation at 10000g for 30 min. A total of 1 mL of Ni-NTA Agarose (Qiagen) was added to the supernatant, and the slurry was mixed gently for 1 h at 4 °C. The slurry was loaded onto an empty column, washed twice with 4 mL of Buffer A, and eluted with 2 mL of Buffer A containing 300 mM imidazole. Purified proteins were dialyzed into Buffer A with 50% (v/v) glycerol. Yeast PheRS and E. coli AlaRS were purified as described (7). Purified E. coli GlnRS and ValRS were provided by J. Perona (University of California, Santa Barbara, CA). Purified Deinococcus radiodurans AspRS and Chlamydia trachomatis LysRS were a gift from D. So¨ll and G. Raczniak (Yale University, CT). P. Schimmel and L. Nangle (Scripps Institute) provided purified E.coli IleRS, MetRS, TrpRS, and the misacylating T222P mutant ValRS (9). Purified Methanococcus jannaschi ProRS was received from K. Musier-Forsyth (University of Minnesota, MN), as well as from Y. M. Hou and V. Scully (Thomas Jefferson University, PA). EF-Tu from T. thermophilus was overexpressed and purified as described previously (10) with the following exception: Ammonium sulfate was slowly added to 30%, and the sample was centrifuged at 13000g for 15 min to remove the contaminating protein. Ammonium sulfate was then added to the supernatant to 70% to precipitate the EF-Tu as before. EF-Tu Assays. EF-Tu stored in its GDP-bound form was converted to its GTP-bound form immediately prior to use. The GTP-bound form of EF-Tu was obtained by incubating 0.5 µM EF-Tu in 50 mM Hepes (pH 7.0), 20 mM MgCl2, 0.5 M NH4Cl, 5 mM DTT, 20 µM GTP, 0.6-3 mM phosphoenolpyruvate, and 50 µg/mL pyruvate kinase (Buffer B) at 37 °C for 3 h. Dissociation rate constants of the EF-Tu‚GTP‚aa-tRNA complex were determined using a modified version of a ribonuclease protection assay (1). Ternary complex was formed by incubation of 0.5 µM EF-Tu‚GTP with 150b 11 1.9 62 8.1 35 17 15 15 3.6 17
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