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Interaction of Elongation Factor 1 with Aminoacylated Brome Mosaic Virus and Trna's MARCEL BASTIN and TIMOTHY C

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Interaction of Elongation Factor 1 with Aminoacylated Brome Mosaic Virus and Trna's MARCEL BASTIN and TIMOTHY C JOURNAL OF VIROLOGY, OCt. 1976, p. 117-122 Vol. 20, No. 1 Copyright 0 1976 American Society for Microbiology Printed in U.S.A. Interaction of Elongation Factor 1 with Aminoacylated Brome Mosaic Virus and tRNA's MARCEL BASTIN AND TIMOTHY C. HALL* Biophysics Laboratory ofthe Graduate School, and Department ofHorticulture,* College ofAgricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706 Received for publication 26 April 1976 Tyrosylated Brome mosaic virus RNA was found to interact with a binary complex of wheat germ elongation factor 1 and [3H]GTP. Increasing amounts of the aminoacylated viral RNA proportionately reduced radioactivity bound to a nitrocellulose filter, as has previously been noted by others for the charged forms of tobacco mosaic virus, turnip yellow mosaic virus, and tRNA's. However, Sephadex chromatography of the products showed that instead of forming the ternary complex elongation factor-GTP-aminoacyl RNA, the viral RNA caused release of GTP from its complex with elongation factor. Acetylated tyrosyl Brome mosaic virus RNA did not react with the binary complex, and only a slight degree, if any, of stabilization of tyrosine bound to viral RNA was observed after interaction with elongation factor 1. Although such interactions are similar to the reaction of elongation factor with aminoacyl-tRNA, the release of GTP is different and accentuates the possible role for aminoacylation in transcription rather than in translation events. Several plant viral RNAs can accept a spe- MATERIALS AND METHODS cific amino acid in a tRNA-like manner. For EF 1 assay. Wheat germ EF 1 will bind GTP to example, the RNA of turnip yellow mosaic vi- form a binary complex, EF 1-GTP, which interacts rus (TYMV) can be esterified with valine by with aminoacyl-tRNA to form the ternary complex Escherichia coli valyl-tRNA synthetase (19, 23, EF 1-GTP-aminoacyl-tRNA (8, 25). The binary com- 29). Tobacco mosaic virus (TMV) RNA can plex is retained on nitrocellulose filters, whereas the serve as a substrate for the yeast histidyl-tRNA ternary complex, or free GTP, passes through. This synthetase (22), and the four RNAs of brome is the basis of a sensitive assay technique wherein mosaic virus (BMV) can accept tyrosine in the complexed radioactive GTP bound to a nitrocellulose presence of a wheat tRNA synthetase (11). membrane filter (Millipore HAWG, 0.45-,um pore size, 25 mm) is measured (6). One unit of activity The significance of aminoacylation of viral was defined as the amount of enzyme needed to RNA is still obscure. The possibility of a tRNA complex 1 pmol of GTP after 10 min ofincubation at (amino acid donor) function for valyl-TYMV 0°C under the following conditions. Partially puri- RNA has been suggested (10), but experiments fied EF 1 was incubated with 400 pmol of [3H]GTP with tyrosyl-BMV RNAs (5, 27) did not provide (Amersham/Searle, activity diluted to 1 uCi/400 evidence for the transfer of the bound amino pmol) in buffer I (10 mM MgCl2-50 mM NH4Cl-10 acid to nascent peptides. More attractive is the mM Tris-hydrochloride, pH 7.5), the final volume proposal of Litvak et al. (20) that aminoacyla- being 200 Al. After 10 min the reaction mixture was tion is related to replicase binding. Bacterial diluted with 1 ml of buffer I and then filtered through a presoaked membrane filter (Millipore elongation factor (EF)-Tu interacts strongly Corp.). The filter was washed four times with 1 ml of with aminoacyl-tRNA (9, 25) and is known to cold buffer I and then dried and counted in a scintil- constitute, like the other elongation factor EF- lation counter with an efficiency of 29%. Ts, one of the four subunits of Q13 replicase (3, Products of the interaction of EF 1, GTP, and 17). Similarly, aminoacylated, but not un- viral RNA were also analyzed by chromatography charged, TMV and TYMV RNAs were shown to on Sephadex G-75. Reaction mixtures were applied bind eukaryotic EF 1 (20), which is functionally to the top of columns (1 by 54 cm) equilibrated with equivalent to bacterial EF-Tu -Ts (7). buffer I. The columns were run at 0°C with a flow In this paper we extend the finding rate of 12 ml/h; fractions of 1 ml were collected. of Litvak Purification of EF 1. Forty grams of wheat germ et al. (20) to the RNAs of BMV, but show that, (General Mills, Vallejo, Calif.) was blended five unlike the interaction of aminoacyl-tRNA with times for 10 s at 2-min intervals with 200 ml of EF 1, the aminoacyl-viral RNA causes the extraction buffer (2 mM MgCl2-2 mM CaCl2-50 mM breakdown of the binary complex EF 1-GTP to KCl-5 mM mercaptoethanol-25 mM Tris-hydrochlo- release the enzyme-bound GTP. ride pH 7.6). The extract was first centrifuged at 117 118 BASTIN AND HALL J.