Escherichia coli 30S mutants lacking protein S20 are defective in translation initiation

Biochimica et Biophysica Acta, 1050 (1990) 93-97 Elsevier

93

BBAEXP 92097

Escherichia coli 30S mutants lacking protein $20 are defective in translation initiation Frank G~Stz 1, Eric R. Dabbs i and Claudio O. Gualerzi 1,2 J Max-Planck-lnstitut fiir Molekulare Genetik, Berlin, (Germany) and 2 Laboratory of Genetics, DBC, University of Camerino, Camerino (MC) (Italy) (Received 16 May 1990)

Key words: Protein synthesis (prokaryotic); Ribosomal protein mutant: Translation initiation; Ribosome

The 30S ribosonml subunits derived from Escherichia coli TAIl4, a temperature-sensitive mutant lacking ribosomal protein 820, were shown to be defective in two ways: (a) they have a reduced capacity for association with the 50S ribosomal subunit which results in the impairment of most of the functions requiring a coordinated interaction between the two subunits; (b) they are defective in functions which do not require their interaction with the large subunit (i.e., the formation of ternary complexes with aminoacyl-tRNAs and templates, including the formation of 30S initiation complexes with tMet-tRNA and mRNA). The 30S (-820) subunits seem to interact normally with both template and aminoacyl-tRNA individually, but appear to be impaired in the rate-limiting isomerization step leading to the formation of a codon-anticodon interaction in the P site.

Introduction The complement of ribosomal proteins has been conserved almost completely during the evolutionary diversification of eubacteria, as evidenced by the 1:1 correspondence of these proteins in Escherichia coli and Bacillus subtilis. It is therefore surprising that mutants of E. coil lacking any one of 15 ribosomal proteins have been isolated, indicating that these proteins are non-essential. In most cases, however, these spontaneously selected mutants grow more slowly than the wild-type suggesting some impairment in organeUe function. Mutants lacking ribosomal protein $20 have been obtained in several selections [1-3]. While anti-S20 cross-reacting material (crm) is generally detected in these mutants, in one case, a range of immunological techniques failed to reveal the presence of any anti-S20 crm [4]. The lesion responsible was mapped to the structural gene for protein $20, rpsT, and this enabled the mutation to be transferred into a wild-type background by P1 transduction to give rise to TAl14. The 30S ribosomal subunits derived from this mutant have been shown, by two separate methods, to be severely

Abbreviations: crm, cross-reacting material; WT, wild type.

Correspondence: C. Gualerzi, Laboratory of Genetics, Department of Cell Biology, University of Camerino, 1-62032 Camerino (MC), Italy.

affected in their capacity to associate with the 50S subunit, especially at low M g 2+ concentrations [5]. In this paper, we present evidence that the S20-1acking 30S subunits of E. coil T A l l 4 are also impaired in their capacity to carry out template-dependent binding of aminoacyl-tRNA and to form two intermediates of the translational initiation pathway, i.e., the 30S and the 70S initiation complexes. Materials and Methods Buffers Buffer A: 11 mM Tris-HC1 (pH 7.7), 100 mM NH4C1, 15 mM magnesium acetate, 1 mM dithiothreitol. Buffer B: 50 mM Tris-HC1 (pH 7.7), 150 mM NHaC1, 7 mM magnesium acetate, 1 mM dithiothreitol. Buffer C: 20 ram triethanolamine-HC1 (pH 7.8), 100 mM NH4C1, 15 mM magnesium acetate, 6 mM fl-mercaptoethanol. Buffer D: 20 mM triethanolamine-HCl (pH 7.8), 30 mM KC1, 15 mM magnesium acetate, 6 mM /3-mercaptoethanol. Cells Wild-type (WT) E. coil CP78 and mutant strain TAl14 [3] missing ribosomal protein $20 were grown at 37 °C in 60 1 culture medium containing (per liter): 9.2 g K2HPO 4, 2.7 g KH2PO 4, 0.5 g Na 3 citrate, 0.1 g MgSO4 • 7H20, 10 g tryptone, 5 g yeast extract and 5 g

0167-4781/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

94 glucose. When the culture reached A650 ~ 1, the cells were harvested by continuous-flow centrifugation. The generation time at 37 °C was approx. 20 min for WT and 100 min for the mutant E. coli cells. Screening strain T A l l 4 for the presence of revertants or contaminants was routinely carried out by incubation at 42°C which is lethal for this strain.

