Proc. Nat. Acad. Sci. USA Vol. 69, No. 12, pp. 3602-3605, December 1972

Release of Polypeptide Chain Initiation Factor IF-2 During Initiation Complex Formation ( synthesis/E. coli/GTP hydrolysis//fMet-tRNA) ARTHUR H. LOCKWOOD, PROBIR SARKAR, AND UMADAS MAITRA Department of Developmental Biology and , Division of Biological Sciences, Albert Einstein College of Medicine, Bronx, New York 10461 Communicated by B. L. Horecker, October 3, 1972

ABSTRACT Polypeptide chain initiation factor IF-2 electrophoresis. Salt-washed ribosomes were prepared from E. binds to 30S ribosomal subunits. This binding is enhanced ribosomes exist primarily as dissociated sub- by IF-1 and IF-3. During GTP-dependent formation of a coli Q13. Such 70S initiation complex, IF-2 is released from the . units. The preparation of f [3H]Met-tRNA, as well as the During 70S initiation complex formation dependent on source of all reagents, has been described (9). the methylene analogue of GTP, GMPPCH2P, IF-2 is not released, but remains bound to the 70S ribosome. This re- Assay of fMet-tRNA and IF-2 Binding to Ribosomes. Reac- sult suggests that IF-2 release requires GTP hydrolysis. tion mixtures (0.125 ml) contained 50 mM Tris HCl (pH 7.8), In agreement with this presumed requirement, IF-2 80 mM NH4CI, 10 mM magnesium acetate, and 4 mM functions catalytically with GTP, but stoichiometrically 2-mercaptoethanol. In addition, the complete system con- with GMPPCH2P, in bringing about 70S initiation complex formation. tained 2 mM GTP, 3 A2w units of salt-washed ribosomes, in- itiation factors (usually 0.15 ,ug of IF-1, 0.15 ,ug of IF-3, and IF-2, one of the bacterial polypeptide chain initiation factors, 0.05 Mg of IF-2), 0.04 A260 units of poly(U,G) [base ratio 3: 1], is essential for the binding of both GTP and fMet-tRNA in a and 1 A200 unit of f[3H ]Met-tRNA (containing about 40 pmol 30S initiation complex (1). This complex contains a 30S ribo- of methionine, of which 20 pmol were present in fMet-tRNA). somal subunit, mRNA, fMet-tRNA, and GTP (2,3). The com- Omission or replacement of components or variation in their bination of a 50S-subunit with the 30S complex results in the amounts are indicated. After incubation for 15 min at 250, hydrolysis of GTP to GDP and Pi, concomitant with the for- the reaction mixtures were chilled and diluted with 3 ml of mation of a functional 70S initiation complex (2, 3). It has cold reaction buffer. f[3H ]Met-tRNA bound to ribosomes was become clear that IF-2 is responsible for hydrolysis of GTP in determined by Millipore filtration (10). Alternatively, 0.1 ml the initiation process. In fact, under conditions uncoupled of the reaction mixture was layered onto a 5-ml linear 5-20% from initiation, IF-2 catalyzes the hydrolysis of GTP to GDP (w/v) sucrose gradient containing reaction buffer, and centri- and Pi in the presence of both ribosomal subunits (4, 6). This fuged for 75 min at 55,000 rpm at 40 in a Spinco SW65 rotor. activity can be coupled to initiation complex formation (6). 0.2-ml fractions were collected and radioactivity was deter- Hence, IF-2 participates in the formation of a 30S initiation mined by counting aliquots in Bray's counting solution. The complex, as well as the subsequent transformation of this location of ribosomes was determined by monitoring the ab- species into a 70S initiation complex. sorbancy at 260 nm. Gradient fractions were assayed for IF-2 Recent studies have demonstrated that IF-2 functions activity by addition of 50-,ul aliquots to the complete reaction stoichiometrically during 30S initiation complex formation, mixture described above, from which IF-2 had been omitted. but catalytically during 70S initiation complex formation After incubation for 30 min at 370, reaction mixtures were (2, 7), i.e., in the presence of both 30S and 50S subunits. chilled and diluted with 3 ml of cold reaction buffer. Bound Hence, it would appear likely that IF-2 binds to a 30S subunit f [3H ]Met-tRNA was determined by Millipore filtration (10). and is released upon addition of a 50S ribosomal subunit. The Under these conditions, fMet-tRNA binding to ribosomes was released factor would then be able to catalyze another round completely dependent on added IF-2. of initiation. Examination of crude cell extracts reveals that IF-2 activity is localized on 30S subunits (8), and that no IF-2 RESULTS (nor other initiation factors) are found on 70S ribosomes. The ability of increasing amounts of IF-2 to promote poly- In this communication, we provide a direct demonstration (U,G)-dependent formation of a 70S initiation complex was that IF-2 binds to 30S subunits. Upon formation of a 70S in- measured in the presence of either GTP or its nonhydrolyzable itiation complex, IF-2 is released from the ribosomes. In con- analog, 5'-guanylylmethylene diphosphonate (GMPPCH2P) trast to IF-1 and IF-3, release of IF-2 requires the hydrolysis (Fig. 1). In the presence of 2 mM GTP, IF-2 functions cata- of GTP. lytically in binding fMet-tRNA to the initiation complex. For example, 1 pmol of IF-2 causes the binding of 6.8 pmol of MATERIALS AND METHODS fMet-tRNA. In contrast, when 2 mM GMPPCH2P replaces Initiation factors were purified (9) from Escherichia coli MRE- GTP, IF-2 functions in a less than stoichiometric fashion: 1 600. IF-1 and IF-3 were homogeneous, while IF-2 was about pmol of IF-2 causes the binding of only 0.8 pmol of fMet- 90% pure by the criteria of native and Na dodecyl S04 disc-gel tRNA. At all levels of IF-2, only about half as much fMet- 3602 Downloaded by guest on October 1, 2021 Proc. Nat. Acad. Sci. USA 69 (1972) Release of IF-2 from Ribosomes 3603

