- Email: [email protected]
[39] Function of the ribosomal protein S1 in initiation and elongation of bacterial protein synthesis
426
INITIATION OF PROTEIN SYNTHESIS
[39]
Remarks
There are two procedures available to isolate the ribosomal protein S l, starting either from 30 S ribosomal subunits or from 70 S ribosomes. If a zonal rotor is available and an effective separation of ribosomal subunits has been worked out, the 30 S method is the procedure of choice. From about 100 g ofE. coli (wet weight), roughly 10 mg of S 1 in about 90% purity may be obtained in 6 working days. Starting from 70 S, the procedure is straightforward, using only simple chromatographic procedures; the yields, however, are lower, and S I may still be contaminated with other ribosomal proteins. Aside from that, many oppose prolonged urea treatment in the isolation of proteins. The material eluted in the void volume of the DEAE column (Fig. 2A) contains other poly(U) binding proteins, but no S1.
[39] F u n c t i o n o f t h e R i b o s o m a l P r o t e i n S 1 in Initiation and Elongation of Bacterial Protein Synthesis By N. Q. KHANH, R. LINDE, U. MANDERSCHIED, and H. G. GASSEN
The ribosomal protein S1 is required for the translation of natural messengers like MS2 RNA from phage. Although its role in the initiation step of protein synthesis is well documented and partially understood on a theoretical basis, its function in the elongation cycle is evident but remains to be clarified from a mechanistic point of view. Protein S I can form a specific 30 S-mRNA initiation complex without prior binding of fMet-tRNA Met-GTP'IF-2 to the 30 S s u b u n i t / D a t a from different laboratories confirm that S1 is required for MS2 or RI7 RNAdependent flVlet-tRNA binding, but not for the AUG-dependent stimulation of fMet-tRNA binding to ribosomes? '4 However, discrepancies in the molecular interpretation still exist. Van Dieijen et al. have postulated that S1 recognizes an initiation type of tertiary structure of the m R N A / whereas others have concluded from their experiments that S 1 binds to the 3' end of the 16 S RNA, thereby changing its conformation so that a l For a recent review, see J. Steitz, "Biological Regulation and C o n t r o l " Plenum, N e w York, in press. 2 G. V a n Dieijen, P. H. V a n Knippenberg, and J. Van Duin, Fur. J. Biochem. 64, 511 (1976). W. Szer, J. M. H e r m o s o , and S. Leffler, Proc. Natl. Acad. Sci. U.S.A. 72, 2325 (1975). 4 j. E. Sobura, M. R. C h o w d h u r y , D. A. Hawley, and A. J. Wahba, Nucl. Acids Res. 4, 17 (1977).
M E T H O D S 1N E N Z Y M O L O G Y , VOL. LX
Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181960-4
[39]
FUNCTION OF PROTEIN S I
427
recognition-type double-strand formation with the intercistronic sequence of a mRNA can occur? The examination of S l function in elongation proves to be more difficult because defined functions like coded AA-tRNA binding or translocation are not lethally affected by omission of S 1, but can be restored in higher Mg "+ concentrations2 Furthermore, interpretations are obscured by the fact that most components of the assay, i.e., for the poly(U)-dependent poly(Phe) synthesis, 30 S and 50 S subunits or the 100,000 g supernatant (SI00) are contaminated with S1. Thus, a comparison of the data from different laboratories is hampered by the fact that the so-called S 1-depleted 30 S ribosomes, 30 S(-S 1), are prepared by nonstandardized methods and that a S100 is used as an enzyme source, where SI has been inactivated by the addition of anti-S 1 IgG. The use of these techniques has led to widespread variation in experimental results and lively discussions on their interpretations in terms of S1 function. Tests described in the following were therefore done with anti-S1 IgG depleted 30 S(-S l) ribosomal subunits and purified initiation and elongation factors. Materials
Buffer I: 50 mM Tris. HCI, pH 7.5,200 mM NH4CI, 20 mM MgCI2, 2 mM DTE Buffer II: 10 mM Tris-HCI, pH 7.2, 10 mM MgCI2, 50 mM NH4Cl Buffer III: 10 mM Tris.HCl, pH 7.6, 15 mM MgCI2 Mix 1:500 mM Tris-HCI, pH 7.8, 1M NH4CI, 40 mM Mg(OAc)z, 10 mM DTE, 10 mM GTP Mix 2:100 mM Tris .HCI, pH 7.2, 40 mM MgClz, 300 mM NH4C1, 10 mM mercaptoethanol Mix 3:100 mM Tris. HC1, pH 7.5,400 mM NH4C1, 40 mM Mg(OAc)z, 2 mM GTP, l0 mM ATP, 25 mM PEP, 2 mM DTE, 0.1 mg/ml pyruvate kinase Protein S1 dissolved in 80 mM Tris .HCI, pH 7.4, l/zl ^ 23 pmol of S l, EF-Tu.GDP (1 /~1 ~ 40 pmol), EF-G (1 p.l = 24/~g) tRNA from E. coli was charged to 2.5% with [:~H]Met (specific activity, 1000 Ci/mol, and 100% formylated f[aH]Met-tRNA Charging, with [aH]Phe (specific activity, 1000 Ci/mol) was 1.8%, of tRNA Polyuridylate, 18 Ae~0 units/ml, chain length 30-50, U4, 120 A2,o units/ml [aH]polyuridylate, 5 A.,~0 units/ml; specific activity 6 Ci/mol per pU residue '~ A. E. Dahlberg and J. E. Dahlberg, Pro('. Natl. Acad. Sci. U.S.A. 72, 2490 (1975). G. Van Dieijen, C. J. Van der Laken, P. H. Van Knippenberg, and J. Van Duin, J. Mol. Biol. 93, 351 (1975).
