Proc. Natl. Acad. Sci. USA Vol. 74, No. 2, pp. 437-441, February 1977 Biochemistry Isomeric aminoacyl-tRNAs are both bound by elongation factor Tu [CTP(ATP).tRNA nucleotidyltransferase/aminoacyl-tRNA deacylation/protein biosynthesis/positional specificity] SIDNEY M. HECHT, KIM HocK TAN, A. CRAIG CHINAULT, AND PAOLO ARCARI Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Mass. 02139 Communicated by Paul C. Zamecnik, September 10, 1976 ABSTRACT Recent suggestions that elongation factor Tu of the latter to the ribosomal A-site (10). The binary complex (EF-Tu) is specific for 2'-0-aminoacyl-tRNA, as compared with the 3'-isomer, prompted us to assay [3Hlaminoacyl-tRNAs from (EF-Tu-GTP) does not bind deacylated or N-acylated amino- Escherichia coli terminating in 2'- or 3'-deoxyadenosine for acyl-tRNAs (11, 12) to a significant extent; that ternary complex binding to EF-Tu to determine the possible positional specificity formation involves the aminoacyl moiety of tRNA is further of the factor. Binding of modified aminaoc l-tRNAs to EF- suggested by the greatly reduced rate of chemical hydrolysis TuGTP was measured both as a function of the ability of EF- of bound aminoacyl-tRNA (13, 14). This report deals with the TuGTP to diminish the rate of chemical deacylation of binding of aminoacylated Escherichia coli tRNAs [3H]aminoacyl-tRNAs and by gel filtration of the individual terminating ternary complexes. Fifteen different tRNA isoacceptors were in 2'- or 3'-deoxyadenosine to EF-Tu from E. coli, as measured tested by the deacylation procedure, including three (tRNAAsP, both by the ability of the factor to retard chemical deacylation tRNACYs, and tRNATYr) for which isomeric modified amino- of the modified aminoacyl-tRNAs and by gel filtration studies. acyl-tRNAs were available. All of the modified aminoacyl- The results, which are not consistent with conclusions reached tRNAs were protected from deacylation, although generally to recently on the basis of fragment reaction studies (15, 16) and a lesser extent than the corresponding unmodified species. experiments Six modified tRNA isoacceptors (including tRNATrP and utilizing positionally defined but nonisomeric tRNATYr, fQr which both modified aminoacyl-tRNAs were ac- tRNAs (17, 18), are presented for 16 different tRNA isoaccep- cessible by enzymatic aminoacylation) were used in gel filtra- tors, including four for which both aminoacyl-tRNA isomers tion experiments to permit direct measurement of the individual were accessible enzymatically. aminoacyl-tRNA-EF-Tu-GTP complexes. These experiments were also done in the presence of equimolar amounts of the corresponding unmodified [14Claminoacyl-tRNAs, and the rel- MATERIALS AND METHODS ative affinities for a limiting amount of EF-TuGTP were mea- [3H]Arginine, aspartate, glutamate, glycine, isoleucine, phe- sured. The results were completely consistent with those ob- nylalanine, threonine, tryptophan, valine, cordycepin, 2'-dATP, tained by the deacylation procedure and indicated that EF-Tu can bind to both positionalisomers of aminoacyl-tRNA with no and GDP were purchased from New England Nuclear; [3H] obvious preference for either. alanine, leucine, and serine were from ICN. [3H]Lysine and asparagine were from Schwarz/Mann and [3H]cystine from Although aminoacyl-tRNA is thought to consist of a mixture Amersham/Searle. Phosphoenolpyruvate and pyruvate kinase of 2'- and 3'-O-aminoacyl species which equilibrate rapidly, were obtained from Sigma Chemicals, as were CTP, ATP, GTP, the specificity generally associated with enzymic processes GDP, 2'-dATP, and cordycepin [which was converted to 3'- makes it not unlikely that single isomers of tRNA may be uti- dATP chemically (19,20)]. Aminoethyl-cellulose (Sigma) was lized exclusively for certain of the partial reactions of protein converted to (acetylated) DBAE-cellulose (N-[N'-(m-dihy- biosynthesis.' The rapid equilibration of the two isomers of droxyborylphenyl)succinamy]aminoethyl-cellulose) by known aminoacyl-tRNAs has precluded a determination of positional methods (ref. 21 and references therein). DEAE-cellulose was specificities involved in such transformations, but data of this from Whatman, glass fiber disks from Schleicher and Schuell, type are now accessible by the use of certain modified tRNAs, and nitrocellulose filters (45 ,m) from Millipore. Unfraction- e.g., those terminating in 2'- and 3'-deoxyadenosine (1-6). ated E. coli tRNA and partially fractionated E. coil amino- Results obtained from the study of suchmodified tRNAs have acyl-tRNA synthetase were prepared as described (9); venom suggested, e.g., that tRNAs are aminoacylated exclusively on phosphodiesterase was from Boehringer Mannheim. The EF-Tu a single (2' or 3') OH group (7-9). The use of isomeric phenyl- was isolated from E. colh by a published method (22); the factor alanyl-tRNAs has also permitted the study of positional speci- was shown to have a molecular weight of approximately 42,000 ficities involved in the binding of (N-acetyl)phenylalanyl-tRNA and to be