E 10 0 a. 0 ZB8 0 m 10 4 6 a.

o B z 622 -10

I 5 n 1 2 3 4 5 IF-2 (pmol) FIG. 1. Effect of GTP or GMPPCH2P on IF-2-dependent binding of fMet-tRNA to ribosomes. Reaction mixtures were as in -5 Methods, except that IF-2 was varied as indicated and 25 pmol of f[3H]Met-tRNA was used. Incubation was for 10 min at 37°. 10 (-4*), 2 mM GTP; (O-O), 2 mM GMPPCH2P.

cy5~ ~ ~ ~ tRNA is bound (on a molar basis) as IF-2 is added. The in- ability of IF-2 to bring about even a stoichiometric amount of 5 10 15 20 fMet-tRNA binding in the presence of GMPPCH2P could be FRACTION NUMBER due to the presence of a substantial fraction of the ribosome FIG. 2. Association of IF-2 with 30S subunits. 1 ,Ag of IF-2 preparation that can bind IF-2, but can not participate in was used and centrifugation was for 65 min at 55,000 rpm. Cen- overall initiation complex formation. Alternatively, the com- trifugation is from right to left. (A) 1 ,ug of IF-2; (B) 1 jg of IF-2 plex formed with GMPPCH2P could be somewhat unstable to and 3 A260 units of ribosomes; (C) 1 gg of IF-2, 3 A260 units of ribo- Millipore filtration. These results suggest that, in the pres- somes, 0.3 jug of IF-1, and 0.3 ,ug of IF-3. ence of GTP, IF-2 dissociates from the ribosome at some point in the initiation process. Released IF-2 could then recycle to (Fig. 3B). In contrast, IF-2, which was originally bound to the catalyze further rounds of initiation. In the presence of 30S subunit, is now found mainly at the position of free fac- GMPPCH2P, IF-2 does not appear to dissociate, but remains tor. In addition, no IF-2 is present in the 70S region of the bound to the initiation complex. gradient (Fig. 3B). Hence, it is evident that IF-2 is released Consequently, we directly determined the binding and re- from the ribosome upon formation of a 70S initiation complex. lease of IF-2 by sucrose gradient analysis. IF-2 promotes the Because IF-2 cannot act catalytically in- the presence of formation of the 30S initiation complex. Hence, we first ex- GMPPCH2P, it was of interest to determine whether IF-2 amined the interaction of IF-2 with ribosomes (Q13, mainly 30S and SOS subunits) under conditions where 70S complex z formation does not occur, i.e., in the absence of fMet-tRNA 0 and mRNA. In the absence of ribosomes, IF-2 sediments as a 0 70S 50S 30S IF-2 single peak near the top of the gradient (Fig. 2A). If IF-2 is cL incubated with ribosomes at 25° before centrifugation, some 4a- I I IiE a. IF-2 activity is detectable in the 30S region (Fig. 2B). How- cr 10 1000 there is 11 ever, considerable trailing toward the position of free 4 factor. When the incubation mixture also contains IF-1 and zw E IJ 5 500 IF-3, most of the IF-2 activity sediments with the 30S subunit 0 (Fig. 2C). In no instance is IF-2 activity found in the 50S or =