428
INITIATION OF PROTEIN SYNTHESIS
[39]
MS2 RNA, 100 A26o units/ml (Boehringer) Sepharose 4B CNBr-activated, Sephadex A-50, Sephadex G-150 (Pharmacia) Dithioerytritol (DTE) (Serva, Heidelberg) Nitrocellulose filter BA85, 2.5 cm, 0.45/xm (Schleicher & Schfill) Glass-fiber filter GF/A 2.5 cm (Whatman) P r e p a r a t i o n of the C o m p o n e n t s for the T e s t Systems The 70 S ribosomes, 30 S subunits, and protein S1 are prepared as described in this volume [38], following published procedures. Initiation factors IF-l.3 and IF-2 and elongation factors EF-Tu and EF-G are prepared as described. 7,s The proteins are free of nucleases examined by a procedure given in this volume [38]. Polyuridylate and oligouridylates are synthesized with polynucleotide phosphorylase (EC 2.7.7.8). 30 S(-S1) subunits are prepared by four different methods and compared according to function: low-salt treatment [Tal's procedure 9 (I)], repeated washings with 1M NH4C1 [Wahba's procedure 1°(II)], separation of 30 S(-S l) from 30 S by chromatography over Sepharose 6B equilibrated with 1 M NH4CI [our procedure (liD], and removal of S 1 by chromatography of the ribosomes over a Sepharose 4B (anti-S! IgG) column [our procedure (IV)]. I. A fast working and effective procedure, which removes up to 90% of S1 from 30 S ribosomes and minor amounts of other ribosomal components. 30 S(-S l) prepared by this method have a high residual activity in the poly(U)-dependent Phe-tRNA binding. II. This procedure removes Sl to 100%, but many other ribosomal proteins, too. 30 S(-S1) cannot be reconstituted into active 30 S by the addition of S1 (at least not in our hands). Ill. A fast and effective procedure for the isolation of smaller amounts of 30 S(-S l) (100 A 260units per run), but reconstitution is only 40-50% in the poly(U)-Phe-tRNA binding test. IV. In our laboratory, the method of choice. S 1 is removed by more than 90%, and the subunits can be reconstituted to 100% by addition of Sl. A limitation of the procedure lies in the low capacity of the anti-Sl IgG column and the fast inactivation of the ribosomes. The experiments described in the following section were done with 30 S(-S1) prepared by method IV. r D. P. Suttle, M. A. Haralson, and J. M. Ravel,Biochem. Biophys. Res. Commun. 51,376 (1973). 8K.-I. Arai, M. Kawita, and Y. Kaziro, J. Biol. Chem. 247, 7029 (1972). 9 M. Smolarskyand M. Tal, Biochim. Biophys. Acta 213, 401 (1976). ~0A. J. Wahba, M. J. Miller, A. Niveleau,T. A. Landers,G. G. Carmichael, K. Weber,D. A. Hawley, and L. I. Slobin,J. Biol. Chem. 249, 3314 (1974).
[39]
FUNCTION OF PROTEIN S!
429
RECONSTITUTION OF DEPLETED 30 S ( - S I ) RIBOSOMES WITH PROTEIN S I - P H E - T R N A BINDING a
l II Ill IV
Method
30 S(-SI) (pmol)
Residual activity (%)
30 S(-SI) + S (pmol)
Reconstituted activity (%)
"Tal'" "Wahba'" Sepharose 6B S e p h a r o s e 4B anti-Sl
10.0 1.5 2.0 4. I
77 12 15 32
II .0 2.0 5.0 i3.0
85 15 39 100
" Phe-tRN A, 13 pmol, is bound to 25 pmol of native 30 S subunits using saturating a m o u n t s of poiyuridylate.
The table lists the residual poly(U)-coded Phe-tRNA binding to 30 S(-SI) and the reconstitution by added SI.
P r e p a r a t i o n of Antibodies against $1 Rabbits are injected with S1 (0.5 mg SI, prepared as described in this volume [38], method A) using Freund's complete adjuvant. ~j The 7 S IgG fraction from the crude serum (53 ml) is enriched by (NH4)2SO 4 precipitation, Sephadex A-50 and Sephadex G-150 chromatography as described. ~ Nuclease-free IgG (800 rag) is obtained with 2% anti-S 1 IgG (Heidelberger assay).
R e m o v a l of S1 from 30 S S u b u n i t by a S e p h a r o s e 4B anti-S1 IgG Column IgG (600 rag) is incubated with 15 g of CNBr-activated Sepharose 4B following the instructions given by the producer; 96% of the input protein is bound to the matrix. From this a theoretical capacity of 1000A,,~0 units of 30 S may be calculated. 30 S, 320A 2~ounits (activated at 37 ° for 10 rain in buffer I), is applied to the Sepharose 4B anti-Sl IgG column ( l cm × 50 cm) at 37 °. When the 30 S subunits are distributed to the column material, a 10-rain adsorption step at 37 ° is required before eluted protein is reapplied to the column. This procedure is repeated at 0 °. Elution is started with buffer I , and 60 A 2~ounits of nonadsorbed 30 S are obtained (Fig. 1). This is followed by elution with buffer I + 800 m M NH4CI; 190A2no units of 30 S(-S 1) ribosomal subunits are eluted, which are readjusted to buffer I concentration. The 30 S(-S 1) are pelleted at 50,000 11 G. St6ffler and H. G. Wittmann, Proc. Natl. Acad. Sci. U.S.A. 65, 2283 (1971). 1~ N. Harboc and A. Ingild, Scand. J. l m m u n o l . 2, Suppl. 1, 161 (1973).
430
[39]
INITIATION OF PROTEIN SYNTHESIS
3-
A260-U~ml
2-
1-
Obuffer
,
I
5~3
+0.8MNH/~CI 100
150
200
ml
Fro. 1. Elution profile of 30 S(-S 1) ribosomal subunits from a Sepharose 4B anti-SI IgG column ( 1 cm × 50 cm). The flow rate is 1 ml/min. A 26ounits are monitored with a flow-through U V spectrophotometer. The 30 S(-S 1) peak (II) is sampled directly into centrifuge tubes. Peak III is pooled for SI work-up.
rpm for 8 hr (Ti 50, 224,000 g). The pellet is dissolved in buffer I to a concentration of280A 260units/ml. The column material is regenerated with 1 M K B r - 2 mM DTE; 70Az~0 units of 30 S and protein SI are eluted at this step. The column kept at 0° may be re-used 10-fold with similar efficiency. R e c o n s t i t u t i o n of 30 S(-S1) b y the Addition of P r o t e i n $1 '3 Reconstitution system for Phe-tRNA binding: 10 tzl of 30 S(-SI) (18 pmoi//A in buffer I), 15/xl of $1 (23 pmoi//zl), 80 ~1 of buffer I, and 5/A of H20. The final volume was 110/zl with a total Mg 2+ concentration of 16.4 m M and a 30 S(-SI) to Si molar ratio of 1:2. Incubation was at 37 ° for 25 mm. Reconstituted subunits may be stored at 0 ° for 24 hr. Of this solution, 20/zl contain 32.7 pmol of 30 S. 30 S(-S 1) + S 1 used for poly(Phe) synthesis are reconstituted at a ratio of 30 S(-S1) to S1 of 1:0.8. F u n c t i o n of S1 in the Initiation Step The function of protein S 1 in the initiation was carefully investigated by Van Dieijen et al. ,2 Szer et al. 2 and Sobura et al. 4 They all concluded from their data that the AUG-stimulated binding of fMet-tRNA is not dependent ,3 M. Laughrea and P. B. Moore, J. Mol. Biol. 112, 399 (1977).
