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

Home , Elongation factor

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 phosphate 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). The acetylated deriva- None 18.15 tive of aminoacylated RNA was prepared by the Tyrosylated 3'-terminal fragment of 1.53 method of Lapidot et al. (18). 159 nucleotides (11.7 pmol) Tyrosylated 3'-terminal fragment of 0.74 159 nucleotides (23.3 pmol) RESULTS Tyrosylated BMV RNA (20.5 pmol) 1.62 Reaction of EF 1 with tRNA's and viral Acetylated tyrosyl-BMV RNA (20 19.27 RNAs. In the absence of viral RNA or tRNA, pmol) EF 1 and [3H]GTP formed a binary complex a The EF 1-GTP complex was formed as described. that was quantitatively retained on the mem- The amount of [3H]GTP bound to EF 1 was deter- brane filter (Millipore Corp.). Addition of in- mined by the Millipore filtration technique. VOL. 20, 1976 AMINOACYL-VIRAL RNA-EF 1 INTERACTIONS 119 Sephadex chromatography of reaction of the 14C radioactivity associated with EF 1 products. The interaction oftyrosyl-BMV RNA was eluted from the column. The reasons for with EF 1-GTP was further analyzed by gel this are not known. Nevertheless, the EF 1- filtration. Sephadex chromatography has been BMV RNA complex was quantitatively re- useful in detecting the formation ofthe ternary tained on nitrocellulose membranes, and hence complex EF 1-GTP-aminoacyl-tRNA (6, 8, 25). the Millipore filtration method could be used Incubation of [3H]GTP in the presence of EF 1, for the detection ofRNA-bound [14C]tyrosine. followed by chromatography on Sephadex G-75, Incubation of [14C]tyrosyl-BMV RNA with resulted in the appearance of 3H radioactivity the EF 1-GTP complex in buffer I at 30°C re- in the void volume (Fig. 2A). The amount of sulted in a first-order deacylation of the RNA radioactivity detected in this manner was con- with a half-life of 9.8 h. A similar deacylation sistent with that retained on the nitrocellulose rate was observed when the incubation was membrane. performed without EF 1-GTP. However, at 0°C However, when the incubation was per- the deacylation rate was decreased, and after 24 formed in the presence of tyrosylated BMV h 27% of the tyrosine bound to viral RNA was RNA, there was a decrease of [3H]GTP in the released, compared with only 9% in the pres- EF 1-GTP complex which eluted in the void ence ofthe EF 1-GTP complex. Thus, it is possi- volume. A concentration of some 20 pmol of ble that some stabilization of the aminoacyl tyrosyl-BMV RNA, which virtually eliminated bond results from the interaction ofaminoacyl- the binding of the EF 1-GTP complex to the ated viral RNA with EF 1, but not to the same membrane filter (Table 1), caused a complete extent as has been found for aminoacylated disappearance of [3H]GTP counts from the void tRNA after reaction with EF 1-GTP (2). volume (Fig. 2B). Analysis of material filtered through the nitrocellulose membrane on Seph- adex G-75 revealed that all the 3H radioactivity DISCUSSION eluted with the free GTP peak (Fig. 2C). Thus, The interaction of tyrosylated BMV RNA instead of forming a stable ternary complex with the EF 1-GTP complex resembled that of analogous to the EF 1-GTP-aminoacyl-tRNA aminoacyl-tRNA, since neither nonaminoac- complex, the aminoacylated viral RNA caused ylated RNA nor acetylated aminoacyl RNA re- a breakdown of the binary complex EF 1- acted with the complex. Also, a slight degree of [3H]GTP. No release of [3H]GTP was observed stabilization of the aminoacyl bond of tyrosyl- by treating the binary complex either with non- BMV RNA was indicated after being complexed aminoacylated BMV RNA (Fig. 2D) or with with EF 1. However, instead offorming a stable acetylated tyrosyl-BMV RNA (Fig. 2E). ternary complex analogous to those found with To see if the release of GTP from the binary aminoacyl-tRNA (9, 25), the aminoacyl-viral complex was a genuine property of the amino- RNA caused the breakdown of the binary com- acyl-viral RNA, or perhaps caused by an unu- plex EF 1-GTP. The same phenomenon has sual behavior of the wheat elongation factor been observed withE. coli EF-Tu-GTP complex preparation, a similar experiment was per- and chemically synthesized 2'(3')-O-amino- formed with wheat germ tyrosyl-tRNA. Figure acyldinucleotide phosphates (CpA-Phe, CpA- 2F shows a typical elution pattern of the ter- Pro, and CpA-Asp) with the nucleotide se- nary complex EF 1-[3H]GTP-tyrosyl-tRNA quence of the 