70S region of the gradient. Hence, IF-2 binds to 30S ribosomal B subunits; this binding is strongly stabilized by the other two initiation factors. 4 I We next determined that the factor is released during 70S E 10 1000 'I. 0a- initiation complex formation and what conditions are required us for release to occur. IF-2 was first incubated with all the com- I- 5 500 ponents necessary for GTP-dependent initiation except Poly(UG). Under these conditions an initiation complex does not form, as shown by the absence of f [3H]Met-tRNA in the CL 5 10 15 20 30S or 70S region of the gradient (Fig. 3A). Considerable IF-2 FRACTION NUMBER is associated with the 30S ribosomal subunit, in agreement FIG. 3. Binding of IF-2 to ribosomes during GTP-dependent with the results presented in Fig, 2. Addition of poly(UG) 70S initiation complex formation. 1 ug of IF-2 was used. (A) allows substantial formation of a 70S initiation complex, as Without poly(UG); (B) 0.04 A260 unit of poly(U,G); (0 0), shown by the presence of f[3H ]Met-tRNA in the 70S region IF-2 activity; (O- - -0), f['H] Met-tRNA. Downloaded by guest on October 1, 2021 3604 Biochemistry: Lockwood et al. Proc. Nat. Acad. Sci. USA 69 (1972) 70S ribosome. These results account for the observation that