[39]
FUNCTION OF PROTEIN SI
431
on S 1, but S 1 requirement for the initiator-tRNA binding can be shown for RI7 RNA and MS2 RNA. TM A U G - D e p e n d e n t Binding o f f M e t - t R N A to 70 S(30 S + 50 S) a n d to 70 S(-S1) (30 S(-S1) + 50 S) R i b o s o m e s
Assay System. Each reaction mixture contains in a total volume of 50 /zl: 10/zl of IF-i.3 (50/zg/ml), 10/zl of IF-2 (100/zg/ml) (plastic pipettes must be used, since IFs stick to glass); 2/zl of 30 S or 30 S(-S 1) (34 pmol), 2 p,1 of 50 S (40 pmol), 10/zl of fMet-tRNA (50 pmol, specific activity 1000 Ci/mol), 6/zl of Mix 1 and varying concentrations of AUG (see Fig. 2A). The reaction is started by the addition o f f M e t - t R N A and placing theplastic tubes into a water bath at 25 °. After 10 min the reaction is terminated by the addition of 1 ml of ice-cold buffer II. The solution is filtered through nitrocellulose filters; the filters are washed 5 times with 1 ml of buffer II each and dried with the aid of an infrared lamp, and the radioactivity is determined in toluene-0.5% PPO + 0.02% POPOP. MS2 R N A - D e p e n d e n t Binding of fMet-tRNA
Assay System. For the MS2 RNA-dependent binding o f f M e t - t R N A the reaction volume is reduced to 30/xl in order to increase the RNA concentration per assay. It contains 2 /xl of IF-i.3 (250 /xg/ml), 2 /zl of IF-2 (500 /zg/ml), 2/zl of 30 S or 30 S(-S1) (34 pmol), 2/xl of 50 S (40 pmol), 6/xl of fMet-tRNA (30 pmol), 3/zl of Mix I and varying amounts of MS2 RNA ( 100 A,,60 units/ml). MS2 RNA, 30 S or 30 S(-S 1), IF- 1.3, and Mix 1 are preincubated at 37 ° for 5 min. After the addition of the remaining components, incubation is continued for an additional 20 min (Fig. 2B). F u n c t i o n of S1 in the P o l y u r i d y l a t e - a n d Oligouridylate-Stimulated P h e - t R N A Binding to R i b o s o m e s An easy to perform test for the function of protein S1 in the coded binding of an aminoacyl-tRNA is the oligo- or polyuridylate-stimulated binding of Phe-tRNA to 30 S or 70 S ribosomes. Whereas poly(U)-coded Phe-tRNA is markedly impaired by the absence of S1, the Ua-coded Phe-tRNA binding is S1 independent. These data closely resemble the fMet-tRNA binding and may point to an S1 polyuridylate interaction outside the decoding site. 15 ~4j. Steitz, Nature (London) 224, 957 (1969). ~'~H. G. Gassen, R. Linde, N. Q. Khanh, R. Lipecky, and J. Kohlschein, "Translation of Natural and Synthetic Templates," p. 79. Poznan Univ. Press, Poznan, 1977.
432
INITIATION OF PROTEIN SYNTHESIS
~20-
[39]
6-
.o
4-
z rY
1o :E
2
10 AUG [nmol]
!
!
i
25
50
75
MS2 RNA [pmoll
FIG. 2. Binding of fMet-tRNA to 70 S and to 70 S ribosomes depleted of protein S1 in response to AUG and MS2 RNA. Reconstitution by S 1addition is not done in this experiment because fMet-tRNA binding is very sensitive to Mg2+. The concentration used in this assay was 5 mM Mg2+. © O, 50 S + 30 S; O------O, 50 S + 30 S(-SI). B i n d i n g of [3H]Polyuridylate to 30 S a n d 30 S(-1) R i b o s o m a l S u b u n i t s In order to examine whether polyuridylate binding requires complete 30 S subunits, binding of poly(U) to either 30 S or 30 S(-1) is examined by the nitrocellulose filter method. The a b o v e question was originally a p p r o a c h e d by Van Duin et al. using sucrose density gradient centrifugation. 1~Whereas an S 1 effect can be d e m o n s t r a t e d by this method under our conditions no S 1 d e p e n d e n c e for polyuridylate binding to 30 S ribosomes can be found (Fig. 3). A s s a y . 30 S or 30 S(-1) are reactivated at 37 ° for 25 min in buffer I. T w o microliters of 30 S or 30 S(-1) (30 pmol) are incubated with increasing a m o u n t s of [3H]poly(U) (5Az~0 units/ml) in 1.0 ml of buffer III for 3 min at 0 °. The solutions are filtered through nitrocellulose filters. The filters are rinsed 3 times with 6 ml each of the same buffer (0°)dried and the radioactivity is determined. The results of this experiment are listed in Fig. 3. P o l y ( U ) a n d U 4 - D e p e n d e n t B i n d i n g of P h e - t R N A to 70 S(30 + 50 S) A s s a y . 30 S, 30 S(-SI), and 30 S(-1) + S1 are reactivated in buffer I according to Zamir. ,7 The following reaction mixture is incubated at 0 ° for 40 min: 10 tzl of [3H]Phe-tRNA (30 pmol), 20/zl of 30 S, or 30 S(-S1) or 30 S(-SI) + SI (30-35 pmol), 50/zl of Mix 2, varying amounts of poly(U) (20 A2~o units/mi), of U4 (120A2~o units/ml), and H,,O to 100/zl. The reaction
16j. Van Duin and C. G. Kurland, Mol. Gen. Genet. 109, 169 (1970). ,r A. Zamir, R. Miskin, and D. Elson, J. Mol. Biol. 60, 347 (1971).
[39]
FUNCTION OF PROTEIN S l
433
/
c
,'
}L"
5 v
5
10
blg2+[mM] Y
0.96
0.6"
0.32
0.55
275
5.50
poly(U) [nmol pU]
FIG. 3. Binding of [3H]poly(U) to 30 S and 30 S(-SI) ribosomes as measured by the nitrocellulose filter assay. The inset shows the Mg2+dependence at a nonsaturating concentration of polyuridylate. No difference between 30 S and 30 S(-SI) can be found, which exceeds the variability of the method. O, 30 S; O, 30 S(-S1), procedure It (Tal); A, 30 S(-S1), procedure IV). mixture is filtered through nitrocellulose filters which are rinsed 3 times with 6 ml of ice-cold buffer II. Further treatment is as described. For results see Figs. 4A and 5A. P o l y ( U ) - a n d U 4 - D e p e n d e n t B i n d i n g of P h e - t R N A to 70 S(30 + 50 S) a n d to 70 S(-S1) (30 S(-S1) + 50 S) R i b o s o m e s
Assay. 30 S and 50 S ribosomal subunits are activated separately in buffer I for 25 min at 37 °. Otherwise the conditions are the same as described for the 30 S, except that 3/zl of 50 S (47 pmol) are added. Ratio 30 S:50 S = 1:1.3-1.5. The results are shown in Figs. 413 and 5B. S l F u n c t i o n in P o l y ( U ) - D e p e n d e n t P o l y ( P h e ) S y n t h e s i s Poly(U)-dependent poly(Phe) synthesis may be used as a model system, when a factor-dependent initiation step is not required. A Mg '-'÷concentration of 10 m M is sufficient to override the initiation phase. Assay. The reaction mixture contains, in a total volume of 100/A: 20/A of [3H]Pbe-tRNA (66 pmol)., i/zl of E F - T u . G DP (40 pmol), 2/zl of E F - G (48 /~g), 20 tzl of 30 S or 30 S(-S I) or 30 s(-S I) + S I (32 pmol), 3 pJ of 50 S (47 pmol), 25 tzl of Mix 3, varying amounts of poly(U) (18 A,_,,0 units/ml) and H.,O to 100/zl. 30 S:50 S = 1:1.3, 30 S(-SI):S1 = 1:0.8. The mixture is incubated at.37 ° for 25 rain.