3'-terminus of aminoacyl-tRNA from a Sephadex G-75 column. Unlike the in- (26). We did not examine if the released GTP teraction with aminoacylated viral RNA, there was hydrolyzed, but in the case of GTP release was no reduction of tritiated nucleotide in the by interaction with the aminoacyldinucleotides void volume, even when higher concentrations no hydrolysis of GTP has been detected (S. of tyrosyl-tRNA were used. On the contrary, Chladek, personal communication). but in agreement with Ravel et al. (24), it was As in the case ofthe aminoacyldinucleotides, observed that aminoacyl-tRNA improved the the specificity of the interaction indicated that recovery of the EF 1-GTP complex after chro- the aminoacylated viral RNA reacted with the matography on Sephadex. elongation factor site where the 3'-terminus of Stability of aminoacyl bond after complex- aminoacyl-tRNA would normally bind. The ing with EF 1. The stability of the aminoacyl release of the enzyme-bound GTP may result bond of tyrosyl-BMV RNA after interaction from an unstable ternary complex EF 1-GTP- with the EF 1-GTP complex was investigated. tyrosyl-BMV RNA, owing to the size or second- Several attempts were made to elute an EF 1- ary structure of the viral RNA. ['4C]tyrosyl-BMV RNA complex from Sephadex Under our experimental conditions, the in- G-75. However, although [14C]tyrosyl BMV teraction between tyrosyl-BMV RNA and EF 1- RNA eluted in the void volume, only a fraction GTP revealed a stoichiometry of about 0.6:1 taj

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120 VOL. 20, 1976 AMINOACYL-VIRAL RNA-EF 1 INTERACTIONS 121 (Fig. 1). This is lower than the interaction with rosyl ribonucleic acid as amino acid donors in protein synthesis. Biochemistry 12:4570-4574. authentic aminoacyl-tRNA (stoichiometry of 6. Ertel, R., N. Brot, B. Redfield, J. E. Allende, and H. 1:1) but is considerably more effective than the Weissbach. 1968. Binding of guanosine 5'-triphos- reaction with 2'-O-aminoacyldinucleotides (26). phate by soluble factors required for polypeptide syn- In this case, the interaction of the most effec- thesis. Proc. Natl. Acad. Sci. U.S.A. 59:861-868. 7. Golinska, B., and A. B. Legocki. 1973. Purification and tive compound (CpA-Phe) with EF Tu-GTP was some properties of elongation factor 1 from wheat only observed at concentrations above 1 ,uM germ. Biochim. Biophys. Acta 324:156-170. (2,000 pmol of compound reacting with 38.5 8. Gordon, J. 1967. Interaction of guanosine 5'-triphos- pmol of EF Tu-GTP). The size of the RNA as phate with a supernatant fraction from E. coli and aminoacyl-sRNA. Proc. Natl. Acad. Sci. U.S.A. well as the concentration used appeared to be 58:1574-1578. important factors, since neither UCCACCA- 9. Gordon, J. 1968. A stepwise reaction yielding a complex Ala (110 nM) (14) nor the 3' halfofE. coli valyl- between a supernatant fraction from E. coli, guano- tRNA (50 nM) interacted with EF-Tu-GTP sine 5'-triphosphate, and aminoacyl-sRNA. Proc. (16). Natl. Acad. Sci. U.S.A. 59:179-183. Although the involvement of other factors 10. Haenni, A-L., A. Prochiantz, 0. Bernard, and F. cannot be ruled out, it appears that the pres- Chapeville. 1973. TYMV valyl-RNA as an amino acid ence of an amino acid at the 3' end of the viral donor valine in protein biosynthesis. Nature (London) RNA may be the only requirement for its inter- New Biol. 241:166-168. 11. Hall, T. C., D. S. Shih, and P. Kaesberg. 1972. Enzyme- action with the EF 1-GTP, complex. Neverthe- mediated binding of tyrosine to brome mosaic virus less, this interaction is as specific as that of ribonucleic acid. Biochem. J. 129:969-976. authentic aminoacyl-tRNA and allows the spe- 12. Hall, T. C., and R. K. Wepprich. 1976. Functional possi- cific binding ofviral RNA to eukaryotic elonga- bilities for aminoacylation of viral RNA in transcription and translation. Ant. Microbiol. Inst. Pasteur tion factor. By analogy with the involvement of 127A:143-152. bacterial elongation factors of Q,B RNA replica- 13. Happe, M., and H. Jockusch. 1975. Phage Q,B replicase: tion (13), this may be an essential step in the cell-free synthesis of the phage-specific subunit and replication of plant RNA viruses (12, 20). Cer- its assembly with host subunits to form active enzyme. Eur. J. Biochem. 58:359-366. tainly, the difference between the formation of 14. Kawakami, M., S. Tanada, and S. Takemura. 