General preparations Ribosomes and ribosomal subunits [5], purified ribosomal protein $20 [6], translation initiation factors IF1, IF2 and IF3 [7], NAc[14C]Phe-tRNA and f[aH]Met-tRNA~et [8] were prepared essentially as described. Amino acid incorporation Poly(U)-dependent polyphenylalanine synthesis was carded out in 0.1 ml of Buffer A containing 2 mM ATP, 0.4 mM GTP, 16 mM phosphoenolpyruvate, 1.8 /~g pyruvate kinase, 15/~g poly(U), 160 l~g E. coil MRE600 tRNA mixture, 2.2 nmol [14C]phenylalanine (Amersham, uniformly labeled) 50 mCi/mmol as well as an optimized amount of an E. coli MRE600 $100 postribosomal fraction. The mixtures also contained 20 pmol each of 30S and 50S ribosomal subunits obtained from E. coil WT CP78 or mutant TAl14 cells, as indicated in each experiment. After incubation at 37 °C for 10 rain, the hot-TCA-insoluble radioactivity present in 75/tl of each sample was determined. Aminoacyl-tRNA binding to ribosomes (a) Enzymatic poly(U)-dependent binding of NAcPhetRNA. The reaction mixtures (0.05 ml of Buffer B) contained 1 mM GTP, 10 /~g poly(U), 50 pmol NAc[14C]Phe-tRNA (780 cpm/pmol), 13 pmol 30S subunits (either WT or mutant), 20 pmol each of IF1, IF2

and IF3 and, when appropriate, 15 pmol of WT 50S subunits. (b) Enzymatic poly(A UG)-dependent binding of fMettRNA. The reaction mixtures were identical to those described above, but for the fact that 10/~g of random poly(AUG) and 1-30 pmol f[3H]Met-tRNA (9200 cpm/pmol) were used instead of poly(U) and NAcPhetRNA, respectively. (c) Non-enzymatic poly(U)-dependent binding o/ NAcPhe-tRNA. The reaction mixtures (0.05 ml in Buffer C) contained 20 #g poly(U), 16 pmol NAc[14C]PhetRNA (780 cpm/pmol), 30 pmol 30S subunits (either WT or mutant).

Binding of polynucleotides to 30S subunits Each reaction mixture (0.05 ml of Buffer D) contained 18/~g of either [3H]poly(U) (1000 cpm//~g) or [32p]poly(AUG) (6300 cpm//Lg) and 0.05-2.0 A260 units of 30S subunits (either WT or mutant) as indicated in Fig. 6A,B. After 15 rain incubation at 2 - 4 ° C , each sample was diluted with 3 ml of Buffer C and the amount of ribosome-bound template was determined by filtration through alkali-treated nitrocellulose filters [9]. Results and Discussion

Protein $20 is a component of the 30S ribosomal subunit but has been occasionally found to be associated with the large subunit from which it has been characterized as protein L26 (see Ref. 10). We w e r e unable, however, to detect $20 in association with the 50S subunits. The absence of ribosomal protein $20 in the E. coli TAl14 mutant was first ascertained by two-dimensional electrophoresis. Compared to the WT 30S (Fig. 1A), ribosomal protein $20 was completely absent in the TP30 fraction obtained from the 30 S

Fig. 1, Two-dimensional e,lvctrophorztic analysis of TP30 from E. coli CP78 (A) and Tl14 (B). Electrophoresis was carried out according to th~ method of C.~yl et al. [11] with an amount of TP30 corresponding to 3 A~,0 units of ribosomal subunits. The arrow indicates the position o protein $20.