z IF-2 acts catalytically with GTP, but stoichiometrically with 0 F 10 1000 GMPPCH2P. U. It is likely that IF-2 is not released from the ribosome until LL . the 30S initiation complex accepts a 50S subunit. This conclu- w 5 500 sion follows from the observations that (i) IF-2 functions ca.0w stoichiometrically in 30S complex formation with either GTP 0 or GMPPCH2P (2); (ii) GTP is intact in the 30S complex. z0 a.E 49 U The IF-2-dependent hydrolysis of GTP occurs only upon of experiments with iso- q 10 1000 < addition 50S subunits (2, 3). Further z lated subunits are necessary to define the requirements for IF-2 binding to 30S ribosomes.- PI1 500 The failure of IF-2 to dissociate from the ribosome during to GMPPCH2P-dependent initiation suggests that release occurs E 1- only subsequent to, or concomitant with, GTP hydrolysis. 500- This mechanism is in contrast to the behavior of IF-1 and > 10 1000 IF-3. Both bind to 30S subunits, IF-1 in the presence of IF-2 I- (11, 12) and IF-3 in the absence of other components (13). IF-1 is released upon formation of a 70S complex with either U.1 500 GTP or GMPPCH2P (12, 13), while IF-3 is released whenever a 70S couple per se is formed-e.g., by elevation of the Mg2+ I I o7 1 ion concentration (13). The requirement for GTP hydrolysis 5 10 15 20 FRACTION NUMBER for IF-2 release is reasonable in view of the absolute require- ment of the IF-2-associated GTPase activity for the presence FIG. 4. Binding of IF-2 to ribosomes during GMPPCH2P- of both ribosomal units (5, 6). dependent formation of 70S initiation complexes. 1 1Ag of IF-2 The 70S complex formed in the presence of GMPPCH2P was used and 2 mM GMPPCH2P replaced GTP. (A) without is unable to transfer fMet into peptide linkage (14). However, poly(U,G); (B) complete; (C) complete plus 2 mM GMPPCH2P as we have discussed elsewhere (2, 7), removal of GMPPCH2P in gradient. (@-4), IF-2 activity; (O--- 0), f[3H]Met- by artificial means reactivates the complex (15). Similarly, tRNA. GTP, bound intact in the 30S complex, can be artificially re- moved without hydrolysis (2, 7). When this is done, formation remains bound to the ribosome when GTP hydrolysis does not of a 70S complex active in peptidyl transfer still ensues upon occur. GTP was replaced by 2 mM GMPPCH2P in reaction addition of 50S subunits. Thus, we feel that the purpose of mixtures containing all the components necessary for initia- GTP hydrolysis during initiation may well be to remove both tion except poly(U,G). The absence of fMet-tRNA from the the nucleotide and IF-2 from the complex, thereby activating 30S or 70S region of gradients indicates that no initiation or unblocking fMet-tRNA for peptidyl transfer. Conversely, complex has formed (Fig. 4A); the majority of IF-2 is detected artificial removal of nucleotide (GTP or GMPPCH2P) without in the 30S region. Upon addition of poly(U,G), a 70S complex hydrolysis may be adequate to bring about release of IF-2 forms, as shown by the binding of f[3H ]Met-tRNA in the from the ribosome. 70S region (Fig. 4B). Strikingly, IF-2 is now found in the The behavior of IF-2 during initiation can be summarized 50S-70S region of the gradient. If 2 mM GMPCCH2P is by the following scheme: included in the sucrose gradient, the binding of IF-2 is stabil- IF-i. IF-3 ized further, resulting in a distinct peak of IF-2 activity at 70S (1) IF-2 + 30S + (GTP or GMPPCH2P) + mRNA i- 0A (Fig. 4C). However, substantial IF-2 activity is still observed IF-2. (GTP or GMPPCH2P) .30S - fMet-tRNA * mRNA in the 50S region. Only a small amount of IF-2 remains bound in the 30S region (Fig. 4C). Preliminary experiments with ra- 30S initiation complex dioactive GMPPCH2P indicate that considerable nucleotide (2) IF-2. GTP * 30S * fMet-tRNA* mRNA + 50S also sediments in the 70S region but, like IF-2, GMPPCH2P trails into the 50S region (data not shown). Thus, in the ab- IF-2 + GDP + Pi + 70S fMet-tRNA-mRNA sence of GTP hydrolysis, IF-2 remains associated with the 70S active 70S initiation complex initiation complex*. (3) IF-2 - GMPPCH2P * 30S * fMet-tRNA* mRNA + 50S

DISCUSSION - 70S* IF-2 - GMPPCH2P - fMet-tRNA * mRNA Polypeptide chain initiation factor IF-2 binds to 30S ribosomal inactive 70S initiation complex subunits. This binding is markedly stabilized by IF-1 and IF-3. During GTP-dependent formation of a 70S initiation The binding and release of IF-2 from ribosomes during in- complex, IF-2 is released from the ribosome. In contrast, during itiation suggests comparison with elongation factors EF-G GMPPCH2P-dependent initiation, IF-2 remains bound to the and EF-Tu. Like IF-2, EF-G and EF-Tu form transient ribo- somal complexes in which GTP participates (IF-2-GTP-70S- * Similar observations have been made by J. Fakunding and fMet-tRNA-mRNA; EF-G-50S-GTP; EF-Tu-GTP-70S- J. W. B. Hershey using a different experimental approach. aminoacyl-tRNA-mRNA)(16-20). Each of the factors has a (Hershey, J. W. B., personal communication). ribosome-dependent GTPase activity (18, 21-23), so that in Downloaded by guest on October 1, 2021 Proc. Nat. Acad. Sci. USA 69 (1972) Release of IF-2 from Ribosomes 3605