434
INITIATION OF PROTEIN SYNTHESIS 20-
z~20 rr
[39]
®
5
I0
s
,o
15
~)
,;
2'o
®
10-
poly[U) [nmol pU] FIG. 4. Poly(U)-stimulated Phe-tRNA binding to 30 S (A) and 70 S ribosomes (B). In both cases depleted 30 S(-SI) are inactive. Activity can be restored to 100% by the addition of S I in a r a t i o o f 3 0 S t o S l ofl:2.(A) O, 30S; @,30S(-SI); A, 30S(-SI) + Sl. (B) O, 50S + 30S;@, 50 S + 30 S(-S1); A, 50 S + 30 S(-S1) + S1.
Fifty microliters of the solution are pipetted to GF/A filters, which are treated at 80° in 10% TCA for 10 min. They are then rinsed in ethanol, ethanol-ether 1:1, and ether. The results from these experiments are listed in Fig. 6. Remarks The procedures described for the preparation of 30 S(-S 1) have different advantages and limitations. The most efficient procedure is the preparation according to Tal. Large quantities of 30 S(-S 1) can be prepared; only small amounts of other ribosomal components are removed--as monitored by gel electrophoresis; the 30 S(-Sl) are stable, and full activity of 30 S can be restored by the addition of S 1. The method, however, has the disadvantage that the residual activity of the 30 S(-SI) in Phe-tRNA binding is high (Table I). The Sepharose 4B anti-SI IgG method renders the best results in inactivation and reactivation of 30 S ribosomal subunits; however, it is time consuming and tedious, the capacity of the column is low, and the 30 S(-S 1) are fully active only for 24-48 hr at 0°. For crucial experiments where the stoichiometric involvement of S1 is unclear 30 S(-S1) should be prepared in this manner in spite of these limitations. Effective reconstitution of 30 S(-S1) with protein S1 depends on the
[39]
FUNCTION OF PROTEIN S l
435
L®
O_
"~
I
i
,
10
2o
10 '
20 '
i
30
i
LO '
U4 [nmol} FIG. 5. U4-stimulated Phe-tRNA binding to 70 S and 70 S-depleted S1 ribosomes. (A) U4-coded Phe-tRNA binding is not affected by omission of S1. S1 addition is inhibitory. In separate experiments (data not shown) we found that functional reconstitution of S 1 requires the presence of oligouridylate n > 12, O, 30 S" 0 , 30 S(-SI);/x, 30 S(-SI) + SI. (B) SI effect on 70 S ribosomes. Only a minor S 1 effect can be seen. O, 50 S + 30 S; 0 , 50 S + 30 S(-S 1): A, 50 S + 30 S(-SI) + SI.
30o
20-
I0.
poly(U) [pmol pU] FIG. 6. Function of S 1 in the poly(U)-dependent poly(Phe) synthesis. The residual activity of the 30S(-S 1) + 50 S ribosomes should be due to S 1 contamination of the 50 S. The optimal 30 S to S 1 ratio is 1:0.8. Even a small excess of S 1 is strongly inhibitory. Amounts of poly(Phe) synthesized are calculated from 50 kd of incubation mixture. O, 50 S + 30 S; O, 50 S + 30 S(-S1); A, 50 S + 30 S(-S1) + SI.
436
I N I T I A T I O N OF P R O T E I N S Y N T H E S I S
[40]
presence of oligouridylate n > 12. Addition of S 1 to an U4-coded binding of Phe-tRNA is inhibiting. The use of S I00 devoid of functional S1 by the addition of anti-Sl IgG should be avoided, since other proteins from the S 100 may partially restore 30 S function. The initiation and elongation factors used in the experiments were free of S1 as checked with anti-S1 IgG in an Ouchterlony assay. The 50 S subunit contained, however, residual S1 activity (Ouchterlony). This contamination seems to be responsible for the partial synthesizing activity of 30 S(-S 1) + 50 S in the poly(Phe) system. The Mg 2+ concentration and the ratio of S 1 over 30 S is very crucial for the reconstitution of 30 S activity. Whereas 20 mM Mg 2÷and 30 S: S 1 = 1:2 are optimal for Phe-tRNA binding, this excess of S I is strongly inhibitory for poly(Phe) synthesis. Here 10 mM Mg 2+ and a ratio of 30 S:SI of 1:0.8 was best. These conditions were found to be optimal by other authors as well. The excellent system for S1 function would be the factor-dependent translation, i.e., no S I00 of MS2 RNA into coat protein. This system does not work for us at present. S1 dependence on proper MS2 RNA coat protein translation was shown by Van Dieijen et al., but using S100 + anti-S 1 lgG.'8 All data from initiation as well as elongation step experiments point toward a function of protein S 1 in the binding of the mRNA to the small subunit. This binding should not occur in the decoding site of the 30 S ribosome (Fig. 5A). A direct influence of S 1 on the binding ofpolyuridylate to 30 S(-SI) under equilibrium conditions cannot be shown. ~8 G. Van Dieijen, P. H. Van Knippenberg, J. Van Duin, B. Koekman, and P. H. Pouwels, Mol. Gen. Genet. 153, 75 (1977).
[40] F u n c t i o n o f R i b o s o m a l P r o t e i n S1 i n t h e A s s e m b l y of t h e 30 S I n i t i a t i o n C o m p l e x
By J. VAN DUIN, G. VAN DIEIJEN, P. ZIPORI, and W. VAN PROOIJEN Ribosomal protein S 1 has recently raised considerable interest because of its unique involvement in the initiation of protein synthesis and in the initiation of Qfl RNA replication.~,2 The molecular mechanism through which S 1 facilitates the binding of either the ribosome or the Qfl replicase to phage RNA is basically unknown. Although there are data that the protein ' G. Van Dieijen, C. J. Van der Laken, P. H. Van Knippenberg, and J. Van Duin, J. Mol. Biol. 93, 351 (1975). 2 R. Kamen, M. Kondo, W. R6mer, and C. Weissmann, Eur. J. Biochem. 31, 44 (1972).