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Fig. 2. Poly(U)-dependent polyphenylalanine synthesis by WT and S20-1acking ribosomes. (A) Time course of Phe incorporation under reaction conditions identical to those described in Materials and Methods with (0) WT and (o) S20-lacking 30S subunits. (B) Recovery of synthetic activity upon incubation (5 rain at 50 o C) of the 30S(-$20) subunits with the indicated amounts of purified $20 protein. The activity of the mutant 30S subunits is expressed as percent of WT 30S controls. (C) Mg2+-dependence of Phe incorporation with (@) WT and (o) S20-lacking 30S subunits. The reaction conditions were those described in Materials and Methods but for the concentration of magnesium acetate which was varied as indicated in the figure. (D) Activity of S20-lacking ribosomes as a function of the Mg 2+ concentration (expressed as percent of WT controls).

change of components in the reaction mixture clearly indicated that the reduction in the synthetic activity is solely attributable to the small subunit of the mutant cells (not shown). The activity of these S20-1acking ribosomes ranges from approx. 20% to 50~ of the controls, depending on the Mg 2+ concentration (Fig. 2D) and is entirely due to the absence of this protein, since full activity can be restored upon incubation of the S20-1acking 30S subunits with purified protein $20 (Fig. 2B). Since it has been shown that 30S(-$20) subunits are impaired in their capacity to associate with 50S subunits [5], their reduced synthetic activity could stem from this defect. The impaired subunit association capacity of 30~(-$20), however, is strongly affected by the Mg 2+ concentration and can be overcome, to a large extent, by high concentrations (i.e., 20 mM) of this cation (Fig. 3). By contrast, as seen in Fig. 2D, recovery of protein synthetic activity at the same Mg 2+ concentration is only marginal (= 40~) suggesting that other 30S activities are also affected by the lack of protein $20. Thus, several partial reactions of translation, exclusively involving the small subunit, were investigated. As seen in Fig. 4, the capacity of 30S(-$20) subunits to bind non-enzymatically NAcPhe-tRNA in response to poly(U) is strongly reduced at all K + and Mg 2+ concentrations tested. Unlike the 30S WT, which shows an optimum activity in NAcPhe-tRNA binding at approx. 120 mM KC1, the activity of 30S(-$20) displays no optimum, but shows a smooth decline as the KC1 concentration is increased (Fig. 4A). Furthermore, the activity of 30S WT reaches a maximum and levels off between 12 and 13 mM Mg 2+, while that of 30S(-$20) continues to increase up to 21 mM Mg 2+ (Fig. 4B). As a consequence, increasing the Mg 2+ concentration from >.

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subunits of the mutant (Fig. 1B); the 50S ribosomal subunits and an acetic acid extract of the post-ribosomal supernatant of E. coli TAl14 were also found to be devoid of $20 (not shown) and, as mentioned above, no immunological reaction with anti-S20 was detected in cell extracts from the T A l l 4 mutant. Furthermore, reconstitution experiments carried out with ribosomal subunits from the T A l l 4 strain and with purified ribosomal protein $20 showed that the protein can reconstitute into the small (but not the large) subunit of the mutant ribosomes (not shown). The translational activity of 50S WT-30S(-S20) couples is strongly reduced in comparison to that of 50S WT-30S WT couples. This can be seen in the experiments shown in Fig. 2 where the time course (Fig. 2A) and the Mg 2+ dependence of poly(U)-dependent polyphenylalanine synthesis (Fig. 2C) were measured; ex-

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Fig. 3. Subunit association capacity of 30S(-$20) as a function of the indicated Mg 2+ concentrations. The subunit association test was carried out using the nitrocellulose filtration method which is based on the subunit association-dependent protection of 30S-bound NAcPhe-tRNA from peptidyl-tRNA hydrola~ [5,12]. The figure presents the association capacity of 30S(-$20) relative to that of WT 30S (redrawn from Ref. 5).