1975. a ternary complex when EF 1-GTP reacts with Properties of alanyl-oligonucleotide, puromycin, and aminoacylated tRNA, and the release of GTP Staphylococcus epidermidis glycyl-tRNA in interac- on addition of aminoacylated viral RNA to EF tion with elongation factor Tu-GTP complex. FEBS Lett. 51:321-324. 1-GTP strongly argues against a translational 15. Kohl, R. J., and T. C. Hall. 1974. Aminoacylation of role for the interaction of viral RNA with elon- RNA from several viruses: amino acid specificity and gation factor. differential activity of plant, yeast, and bacterial synthetases. J. Gen. Virol. 25:257-261. 16. Krauskopf, M., C-M. Chen, and J. Ofengand. 1972. ACKNOWLEDGMENTS Interaction of fragmented and cross-linked Esche- This work was supported by National Science Founda- richia coli valine transfer ribonucleic acid with Tu tion grant BMS 73-07008, Public Health Service grants AI- factor-guanosine triphosphate complex. J. Biol. 11572, AI-01466, and AI-21942 from the National Institute of Chem. 247:842-850. Allergy and Infectious Diseases, CA-15613 from the Na- 17. Landers, T. A., T. Blumenthal, and K. Weber. 1974. tional Cancer Institute, and contract AT-11-1-1033 from the Function and structure in ribonucleic acid phage Q,B Biology Division of the Energy Research and Development ribonucleic acid replicase. The roles of the different Administration. subunits in transcription of synthetic templates. J. We thank P. Kaesberg and W. Zagorski for their advice Biol. Chem. 249:5801-5808. and encouragement. 18. Lapidot, Y., N. De Groot, and I. Fry-Shafrir. 1967. Modified aminoacyl-tRNA. II. A general method for the preparation of acylaminoacyl-tRNA. Biochim. Biophys. Acta 145:292-299. LITERATURE CITED 19. Litvak, S., D. S. Carrm, and F. Chapeville. 1970. TYMV 1. Bastin, M., R. Dasgupta, T. C. Hall, and P. Kaesberg. RNA as a substrate ofthe tRNA nucleotidyltransfer- 1976. Similarity in structure and function of the 3'- ase. FEBS Lett. 11:316-319. terminal region ofthe four brome mosaic viral RNAs. 20. Litvak, S., A. Tarrag6, L. Tarrago-Litvak, and J. E. J. Mol. Biol. 103:737-745. Allende. 1973. Elongation factor-viral genome inter- 2. Beres, L., and J. Lucas-Lenard. 1973. Studies on the action dependent on the aminoacylation of TYMV fluorescence of the Y base of yeast phenylalanine and TMV RNAs. Nature (London) New Biol. 241:88- transfer ribonucleic acid. Effect of pH, aminoacyla- 90. tion, and interaction with elongation factor Tu. Bio- 21. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. chemistry 12:3998-4002. Randall. 1951. Protein measurement with the Folin 3. Blumenthal, T., T. A. Landers, and K. Weber. 1972. phenol reagent. J. Biol. Chem. 193:265-275. Bacteriophage Qf8 replicase contains the protein bio- 22. Oberg, B., and L. Philipson. 1972. Binding of histidine synthesis elongation factors EF Tu and EF Ts. Proc. to tobacco mosaic virus RNA. Biochem. Biophys. Res. Natl. Acad. Sci. U.S.A. 69:1313-1317. Commun. 48:927-932. 4. Bockstahler, L. E., and P. Kaesberg. 1965. Isolation 23. Pinck, M., P. Yot, F. Chapeville, and H. M. Duranton. and properties ofRNA from bromegrass mosaic virus. 1970. Enzymatic binding of valine to the 3' end of J. Mol. Biol. 13:127-137. TYMV-RNA. Nature (London) 226:954-956. 5. Chen, J. M., and T. C. Hall. 1973. Comparison oftyrosyl 24. Ravel, J. M., R. L. Shorey, S. Froehner, and W. Shive. transfer ribonucleic acid and brome mosaic virus ty- 1968. A study ofthe enzymatic transfer to aminoacyl- 122 BASTIN AND HALL J. VIROL. RNA to Escherichia coli ribosomes. Arch. Biochem. 27. Shih, D. S., P. Kaesberg, and T. C. Hall. 1974. Messen- Biophys. 125:514-526. ger and aminoacylation functions of brome mosaic 25. Ravel, J. M., R. L. Shorey, and W. Shive. 1967. Evi- virus RNA after chemical modification of 3' termi- dence for a guanine nucleotide-aminoacyl-RNA com- nus. Nature (London) 249:353-355. plex as an intermediate in the enzymatic transfer to 28. Shih, D. S., L. C. Lane, and P. Kaesberg. 1972. Origin aminoacyl-RNA to ribosomes. Biochem. Biophys. of the small component of brome mosaic virus RNA. Res. Commun. 29:68-73. J. Mol. Biol. 64:353-362. 26. Ringer, D., and S. Chlidek. 1975. Interaction ofelonga- 29. Yot, P., M. Pinck, A-L. Haenni, H. M. Duranton, and tion factor Tu with 2'(3')-O-aminoacyloligonucleo- F. Chapeville. 1970. Valine-specific tRNA-like struc- tides derived from the 3' terminus of aminoacyl- ture in turnip yellow mosaic virus RNA. Proc. Natl. tRNA. Proc. Natl. Acad. Sci. U.S.A. 72:2950-2954. Acad. Sci. U.S.A. 67:1345-1352.