METHODS IN ENZYMOLOGY, VOL. LX
Copyright ~) 1979by Academic Press, Inc. All rights of reproduction in any form reserved. 1SBN 0d 2-181960-4
INITIATION OF PROTEIN SYNTHESIS
[39]
Remarks
There are two procedures available to isolate the ribosomal protein S l, starting either from 30 S ribosomal subunits or from 70 S ribosomes. If a zonal rotor is available and an effective separation of ribosomal subunits has been worked out, the 30 S method is the procedure of choice. From about 100 g ofE. coli (wet weight), roughly 10 mg of S 1 in about 90% purity may be obtained in 6 working days. Starting from 70 S, the procedure is straightforward, using only simple chromatographic procedures; the yields, however, are lower, and S I may still be contaminated with other ribosomal proteins. Aside from that, many oppose prolonged urea treatment in the isolation of proteins. The material eluted in the void volume of the DEAE column (Fig. 2A) contains other poly(U) binding proteins, but no S1.
[39] F u n c t i o n o f t h e R i b o s o m a l P r o t e i n S 1 in Initiation and Elongation of Bacterial Protein Synthesis By N. Q. KHANH, R. LINDE, U. MANDERSCHIED, and H. G. GASSEN
The ribosomal protein S1 is required for the translation of natural messengers like MS2 RNA from phage. Although its role in the initiation step of protein synthesis is well documented and partially understood on a theoretical basis, its function in the elongation cycle is evident but remains to be clarified from a mechanistic point of view. Protein S I can form a specific 30 S-mRNA initiation complex without prior binding of fMet-tRNA Met-GTP'IF-2 to the 30 S s u b u n i t / D a t a from different laboratories confirm that S1 is required for MS2 or RI7 RNAdependent flVlet-tRNA binding, but not for the AUG-dependent stimulation of fMet-tRNA binding to ribosomes? '4 However, discrepancies in the molecular interpretation still exist. Van Dieijen et al. have postulated that S1 recognizes an initiation type of tertiary structure of the m R N A / whereas others have concluded from their experiments that S 1 binds to the 3' end of the 16 S RNA, thereby changing its conformation so that a l For a recent review, see J. Steitz, "Biological Regulation and C o n t r o l " Plenum, N e w York, in press. 2 G. V a n Dieijen, P. H. V a n Knippenberg, and J. Van Duin, Fur. J. Biochem. 64, 511 (1976). W. Szer, J. M. H e r m o s o , and S. Leffler, Proc. Natl. Acad. Sci. U.S.A. 72, 2325 (1975). 4 j. E. Sobura, M. R. C h o w d h u r y , D. A. Hawley, and A. J. Wahba, Nucl. Acids Res. 4, 17 (1977).
M E T H O D S 1N E N Z Y M O L O G Y , VOL. LX
Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181960-4
[39]
FUNCTION OF PROTEIN S I
427
recognition-type double-strand formation with the intercistronic sequence of a mRNA can occur? The examination of S l function in elongation proves to be more difficult because defined functions like coded AA-tRNA binding or translocation are not lethally affected by omission of S 1, but can be restored in higher Mg "+ concentrations2 Furthermore, interpretations are obscured by the fact that most components of the assay, i.e., for the poly(U)-dependent poly(Phe) synthesis, 30 S and 50 S subunits or the 100,000 g supernatant (SI00) are contaminated with S1. Thus, a comparison of the data from different laboratories is hampered by the fact that the so-called S 1-depleted 30 S ribosomes, 30 S(-S 1), are prepared by nonstandardized methods and that a S100 is used as an enzyme source, where SI has been inactivated by the addition of anti-S 1 IgG. The use of these techniques has led to widespread variation in experimental results and lively discussions on their interpretations in terms of S1 function. Tests described in the following were therefore done with anti-S1 IgG depleted 30 S(-S l) ribosomal subunits and purified initiation and elongation factors. Materials
Buffer I: 50 mM Tris. HCI, pH 7.5,200 mM NH4CI, 20 mM MgCI2, 2 mM DTE Buffer II: 10 mM Tris-HCI, pH 7.2, 10 mM MgCI2, 50 mM NH4Cl Buffer III: 10 mM Tris.HCl, pH 7.6, 15 mM MgCI2 Mix 1:500 mM Tris-HCI, pH 7.8, 1M NH4CI, 40 mM Mg(OAc)z, 10 mM DTE, 10 mM GTP Mix 2:100 mM Tris .HCI, pH 7.2, 40 mM MgClz, 300 mM NH4C1, 10 mM mercaptoethanol Mix 3:100 mM Tris. HC1, pH 7.5,400 mM NH4C1, 40 mM Mg(OAc)z, 2 mM GTP, l0 mM ATP, 25 mM PEP, 2 mM DTE, 0.1 mg/ml pyruvate kinase Protein S1 dissolved in 80 mM Tris .HCI, pH 7.4, l/zl ^ 23 pmol of S l, EF-Tu.GDP (1 /~1 ~ 40 pmol), EF-G (1 p.l = 24/~g) tRNA from E. coli was charged to 2.5% with [:~H]Met (specific activity, 1000 Ci/mol, and 100% formylated f[aH]Met-tRNA Charging, with [aH]Phe (specific activity, 1000 Ci/mol) was 1.8%, of tRNA Polyuridylate, 18 Ae~0 units/ml, chain length 30-50, U4, 120 A2,o units/ml [aH]polyuridylate, 5 A.,~0 units/ml; specific activity 6 Ci/mol per pU residue '~ A. E. Dahlberg and J. E. Dahlberg, Pro('. Natl. Acad. Sci. U.S.A. 72, 2490 (1975). G. Van Dieijen, C. J. Van der Laken, P. H. Van Knippenberg, and J. Van Duin, J. Mol. Biol. 93, 351 (1975).