96 5 to 21 mM, the activity of 30S(-$20) increases from less than 20% to approx. 60% of 30S WT subunits without, however, reaching full recovery (Fig. 4C); this behavior seems to resemble more closely that displayed by overall protein synthesis (Fig. 2D) than that seen in subunit association activity (Fig. 3). Under conditions resembling those of translation initiation, the ribosomal binding of NAcPhe-tRNA is also impaired in S20-1acking 30S subunits. The reduction of activity under these conditions is even more pronounced than in the previous case as seen from the time course of NAcPhe-tRNA binding in the absence of initiation factors (Fig. 4D), in the presence of IF1, IF2 and IF3 (Fig. 4E) or with only IF1 and IF2 (Fig. ,4F). Similar defects were also observed when WT 50S-30S($20) couples were tested for their template-dependent aminoacyl-tRNA binding under both enzymatic and non-enzymatic conditions (not shown). As with NAcPhe-tRNA, binding of fMet-tRNAfM~t to form 30S and 70S initiation complexes was also severely affected with the S20-1acking 30S subunits; the impairment of this function was not alleviated by increasing amounts of initiation factors (Fig. 5) or fMet-

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1½0 0 time (see) Fig. 4. Poly(U)-dependeat binding of NAcPhe-tRNA to ribosomes. The experimental conditions are described in Materials and Methods. Panels A-C show NAcPhe-tRNA binding under non-enzymatic conditions as a function of the indicated concentrations of (A)KCI and (B) magne~um acetate; (C) activity of S20-11meidng30S as a function of the Mg 2+ concentration relative to that of WT 30S. Panels D - F show the time course of NAc[14C]Phe-tRNA binding under enzymatic conditions. (D) Initiation factors omitted, (E) complete system, (F) IF3 omitted. Symbols: (O) WI" 30S, (o) 30S(-$20).

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2o ~0 60 80 initi0ti0nf0ct0rzofferedIpm0t} Fig. 5. Poly(AUG)-dependentbindingof fMet-tRNAto form30S and 70S initiation complexes.The binding reaction was carried out as a function of inew,asing amounts of initiation factors as indicated on the abscissa. Other conditions were as described in Materials and Methods.(e) 50S WT-30S WT; (o) 50S WT-30S(-S20), (I) 30S WT; (t]) 30S(-$20).

tRNA (not shown). This suggests that the reduced activity of the 30S(-$20) subunits does not stem from a reduced affinity for either initiation factors or initiator tRNA. Formation of 30S initiation complexes, both model (containing NAcPhe-tRNA and poly(U)) and physiological (containing fMet-tRNA and mRNA) [13,14], proceeds through the random-order formation of two binary complexes (30S-template and 30S-aminoacyltRNA) followed by the binding of the second ligand (aminoacyl-tRNA or template, respectively) to form a pre-ternary complex in which the two ribosomal [igands are not yet mutually interacting. A rate-limiting firstorder isomerization of the complex results in codon-anticodon interaction and formation of the 30S initiation complex. To determine which of these steps is affected by the absence of protein $20, we measured the capacity of the 30S subunits to bind radioactive template and the initial rates of 30S initiation complex formation as a function of increasing concentrations of initiator tRNA. Compared to WT 30S, neither the ribosomal binding of poly(U) (Fig. 6A) nor that of poly(AUG) (Fig. 6B) was found to be impaired in the 30S(-$20)subunits; likewise, the affinity of the 30S(-S20) subunit for fMett R N A was found to be the same as that of W T 30S as indicated by their common intercept on the abscissa in the double reciprocal plot of the kinetic data presented in Fig. 6C. The 30S subunits from WT and T A l l 4 cells, on the other hand, differ in the rate at which they carry out the isomerization step, as indicated by the different intercepts on the ordinate (Fig. 6C). Separate e x p e r i m e n t s ( n o t s h o w n ) have also d e m o n strated that the 30S(.S20) subunlts are not o n l y slower than W T 30S in p r o m o t i n g a stable interaction b e t w e e n

fMet-tRNA and template on their matrix, but also allow a more rapid dissociation of the ternary complexes which they form.

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This work was supported in part by grants from the Italian Ministry of Public Education and National Research Council (CNR) to C.O.G. Target project on Biotechnology and Bio-instrumentation.

