fraction (5-10%) of the 70 S and 100 S ribosomes, called "active" ribosomes, participate MESSENGER-RNA ATTACHMENT TO ACTIVE RIBOSOMES*

Transcription

1 430 BIOCHEMISTRY: RISEBROUGH, TISSIARES, AND WATSON PROC. N. A. S. 16 In a current experiment, we have found that a protein fraction isolated from the 105,000 X g supernatant can catalyze a similar RNA synthesis in the presence of four nucleoside triphosphates and the E. coli DNA prepared by the method of Marmur (J. Mol. Biol., 3, 208, 1961). In this system also, the addition of salmine enhances the initial rate of the synthesis and prevents the degradation of the product. The rate is about comparable to that of the crude system. However the reaction practically ceases after ten minutes even if the nucleoside triphosphates are further added at 10-min intervals. The ratio of the synthesized RNA to the DNA added is around 0.5. The result might be explained by assuming a factor necessary for the liberation of the synthesized RNA from the DNA. In the crude system, the RNA would be continuously liberated from DNA in the presence of the "factor;" in the "purified" system, the RNA would not be liberated because of the absence of the "factor." 17 Mandell, J. D., and A. D. Hershey, Anal. Biochem., 1, 66 (1960). 18 Philipson, L., J. Gen. Physiol., 44, 899 (1961 ). 19 Britten, R. J., and R. B. Roberts, Science, 131, 32 (1960). 20 Ishihama, A., unpublished experiment (1961). MESSENGER-RNA ATTACHMENT TO ACTIVE RIBOSOMES* BY R. W. RISEBROUGH, A. TIssIARES, AND J. D. WATSON THE BIOLOGICAL LABORATORIES, HARVARD UTNIVERSITY Communicated by John T. Edsall, January 25, 1962 Several kinds of evidence support the view that metabolically unstable RNA molecules, messenger-rna, carry the information for the synthesis of specific proteins. 1-4 In this concept, messenger-rna (mrna), made on a DNA template, attaches to a ribosome where the synthesis of a protein molecule can then take place. It is likely that the average molecular weight of mrna molecules is at least 2.5 X Thus, attachment to ribosomes should lead to the presence of "heavy ribosomes," which may sediment faster than free ribosomes. Since only approximately 2 per cent of total E. coli RNA is mrna, less than 10 per cent of the ribosomes should have attached mrna. This explains the finding that a correspondingly small fraction (5-10%) of the 70 S and 100 S ribosomes, called "active" ribosomes, participate in protein synthesis by E. coli extracts.6 Only those ribosomes complexed with mrna are able to synthesize protein. Below we report experiments confirming this picture by showing that active ribosomes, in the process of synthesizing protein, have attached mrna and sediment faster than ordinary ribosomes. Materials and Methods.-All experiments employed Escherichia coli Strain B, except those in which mrna was labeled. For these, the pyrimidine requiring strain (B148) (kindly supplied by Dr. Martin Lubin) of E. coli was used. Cells were grown with strong aeration at 37 C in a medium consisting of 10-2 M tris, ph 7.4, 5 X 10-4 M Na2HPO4, 10-4 M CaC12, 10-3 M MgSO M FeCl3 and 5 gm vitamin-free casamino acids, 5 gm NaCl, 1 gm NH4Cl, and 4 ml glycero, per liter of distilled water. Addition of 4 ug uracil/ml to the medium permits B148 growth to about 4 X 108 cells/ml (ODn5o = 0.4). In this medium both B and B148 (in excess uracil) have a generation time, at 37 C, of 30 min. Labeled mrna was prepared either by a sec exposure of C'4-uracil to uninfected cells,4 or by a 1-3 min exposure to cells infected three minutes previously with phage T.2.4 Labeling of uninfected cells was always preceded by a min uracil starvation at 4 X 108 cells/ml, following which 10-20,C of C'4-uracil was added per liter of culture. After 20 sec, the cells were poured on crushed ice to cool rapidly to 1 C, centrifuged and washed in 5 X 10-3 M tris buffer, ph 7.4, containing 10-3 M Mg++. At this stage, the cells (approximately 0.5 gm/liter of medium) were