428
INITIATION OF PROTEIN SYNTHESIS
[39]
MS2 RNA, 100 A26o units/ml (Boehringer) Sepharose 4B CNBr-activated, Sephadex A-50, Sephadex G-150 (Pharmacia) Dithioerytritol (DTE) (Serva, Heidelberg) Nitrocellulose filter BA85, 2.5 cm, 0.45/xm (Schleicher & Schfill) Glass-fiber filter GF/A 2.5 cm (Whatman) P r e p a r a t i o n of the C o m p o n e n t s for the T e s t Systems The 70 S ribosomes, 30 S subunits, and protein S1 are prepared as described in this volume [38], following published procedures. Initiation factors IF-l.3 and IF-2 and elongation factors EF-Tu and EF-G are prepared as described. 7,s The proteins are free of nucleases examined by a procedure given in this volume [38]. Polyuridylate and oligouridylates are synthesized with polynucleotide phosphorylase (EC 2.7.7.8). 30 S(-S1) subunits are prepared by four different methods and compared according to function: low-salt treatment [Tal's procedure 9 (I)], repeated washings with 1M NH4C1 [Wahba's procedure 1°(II)], separation of 30 S(-S l) from 30 S by chromatography over Sepharose 6B equilibrated with 1 M NH4CI [our procedure (liD], and removal of S 1 by chromatography of the ribosomes over a Sepharose 4B (anti-S! IgG) column [our procedure (IV)]. I. A fast working and effective procedure, which removes up to 90% of S1 from 30 S ribosomes and minor amounts of other ribosomal components. 30 S(-S l) prepared by this method have a high residual activity in the poly(U)-dependent Phe-tRNA binding. II. This procedure removes Sl to 100%, but many other ribosomal proteins, too. 30 S(-S1) cannot be reconstituted into active 30 S by the addition of S1 (at least not in our hands). Ill. A fast and effective procedure for the isolation of smaller amounts of 30 S(-S l) (100 A 260units per run), but reconstitution is only 40-50% in the poly(U)-Phe-tRNA binding test. IV. In our laboratory, the method of choice. S 1 is removed by more than 90%, and the subunits can be reconstituted to 100% by addition of Sl. A limitation of the procedure lies in the low capacity of the anti-Sl IgG column and the fast inactivation of the ribosomes. The experiments described in the following section were done with 30 S(-S1) prepared by method IV. r D. P. Suttle, M. A. Haralson, and J. M. Ravel,Biochem. Biophys. Res. Commun. 51,376 (1973). 8K.-I. Arai, M. Kawita, and Y. Kaziro, J. Biol. Chem. 247, 7029 (1972). 9 M. Smolarskyand M. Tal, Biochim. Biophys. Acta 213, 401 (1976). ~0A. J. Wahba, M. J. Miller, A. Niveleau,T. A. Landers,G. G. Carmichael, K. Weber,D. A. Hawley, and L. I. Slobin,J. Biol. Chem. 249, 3314 (1974).
[39]
FUNCTION OF PROTEIN S!
429
RECONSTITUTION OF DEPLETED 30 S ( - S I ) RIBOSOMES WITH PROTEIN S I - P H E - T R N A BINDING a
l II Ill IV
Method
30 S(-SI) (pmol)
Residual activity (%)
30 S(-SI) + S (pmol)
Reconstituted activity (%)
"Tal'" "Wahba'" Sepharose 6B S e p h a r o s e 4B anti-Sl
10.0 1.5 2.0 4. I
77 12 15 32
II .0 2.0 5.0 i3.0
85 15 39 100
" Phe-tRN A, 13 pmol, is bound to 25 pmol of native 30 S subunits using saturating a m o u n t s of poiyuridylate.
The table lists the residual poly(U)-coded Phe-tRNA binding to 30 S(-SI) and the reconstitution by added SI.
P r e p a r a t i o n of Antibodies against $1 Rabbits are injected with S1 (0.5 mg SI, prepared as described in this volume [38], method A) using Freund's complete adjuvant. ~j The 7 S IgG fraction from the crude serum (53 ml) is enriched by (NH4)2SO 4 precipitation, Sephadex A-50 and Sephadex G-150 chromatography as described. ~ Nuclease-free IgG (800 rag) is obtained with 2% anti-S 1 IgG (Heidelberger assay).
R e m o v a l of S1 from 30 S S u b u n i t by a S e p h a r o s e 4B anti-S1 IgG Column IgG (600 rag) is incubated with 15 g of CNBr-activated Sepharose 4B following the instructions given by the producer; 96% of the input protein is bound to the matrix. From this a theoretical capacity of 1000A,,~0 units of 30 S may be calculated. 30 S, 320A 2~ounits (activated at 37 ° for 10 rain in buffer I), is applied to the Sepharose 4B anti-Sl IgG column ( l cm × 50 cm) at 37 °. When the 30 S subunits are distributed to the column material, a 10-rain adsorption step at 37 ° is required before eluted protein is reapplied to the column. This procedure is repeated at 0 °. Elution is started with buffer I , and 60 A 2~ounits of nonadsorbed 30 S are obtained (Fig. 1). This is followed by elution with buffer I + 800 m M NH4CI; 190A2no units of 30 S(-S 1) ribosomal subunits are eluted, which are readjusted to buffer I concentration. The 30 S(-S 1) are pelleted at 50,000 11 G. St6ffler and H. G. Wittmann, Proc. Natl. Acad. Sci. U.S.A. 65, 2283 (1971). 1~ N. Harboc and A. Ingild, Scand. J. l m m u n o l . 2, Suppl. 1, 161 (1973).
430
[39]
INITIATION OF PROTEIN SYNTHESIS
3-
A260-U~ml
2-
1-
Obuffer
,
I
5~3
+0.8MNH/~CI 100
150
200
ml
Fro. 1. Elution profile of 30 S(-S 1) ribosomal subunits from a Sepharose 4B anti-SI IgG column ( 1 cm × 50 cm). The flow rate is 1 ml/min. A 26ounits are monitored with a flow-through U V spectrophotometer. The 30 S(-S 1) peak (II) is sampled directly into centrifuge tubes. Peak III is pooled for SI work-up.
rpm for 8 hr (Ti 50, 224,000 g). The pellet is dissolved in buffer I to a concentration of280A 260units/ml. The column material is regenerated with 1 M K B r - 2 mM DTE; 70Az~0 units of 30 S and protein SI are eluted at this step. The column kept at 0° may be re-used 10-fold with similar efficiency. R e c o n s t i t u t i o n of 30 S(-S1) b y the Addition of P r o t e i n $1 '3 Reconstitution system for Phe-tRNA binding: 10 tzl of 30 S(-SI) (18 pmoi//A in buffer I), 15/xl of $1 (23 pmoi//zl), 80 ~1 of buffer I, and 5/A of H20. The final volume was 110/zl with a total Mg 2+ concentration of 16.4 m M and a 30 S(-SI) to Si molar ratio of 1:2. Incubation was at 37 ° for 25 mm. Reconstituted subunits may be stored at 0 ° for 24 hr. Of this solution, 20/zl contain 32.7 pmol of 30 S. 30 S(-S 1) + S 1 used for poly(Phe) synthesis are reconstituted at a ratio of 30 S(-S1) to S1 of 1:0.8. F u n c t i o n of S1 in the Initiation Step The function of protein S 1 in the initiation was carefully investigated by Van Dieijen et al. ,2 Szer et al. 2 and Sobura et al. 4 They all concluded from their data that the AUG-stimulated binding of fMet-tRNA is not dependent ,3 M. Laughrea and P. B. Moore, J. Mol. Biol. 112, 399 (1977).
