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 (protein synthesis/E. coli/GTP hydrolysis/ribosomes/fMet-tRNA) ARTHUR H. LOCKWOOD, PROBIR SARKAR, AND UMADAS MAITRA Department of Developmental Biology and Cancer, 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 ribosome. 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.