2 VOL. 48, 1962 BIOCHEMISTRY: RISEBROUGH, TISSIPRES, AND WATSON 431 usually frozen and stored at -15'C. Labeling of phage infected cells followed the same procedure, except that longer exposures to the isotope were possible, since phage T2 infection stops simultaneous synthesis of ribosomal and soluble RNA.2-4 A T2 multiplicity of 20 was used to ensure that most bacteria were quickly infected. Usually phage absorption was greater than 90 per cent and the number of uninfected bacteria less than 0.1 per cent..both logarithmically growing B148 cells (in excess uracil) and starved cells were used. When starved cells were used, cold uracil was added with the phage. No differences were detected between these two methods of labeling. The burst size in these experiments averaged 25. The preparation of crude extracts, of sucrose gradients, and the counting procedure for radioactive samples have been described earlier.4' 6 The reaction mixture for the study of amino acid incorporation into proteins consisted of M ATP, M phosphoenol pyruvate (PEP), 40 jg pyruvate kinase (PK), M KCl, M or 0.01 M Mg acetate, M tris buffer, ph 7.4; 0.25 to 5,uC of C14 labeled chlorella protein hydrolysate and 0.5 to 0.7 ml crude bacterial extract, containing 3-6 mg ribosomes, in a total volume of 0.72 to 1.0 ml. Deoxyribonuclease (2 X recrystallized) and ribonuclease (1 X recrystallized) were obtained from the Worthington Biochemical Company, Freehold, New Jersey. C14-urill (7.6,uC/,umole) was a product of the California Corporation for Biochemical Research, Los Angeles. The C'4-chlorella protein hydrolysate (1,uC/5 usg) was supplied by the Volk Radio-Chemical Company, Chicago, Illinois. Results.-(a) Stability of mrna in cell extracts: Extracts containing labeled '15 32S 7 Pt a-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~a 11-.,, ~~~~~~~~~~~~~~501S 1000 ~~~~~~~~~~~~~~~~~~~~~~~~240 to acid souble produts. T2 infcted cellsl,id 70 were exposed to C'4-Uracil 3-5 min following infection. The labeled cells were then har-tuenmr vested and stored frozen, as described under FIG. 2.-Sedimentation in a sucrose gradi- Materials and Methods. Several days later ent of T2 mrna bound to ribosomes. A cellthe frozen cells were ground with alumina and free extract, similar to that described in a cell-free extract prepared at 2 C in 5 X Figure 1, was prepared in 5 X 10-3 M tris 10-3 M tris buffer, ph 7.4, containing 10-2 M buffer, ph 7.4, containing 10-2 M MgSO4. MgSO4. Deoxyribonuclease (10,&sg/ml) was Following addition of DNase and several low then added and the extract clarified of alu- speed centrifugations, 1 ml. of the clarified mina, unbroken cells, and cell debris by two extract was layered on top of a 24 ml sucrose 15-min centrifugations at 10,000 g. The gradient (exponential 5-20%) containing clarified extract was then 'dialyzed 5 hr 10-2 M MgSO4 and 5 X 10-3 M tris buffer, against two changes of 5 X 10-3 M tris buffer, ph 7.4, in a Spinco SW25 swinging bucket ph 7.4, containing 10-2 M MgSO4. Ali- tube. The gradient solution was centrifuged quots were then incubated at 20, 24, and 3 hr at24,000 rpm. The bottom of the tube 37 C for various intervals, after which the was then punctured with a 22 gauge 1/4 inch breakdown reaction was terminated by addi- hypodermic needle, which was sealed to the tion of cold 5% TCA. The remaining TCA tube with grease. Ten-drop samples were precipitable radioactivity was then collected taken to measure the absorption at 2600 X (2 on millipore filters (pore size = 0.65,u) and drops in 3 ml H20) and the 5% TCA precipicounted to at least 1,000 cts. table radioactivity (8 drops).