[39]
FUNCTION OF PROTEIN SI
431
on S 1, but S 1 requirement for the initiator-tRNA binding can be shown for RI7 RNA and MS2 RNA. TM A U G - D e p e n d e n t Binding o f f M e t - t R N A to 70 S(30 S + 50 S) a n d to 70 S(-S1) (30 S(-S1) + 50 S) R i b o s o m e s
Assay System. Each reaction mixture contains in a total volume of 50 /zl: 10/zl of IF-i.3 (50/zg/ml), 10/zl of IF-2 (100/zg/ml) (plastic pipettes must be used, since IFs stick to glass); 2/zl of 30 S or 30 S(-S 1) (34 pmol), 2 p,1 of 50 S (40 pmol), 10/zl of fMet-tRNA (50 pmol, specific activity 1000 Ci/mol), 6/zl of Mix 1 and varying concentrations of AUG (see Fig. 2A). The reaction is started by the addition o f f M e t - t R N A and placing theplastic tubes into a water bath at 25 °. After 10 min the reaction is terminated by the addition of 1 ml of ice-cold buffer II. The solution is filtered through nitrocellulose filters; the filters are washed 5 times with 1 ml of buffer II each and dried with the aid of an infrared lamp, and the radioactivity is determined in toluene-0.5% PPO + 0.02% POPOP. MS2 R N A - D e p e n d e n t Binding of fMet-tRNA
Assay System. For the MS2 RNA-dependent binding o f f M e t - t R N A the reaction volume is reduced to 30/xl in order to increase the RNA concentration per assay. It contains 2 /xl of IF-i.3 (250 /xg/ml), 2 /zl of IF-2 (500 /zg/ml), 2/zl of 30 S or 30 S(-S1) (34 pmol), 2/xl of 50 S (40 pmol), 6/xl of fMet-tRNA (30 pmol), 3/zl of Mix I and varying amounts of MS2 RNA ( 100 A,,60 units/ml). MS2 RNA, 30 S or 30 S(-S 1), IF- 1.3, and Mix 1 are preincubated at 37 ° for 5 min. After the addition of the remaining components, incubation is continued for an additional 20 min (Fig. 2B). F u n c t i o n of S1 in the P o l y u r i d y l a t e - a n d Oligouridylate-Stimulated P h e - t R N A Binding to R i b o s o m e s An easy to perform test for the function of protein S1 in the coded binding of an aminoacyl-tRNA is the oligo- or polyuridylate-stimulated binding of Phe-tRNA to 30 S or 70 S ribosomes. Whereas poly(U)-coded Phe-tRNA is markedly impaired by the absence of S1, the Ua-coded Phe-tRNA binding is S1 independent. These data closely resemble the fMet-tRNA binding and may point to an S1 polyuridylate interaction outside the decoding site. 15 ~4j. Steitz, Nature (London) 224, 957 (1969). ~'~H. G. Gassen, R. Linde, N. Q. Khanh, R. Lipecky, and J. Kohlschein, "Translation of Natural and Synthetic Templates," p. 79. Poznan Univ. Press, Poznan, 1977.
432
INITIATION OF PROTEIN SYNTHESIS
~20-
[39]
6-
.o
4-
z rY
1o :E
2
10 AUG [nmol]
!
!
i
25
50
75
MS2 RNA [pmoll
FIG. 2. Binding of fMet-tRNA to 70 S and to 70 S ribosomes depleted of protein S1 in response to AUG and MS2 RNA. Reconstitution by S 1addition is not done in this experiment because fMet-tRNA binding is very sensitive to Mg2+. The concentration used in this assay was 5 mM Mg2+. © O, 50 S + 30 S; O------O, 50 S + 30 S(-SI). B i n d i n g of [3H]Polyuridylate to 30 S a n d 30 S(-1) R i b o s o m a l S u b u n i t s In order to examine whether polyuridylate binding requires complete 30 S subunits, binding of poly(U) to either 30 S or 30 S(-1) is examined by the nitrocellulose filter method. The a b o v e question was originally a p p r o a c h e d by Van Duin et al. using sucrose density gradient centrifugation. 1~Whereas an S 1 effect can be d e m o n s t r a t e d by this method under our conditions no S 1 d e p e n d e n c e for polyuridylate binding to 30 S ribosomes can be found (Fig. 3). A s s a y . 30 S or 30 S(-1) are reactivated at 37 ° for 25 min in buffer I. T w o microliters of 30 S or 30 S(-1) (30 pmol) are incubated with increasing a m o u n t s of [3H]poly(U) (5Az~0 units/ml) in 1.0 ml of buffer III for 3 min at 0 °. The solutions are filtered through nitrocellulose filters. The filters are rinsed 3 times with 6 ml each of the same buffer (0°)dried and the radioactivity is determined. The results of this experiment are listed in Fig. 3. P o l y ( U ) a n d U 4 - D e p e n d e n t B i n d i n g of P h e - t R N A to 70 S(30 + 50 S) A s s a y . 30 S, 30 S(-SI), and 30 S(-1) + S1 are reactivated in buffer I according to Zamir. ,7 The following reaction mixture is incubated at 0 ° for 40 min: 10 tzl of [3H]Phe-tRNA (30 pmol), 20/zl of 30 S, or 30 S(-S1) or 30 S(-SI) + SI (30-35 pmol), 50/zl of Mix 2, varying amounts of poly(U) (20 A2~o units/mi), of U4 (120A2~o units/ml), and H,,O to 100/zl. The reaction
16j. Van Duin and C. G. Kurland, Mol. Gen. Genet. 109, 169 (1970). ,r A. Zamir, R. Miskin, and D. Elson, J. Mol. Biol. 60, 347 (1971).
[39]
FUNCTION OF PROTEIN S l
433
/
c
,'
}L"
5 v
5
10
blg2+[mM] Y
0.96
0.6"
0.32
0.55
275
5.50
poly(U) [nmol pU]
FIG. 3. Binding of [3H]poly(U) to 30 S and 30 S(-SI) ribosomes as measured by the nitrocellulose filter assay. The inset shows the Mg2+dependence at a nonsaturating concentration of polyuridylate. No difference between 30 S and 30 S(-SI) can be found, which exceeds the variability of the method. O, 30 S; O, 30 S(-S1), procedure It (Tal); A, 30 S(-S1), procedure IV). mixture is filtered through nitrocellulose filters which are rinsed 3 times with 6 ml of ice-cold buffer II. Further treatment is as described. For results see Figs. 4A and 5A. P o l y ( U ) - a n d U 4 - D e p e n d e n t B i n d i n g of P h e - t R N A to 70 S(30 + 50 S) a n d to 70 S(-S1) (30 S(-S1) + 50 S) R i b o s o m e s
Assay. 30 S and 50 S ribosomal subunits are activated separately in buffer I for 25 min at 37 °. Otherwise the conditions are the same as described for the 30 S, except that 3/zl of 50 S (47 pmol) are added. Ratio 30 S:50 S = 1:1.3-1.5. The results are shown in Figs. 413 and 5B. S l F u n c t i o n in P o l y ( U ) - D e p e n d e n t P o l y ( P h e ) S y n t h e s i s Poly(U)-dependent poly(Phe) synthesis may be used as a model system, when a factor-dependent initiation step is not required. A Mg '-'÷concentration of 10 m M is sufficient to override the initiation phase. Assay. The reaction mixture contains, in a total volume of 100/A: 20/A of [3H]Pbe-tRNA (66 pmol)., i/zl of E F - T u . G DP (40 pmol), 2/zl of E F - G (48 /~g), 20 tzl of 30 S or 30 S(-S I) or 30 s(-S I) + S I (32 pmol), 3 pJ of 50 S (47 pmol), 25 tzl of Mix 3, varying amounts of poly(U) (18 A,_,,0 units/ml) and H.,O to 100/zl. 30 S:50 S = 1:1.3, 30 S(-SI):S1 = 1:0.8. The mixture is incubated at.37 ° for 25 rain.