free of nuclease activities. to the ribosomal A- and P-sites and in participation of phenyl- Preparation of Modified E. coli tRNAs. E. coli tRNA alanyl-tRNA as an acceptor in the peptidyltransferase reaction species 2a and 3a (Fig. 1) were prepared as described (2,S9). E. (2,4, 5). coli tRNA (2000 A260 units) was digested with 200Mig of venom Elongation factor Tu (EF-Tu) forms a ternary complex with endonuclease for 30 min. (One A260 unit is that amount of GTP and aminoacyl-tRNA and is essential for proper binding material that gives an absorbance of 1 when dissolved in 1 ml of solvent when the light path is 1 cm.) The recovered tRNA Abbreviations: tRNA-C-CoH, tRNA missing the 3'-terminal adenosine was applied to a DBAE-cellulose column (1.4 x 28 cm) that had moiety; DBAE-cellulose, N-[N'-(m-dihydroxyborylphenyl)succi- been equilibrated with 50 mM morpholine-HCl, pH 8.7, con- namyljaminoethyl-cellulose; nucleoside Q, 7-(4,5-cis-dihydroxy-1- cyclopenten-3-ylaminomethyl)-7-deazaguanosine; Na+-Mes, sodium taining 1 M NaCl and 0.1 M MgCl2. The column was washed 2-(N-morpholino)ethanesulfonate; NH4+-Pipes, ammonium pipera- with 400 ml of the same buffer at 250 to remove those zine-N,N'-bis-(2-ethanesulfonate); EF-Tu, elongation factor Tu; 1 venom-treated tRNAs containing no more than one cis-diol A260 unit, that amount of material that gives an absorbance of 1 when moiety; tRNAs containing 7-(4,5-cis-dihydroxy-1-iyclopen- dissolved in 1 ml of solvent when the light path is 1 cm. ten-3-ylaminomethyl-7-deazaguanosine (nucleoside Q) were 437 Downloaded by guest on September 30, 2021 438 Biochemistry: Hecht et al. Proc. Nati. Acad. Sci. USA 74 (1977) NH2 tRNA-CCCcQ H OR a, R=H 2R HR 2 b. R=FCHR' 3 FIG. 1. Isomeric aminoacyl-tRNAs. recovered by elution with 50 mM Na+-Mes [sodium 2-(N- RESULTS morpholino)ethanesulfonate], pH 5.5, containing 1 M NaCI. Modified tRNAs were prepared by treatment of unfractionated The venom-treated tRNAs lacking and containing nucleoside E. coli tRNAs with venom exonuclease until the tRNA was Q (23) were reconstituted separately with CTP and yeast without significant acceptor activity for phenylalanine, aspartic CTP(ATP):tRNA nucleotidyltransferase to afford the respective acid, or asparagine. The venom-treated tRNAs were then ap- abbreviated tRNAs. Portions of the two tRNA-C-COH samples plied to a DBAE-cellulose column that had been equilibrated were further treated with 2'- or 3-'dATP and CTP(ATP):tRNA with 50mM morpholine-HCl, pH 8.7, containing 1 M NaCl and nucleotidyltransferase to afford tRNA species 2a and 3a, re- 0.1 M MgCl2. The column was washed with the same buffer spectively. The modified tRNAs containing nucleoside Q were solution at room temperature, thus effecting elution of all purified by chromatography on DEAE-cellulose and then on tRNAs not containing nucleoside Q. The remaining four species acetylated DBAE-cellulose, elution with 50 mM morpholine- (tRNAAsP, tRNAASn, tRNAHis, and tRNATYr) containing nu- HCl buffer, pH 8.7, containing 1.0 M NaCl and 0.1 M MgCl2. cleoside Q, which has a css-diol moiety, were washed from the Unreacted tRNA-C-COH was recovered by elution with 50 mM column with 50 mM Na+-Mes buffer, pH 5.5, containing 1 M Na+-Mes, pH 5.5, containing 1.0 M NaCI. The modified tRNA NaCl. The two types of tRNAs (i.e., with and without nucleoside species (2a and 3a) derived from tRNAs not containing nucle- Q) were then treated separately with CTP and CTP(ATP): oside Q were purified on DEAE-cellulose and then applied to tRNA nucleotidyltransferase to afford the respective abbre- DBAE-cellulose columns that had been preequilibrated at 40 viated tRNAs (tRNA-C-COH), and portions of each were further with 50 mM morpholine.HCl buffer, pH 8.7, containing l.0-M treated with 2'- and 3'-deoxyadenosine 5'-triphosphate and NaCl, 0.1 M MgCl2, and 20% dimethylsulfoxide. Elution with yeast CTP(ATP):tRNA nucleotidyltransferase to afford tRNA the same buffer afforded purified tRNA species 2a and 3a, and species 2a and 3a derived from the two types of tRNA. Final unreacted tRNA-C-C~owas recovered by washing at 40 with purification of the tRNAs was effected by chromatography on 50 mM Na+-Mes (pH 5.5) containing 1 M NaCl. (acetylated) DBAE-cellulose; since the conditions required to Deacylation of Aminoacyl4RNAs. The aminoacylation separate tRNA species lacking cis-diols from those containing mixture consisted of 360 ,ul (total volume) of 13 mM NH4+- one such group (e.g., tRNAPhe species 2a from tRNAPh'-C-CoH) Pipes buffer [ammopium piperazine-N,N'-bis-(2-ethanesul- are different from those used to separate species containing one fonate)], pH 7.0, containing 0.13 M KC1, 20 mM MgCl2, 0.7 mM and two cis-diol groups (e.g., tRNAHiS species 2a from tRNA- EDTA, 1.5-8 ,uCi of 3H-labeled amino acid at 2-10 MM con- His-C-COH), the initial fractionation of the venom-treated centration, 8 MM of'each of 18 other unlabeled amino acids tRNAs on DBAE-cellulose facilitated purification of the (except cysteine) to prevent misacylations (24), 2 mM ATP, and modified tRNAs.