- VIROL. 8,000 rpm for 20 min in a Sorvall type GSA rotor and creasing amounts of tyrosyl-BMV RNA caused then ultracentrifuged at 40,000 rpm (Beckman type a proportional reduction in the 3H radioactivity Ti 60) for 90 min to pellet the ribosomes. The upper retained on the me abrnes (Fig. 1). Nonacy- three-fourths of the supernatant were withdrawn lated RNA completely failed to interact with and made to 65% saturation with (NH4)2S04. The pH the EF 1-GTP complex (Fig. 1), as did BMV was kept at 7.7 by the addition of 1 N NH40H. The proteins were pelleted by centrifugation, suspended RNA with the acetylated tyrosine moiety (Ta- in about 30 ml of buffer II (10 mM KCl-5 mM mer- ble 1). The 3'-terminal fragment of 159 nucleo- captoethanol-5% glycerol-20 mM Tris-hydrochlo- tides, cleaved from BMV RNA 4 by partial ride, pH 7.6), and dialyzed overnight with two digestion and aminoacylated with tyrosine, re- changes ofthe same buffer. The solution (37 ml) was acted with the binary complex as efficiently as centrifuged at 10,000 rpm for 10 min in a Sorvall did the intact RNA (Table 1). Thus, the interac- type SS34 rotor and then applied to a DEAE-cellu- tion of EF 1 with RNA from BMV was similar lose column (2.4 by 27 cm) equilibrated with buffer to that observed by Litvak et al. (20) with TMV II. The void volume (78 ml) was discarded. The and TYMV RNAs. Moreover, EF 1 binding to column was eluted with 180 ml of buffer II contain- same as ing 0.1 M KCl, and then (NH4)2SO4 was added to viral RNA showed the specificity did 65% saturation. After stirring for 30 min, the sus- EF 1 binding to aminoacyl-tRNA (9, 25). pension was centrifuged (10,000 rpm for 15 min in a Sorvall type SS34 rotor). The resulting precipitate was dissolved in a small volume of 0.01 M potassium -2 70C phosphate buffer, pH 7.3, containing 5% glycerol '0 and dialyzed overnight against the same buffer. The solution was applied to an equilibrated carboxy- methyl Sephadex (CM Sephadex, C-50 medium, 100 0 6- 0~~~~~~~ to 270 mesh, Pharmacia Fine Chemicals) column IJE (2.0 by 13.5 cm). The column was washed with about 50 ml of 0.03 M phosphate buffer, pH 7.3, and then 10 0 eluted with a gradient (400 ml) of potassium phos- phate buffer between 0.1 M to 0.3 M and a change of 't_ -1 pH from 7.3 to 8.0. Fractions containing EF 1 activ- S4V RNA (p mol) ity were pooled (87 ml) and concentrated by precipi- tation with (NH4)2SO4 (65% saturation). The protein FIG. 1. Interaction of BMV RNA with the EF 1- wheat germ EF 1 precipitate was finally dissolved in 4 to 5 ml ofbuffer GTP complex. Partially purified was incubated with 400 in II, dialyzed for 2 to 3 h, and stored at -20°C as a pmol of[3H]GTP (1 pACi) mM mM mM solution of 20 mg/ml. Protein was measured by the 10 MgCl2-50 NH4Cl-10 Tris-hydro- in a volume 10 method of Lowry et al. (21). A preparation having a chloride, pH 7.5, final of200 p. After specific activity of 33.9 U/mg of protein (about 8.4- min of incubation at 0°C the mixture was diluted and over a fold purified) was used in this work. The activity with 1 ml of buffer filtered presoaked The reaction was in was stable for several months under these condi- nitrocellulose filter. performed presence concentrations tions and was not affected by freezing or thawing. the of various of uncharged Viral RNA and aminoacylation. BMV (Russian BMV RNA (0) or with tyrosyl-BMV RNA (0); 67% were The results are ex- strain) was propagated in barley leaves and purified of the molecules charged. as a BMV RNA con- by the procedure of Shih et al. (28). RNA was iso- pressed function of tyrosylated lated from purified virus by phenol extraction (4). centration. First assay (0); second assay (O). The 3'-terminal fragment of 159 bases was cleaved from BMV RNA 4 by limited digestion with TABLE 1. Interaction oftyrosylated BMVRNAs with ribonuclease Ti and purified by gel electrophoresis EF 1-[3H]GTPa as described previously (1). The preparation of tRNA synthetase from wheat Amount of germ and the conditions for amino acid binding to [3H]GTP re- Addition tained on Milli- viral RNA have been described (11, 15). Although pore filters the tyrosyl residue bound to BMV RNA is labile to (pmol) alkaline hydrolysis, it can be stabilized by acetyla- tion ofthe a-amino group (1).
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