each case GTP is hydrolyzed to GDP and Pi and the factor 6. Dubnoff, J. S. & Maitra, U. (1972) J. Biol. Chem. 247, 2876- and nucleotide dissociate from the ribosomes (18, 19, 21, 24). 2883. However, when GTP hydrolysis is prevented by the use of 7. Lockwood, A. H. & Maitra, U. (1972) Fed. Proc. 31, 1079 Abstr. GMPPCH2P (or, with IF-2, absence of the 5OS subunit), each 8. Dubnoff, J. S., Lockwood, A. H. & Maitra, U. (1972) Arch. of the complexes is stabilized (24-26). Biochem. Biophvs. 149.528-540. Furthermore, each of these complexes has a function in pro- 9. Dubnoff, J. S. & Maitra, U. (1971) in Methods Enzymol. 20, tein synthesis that involves interaction with tRNA. IF-2 and 248-260. EF-Tu promote binding of, respectively, fMet-tRNA and 10. Nirenberg, M. W. & Leder, P. (1964) Science 145, 1399-1402. 11. Thach, R. E., Hershey, J. W. B., Kolakosfky, D., Dewey, K. aminoacyl-tRNAs to ribosomes, while EF-G catalyzes ejec- F. & Remold-O'Donnell, E. (1969) Cold Spring Harbor tion of deacylated tRNA from ribosomes and also transloca- Symp. Quant. Biol. 34, 277-283. tion of peptidyl-tRNA (1). 12. Hershey, J. W. B., Dewey, K. F. & Thach, R. E. (1969) Na- These analogies suggest a general function for GTP in pro- ture 222, 944-947. tein synthesis beyond its putative use as an energy source. We 13. Sabol, S. & Ochoa, S. (1971) Nature New Biol. 234, 233-236. postulate that GTP acts as a steric effector that permits as- 14. Kolakofsky, D., Ohta, T. & Thach, R. E. (1968) Nature 220, 244-247. sociation of each of the factors with ribosomes and, directly 15. Benne, R. & Voorma, H. 0. (1972) FEBS Lett. 20, 447-450. or indirectly, with tRNA. The hydrolysis of GTP to GDP dis- 16. Ravel, J. M., Shorey, R. L. & Shive, W. (1968) Biochem. sociates both factor and nucleotide from the ribosome, not per- Biophys. Res. Commun. 32, 9-14. haps in any energy-dependent process, but simply by conver- 17. Lucas-Lenard, J. & Haenpi, A.-L. (1968) Proc. Nat. Acad. sion of one steric effector-GTP-to another-GDP. Sci. USA 59, 554-560. 18. Lockwood, A. H., Hattman, S., Dubnoff, J. & Maitra, U. This work was supported by grants from the National In- (1971) J. Biol. Chem. 246, 2936-2947. stitutes of Health, the American Heart Association, and the Life 19. Skoultchi, A., Ono, Y., Waterson, J. & Lengyel, P. (1969) Insurance Medical Research Fund. A. H. L. is a predoctoral Cold Spring Harbor Symp. Quant. Biol. 34, 437-454. trainee of the National Institutes of Health; U. M. is a recipient 20. Bodley, J. W. & Lin, G. (1970) Nature 227, 60-61. of a Faculty Research Award of the American Cancer Society. 21. Shorey, R. L., Ravel, J. M., Garner, C. W. & Shive, W. 1. For a review see: Lqcas-Lenard, J. & Lipmann, F. (1971) (1969) J. Biol. Chem. 244, 4555-4564. Annu. Rev. Biochem. 40, 409-448. 22. Gordon, J. (1969) J. Biol. Chem. 244, 5680-5686. 2. Dubnoff, J. S., Lockwood, A. H. & Maitra, U. (1972) J. Biol. 23. Nishizuka, Y. & Lipmann, F. (1966) Arch. Biochem. Bio- Chem. 247, 2884-2894. phys. 116, 344-351. 3. Thach, S. S. & Thach, R. E. (1971) Nature New Biol. 229, 24. Bodley, J. W., Zieve, F. J., Lin, L. & Zieve, S. T. (1970) J. 219-221. Biochem. 245, 5656-5661. 4. Kolakofsky, D., Dewey, K. F., Hershey, J. W. B. & Thach, 25. Skoultchi, A., Ono, Y., Waterson, J. & Lengyel, P. (1970) R. E. (1968) Proc. Nat. Acad. Sci. USA 61, 1066-1070. Biochemistry 9, 508-514. 5. Kolakofsky, D., Dewey, K. F. & Thach, R. E. (1969) Nature 26. Brot, N., Spears, C. & Weissbach, H. (1969) Biochem. Bio- 223,694-697. phys. Res. Commun. 34,843-848. Downloaded by guest on October 1, 2021