References

O

4

T A l l 4 30S subunits and, in turn, the 5-fold reduction in the generation time displayed by "TAll4 cells. Thus, it is conceivable that the reduction of the on-rate of fMet-tRNA binding and the increase in the corresponding dissociation rate results in a critical shortening of the average half-life of the 30S initiation complexes which, coupled with the defect in the ensuing subunit association step, could lead to a severe (or even lethal at the non-permissive temperature) reduction of translation of some essential genes. Experiments are now in progress to test this hypothesis in vivo and in vitro.

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15

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Fig. 6. Template and initiator tRNA bind normally to 30S(-$20), but 30S initiation complex formation proceeds at a reduced rate. Retention of radioactive (A) poly(U) and (B) random poly(AUG) by nitrocellulose filters [9] as a function of increasing amounts of 30S subunits. (C) Initial rate of 30S initiation complex formation as a function of increasing concentrations ((0.5-5.0).10 -8 M) of fMettRNA naet. The reaction was carried out as previously described [13,14]. (It) 30S WT; (o) 30S(-$20).

Protein $20 has been localized in a region of the 30S subunit presumed to be in contact with the 50S subunit, just at the edge of the cleft in which the P-site codonanticodon interaction is believed to occur [15,16]. Thus, this topographical localization appears to be consistent with a defect in both subunit association and codonanticodon interaction at the P-site of the 30S(-$20) subunits. The restricted capacity of the 30S(-$20) subunits to bind peptidyl-tRNA and fMet-tRNA at the P-site, in combination with their faulty subunit association capacity can explain the reduced translational activity of

1 Wittmann, H.G., StSffler, G., Piepersberg, W., Buckel, P., Ruffler, D. and Bt~ck, A. (1974) MoL Gen. Genet. 134, 225-236. 2 Isono, S., Isono, K. and Hirota, Y. (1978) MoL Gen. Genet. 165, 15-20. 3 Dabbs, E. (1978) Mol. Gen. Genet. 165, 73-78. 4 Dabbs, E.R., Hasenbank, R., Kastner, B., Rak, K.H., Wartusch, B. and StSffler, G. (1983) Mol. Gen. Genet. 192, 301-308. 5 GOtz, F., Fleischer, C., Pon, C.L. and Gualerzi, C.O. (1989) Eur. J. Biochem. 183, 19-24. 6 Muto, A., Ehresmann, C., Fellner, P. and Zimmermann, R.A. (1974) J. Mol. Biol. 86, 411-432. 7 Pawlik, R.T., Littlechild, J., Pon, C.L. and Gualerzi, C. (1981) Biochem. Int. 2, 421-428. 80hsawa, H. and Gualerzi, C. (1983) J. Biol. Chem. 258, 150-156. 9 Smolarsky, M. and Tal, M. (1970) Biochim. Biophys. Acta 199, 447-452. 10 Trant, R.R., Lambert, J.M., Boileau, G. and Kenny, J.W. (1980) in Ribosomes: Structure, Function, and Genetics (Chambliss, G., Craven, G.R., Davies, J., Davis, K., Kahan, L. and Nomura, M., eds.), pp. 89-110, University Park Press, Baltimore. 11 Geyl, D., BiSck, A. and Isono, K. (1981) Mol. Gen. Genet. 119, 309-312. 12 Gualerzi, C., Hundertmark, U. and Pon, C.L. (1980) Biochem. Int. 1, 553-560. 13 Gualerzi, C., Risuleo, G. and Pon, C.L. (1977) Biochemistry 16, 1684-1689. 14 Calogero, R.A., Pon, C.L., Canonaco, M.A. and Gualerzi, C.O. (1988) Proc. Natl. Acad. SCi. USA 85, 6427-6431. 15 Capel, M.S., Engelman, D.M., Freeborn, B.R., Kjeldgaard, M., Langer, J.A., Ramakrishnan, V., Schindler, D.G., Sclmeider, D.K., Schoenborn, B.P., Sillers, I.Y., Yabuki, S. and Moore, P.B. (1987) Science 238, 1403-1406. 16 Brimacombe, R. (1988) Biochemistry 27, 4207-4214.