434
INITIATION OF PROTEIN SYNTHESIS 20-
z~20 rr
[39]
®
5
I0
s
,o
15
~)
,;
2'o
®
10-
poly[U) [nmol pU] FIG. 4. Poly(U)-stimulated Phe-tRNA binding to 30 S (A) and 70 S ribosomes (B). In both cases depleted 30 S(-SI) are inactive. Activity can be restored to 100% by the addition of S I in a r a t i o o f 3 0 S t o S l ofl:2.(A) O, 30S; @,30S(-SI); A, 30S(-SI) + Sl. (B) O, 50S + 30S;@, 50 S + 30 S(-S1); A, 50 S + 30 S(-S1) + S1.
Fifty microliters of the solution are pipetted to GF/A filters, which are treated at 80° in 10% TCA for 10 min. They are then rinsed in ethanol, ethanol-ether 1:1, and ether. The results from these experiments are listed in Fig. 6. Remarks The procedures described for the preparation of 30 S(-S 1) have different advantages and limitations. The most efficient procedure is the preparation according to Tal. Large quantities of 30 S(-S 1) can be prepared; only small amounts of other ribosomal components are removed--as monitored by gel electrophoresis; the 30 S(-Sl) are stable, and full activity of 30 S can be restored by the addition of S 1. The method, however, has the disadvantage that the residual activity of the 30 S(-SI) in Phe-tRNA binding is high (Table I). The Sepharose 4B anti-SI IgG method renders the best results in inactivation and reactivation of 30 S ribosomal subunits; however, it is time consuming and tedious, the capacity of the column is low, and the 30 S(-S 1) are fully active only for 24-48 hr at 0°. For crucial experiments where the stoichiometric involvement of S1 is unclear 30 S(-S1) should be prepared in this manner in spite of these limitations. Effective reconstitution of 30 S(-S1) with protein S1 depends on the
[39]
FUNCTION OF PROTEIN S l
435
L®
O_
"~
I
i
,
10
2o
10 '
20 '
i
30
i
LO '
U4 [nmol} FIG. 5. U4-stimulated Phe-tRNA binding to 70 S and 70 S-depleted S1 ribosomes. (A) U4-coded Phe-tRNA binding is not affected by omission of S1. S1 addition is inhibitory. In separate experiments (data not shown) we found that functional reconstitution of S 1 requires the presence of oligouridylate n > 12, O, 30 S" 0 , 30 S(-SI);/x, 30 S(-SI) + SI. (B) SI effect on 70 S ribosomes. Only a minor S 1 effect can be seen. O, 50 S + 30 S; 0 , 50 S + 30 S(-S 1): A, 50 S + 30 S(-SI) + SI.
30o
20-
I0.
poly(U) [pmol pU] FIG. 6. Function of S 1 in the poly(U)-dependent poly(Phe) synthesis. The residual activity of the 30S(-S 1) + 50 S ribosomes should be due to S 1 contamination of the 50 S. The optimal 30 S to S 1 ratio is 1:0.8. Even a small excess of S 1 is strongly inhibitory. Amounts of poly(Phe) synthesized are calculated from 50 kd of incubation mixture. O, 50 S + 30 S; O, 50 S + 30 S(-S1); A, 50 S + 30 S(-S1) + SI.
436
I N I T I A T I O N OF P R O T E I N S Y N T H E S I S
[40]
presence of oligouridylate n > 12. Addition of S 1 to an U4-coded binding of Phe-tRNA is inhibiting. The use of S I00 devoid of functional S1 by the addition of anti-Sl IgG should be avoided, since other proteins from the S 100 may partially restore 30 S function. The initiation and elongation factors used in the experiments were free of S1 as checked with anti-S1 IgG in an Ouchterlony assay. The 50 S subunit contained, however, residual S1 activity (Ouchterlony). This contamination seems to be responsible for the partial synthesizing activity of 30 S(-S 1) + 50 S in the poly(Phe) system. The Mg 2+ concentration and the ratio of S 1 over 30 S is very crucial for the reconstitution of 30 S activity. Whereas 20 mM Mg 2÷and 30 S: S 1 = 1:2 are optimal for Phe-tRNA binding, this excess of S I is strongly inhibitory for poly(Phe) synthesis. Here 10 mM Mg 2+ and a ratio of 30 S:SI of 1:0.8 was best. These conditions were found to be optimal by other authors as well. The excellent system for S1 function would be the factor-dependent translation, i.e., no S I00 of MS2 RNA into coat protein. This system does not work for us at present. S1 dependence on proper MS2 RNA coat protein translation was shown by Van Dieijen et al., but using S100 + anti-S 1 lgG.'8 All data from initiation as well as elongation step experiments point toward a function of protein S 1 in the binding of the mRNA to the small subunit. This binding should not occur in the decoding site of the 30 S ribosome (Fig. 5A). A direct influence of S 1 on the binding ofpolyuridylate to 30 S(-SI) under equilibrium conditions cannot be shown. ~8 G. Van Dieijen, P. H. Van Knippenberg, J. Van Duin, B. Koekman, and P. H. Pouwels, Mol. Gen. Genet. 153, 75 (1977).
[40] F u n c t i o n o f R i b o s o m a l P r o t e i n S1 i n t h e A s s e m b l y of t h e 30 S I n i t i a t i o n C o m p l e x
By J. VAN DUIN, G. VAN DIEIJEN, P. ZIPORI, and W. VAN PROOIJEN Ribosomal protein S 1 has recently raised considerable interest because of its unique involvement in the initiation of protein synthesis and in the initiation of Qfl RNA replication.~,2 The molecular mechanism through which S 1 facilitates the binding of either the ribosome or the Qfl replicase to phage RNA is basically unknown. Although there are data that the protein ' G. Van Dieijen, C. J. Van der Laken, P. H. Van Knippenberg, and J. Van Duin, J. Mol. Biol. 93, 351 (1975). 2 R. Kamen, M. Kondo, W. R6mer, and C. Weissmann, Eur. J. Biochem. 31, 44 (1972).
METHODS IN ENZYMOLOGY, VOL. LX
Copyright ~) 1979by Academic Press, Inc. All rights of reproduction in any form reserved. 1SBN 0d 2-181960-4