3 432 BIOCHEMISTRY: RISEBROUGH, TISSIERES, AND WATSON PROC. N. A. S. mrna were incubated at several temperatures and mrna breakdown to acid soluble products measured (Fig. 1). At 40 the extracts were very stable; less than 5 per cent of the mrna became soluble after 24 hours. But at both 250 and 370 there was a marks breakdown,7 which increased with temperature. At 370, over 30 per cent of the ynrna became acid soluble within 30 minutes. The breakdown rate did not vary greatly with the concentration of the extract: dilution of a crude extract, containing 4.5 mg ribosomes/ml, by a factor of 5 decreased only slightly (10%) the initial breakdown rate. Under all conditions, the breakdown rate decreased with time and approximately 25 per cent of the RNA label remained acid insoluble after 18 hours of incubation. Messenger-RNA preparations must thus be kept cold when their attachment to ribosomes is studied. Unless otherwise indicated, all experiments reported below were done at 0-40, including use of sucrose gradients precooled to 20. (b) Sedimentation f ribosomal-bound messenger-rna: In solutions containing 10-2 M Mg++ most mrna is bound to 100 S and 70 S ribosomes. This is illustrated in Figure 2, which shows the sedimentation through a sucrose gradient of a crude extract containing labeled T2 mrna. Experiments with pulse labeled E. coli give a similar pattern. Most of the mrna moves faster than the free ribosomes, suggesting the presence of "heavy" ribosomes sedimenting faster because of associated mrna. The mrna is mainly found in "heavy 100 S ribosomes"; there is always less mrna (per unit of ribosomal RNA) bound to 70 S ribosomes than to 100 S ribosomes. Some variation is observed in the rate at which the complex mrna-ribosomes sediment: though the majority of "heavy" ribosomes sediment.150.2q0,'i0os 20 -OS TOS~~~~~~~~~~~~~~~0 CPM0-5 -0D i oo0s 3"00 TUBE ~~~~~~~~~~~~~~~~~~~~~~~~~~~0 NUMBER ~~~~~~~~~~~~~~~~~~ , o TUBE NUMBER FIG. 3.-Sedimentation of T2 mrna bound TUBE NUMBER to washed ribosomes. A clarified cell-free ex- FIG. 4.-Effect of incubation at 30 C on tract, similar to that described in Figure 2, sedimentation of ribosomal bound T2 mrna. was centrifuged one hr at 40,000 rpm in a 0.5 ml of the same C14 labeled washed ribo- Spinco #40 rotor. The supernatant was dis- some suspension, used in the experiment decarded and the pellet taken up in 5 X 10-3 M scribed in Figure 3, was mixed with 0.25 ml of tris buffer, ph 7.4, containing 10-2 M MgSO4. unlabeled supernatant. The mixture was The resuspended ribosomes were again cen- then incubated for 15 min at 300, layered on a trifuged at 40,000 rpm for one hr, after which pre-cooled sucrose gradient and run and anathe pellet was resuspended in the above tris- lysed as in Figure 2. The unlabeled super- Mg++ buffer. 0.5 ml of the washed ribosome natant was prepared by centrifuging a cellsuspension was layered on a 24 ml sucrose free extract (in 10-3 M Mg++) for 2 hr at gradient and centrifuged for 3 hr at 24,000 40,000 rpm. The upper 2/3 of the supernarpm, then analysed as in Figure 2. tant was used.

4 VOL. 48, 1962 BIOCHEMISTRY: RISEBROUGH, TISSIPRES, AND WATSON 433 about 5-10 S faster than free ribosomes, a variable fraction sediments much faster, suggesting that mrna-complexed ribosomes aggregate more easily than free ribosomes. In Figure 3 is shown a sucrose gradient centrifugation ofa washed ribosome preparation with bound labeled mrna. The ribosomes were Washed once in 10-2 M Mg++ tris buffer and resuspended in the same mixture. Here again, the peak of mrna moves about 10 S faster than free ribosomes: Raising the temperature of either crude extracts or washed ribosomes resuspended in unlabeled supernatant caused a marked change in the sedimentation of labeled mrna. Fiure 4 shows the sedimentation of labeled, washed ribosomes, resuspended in cold supernatant and incubated 15 minutes at 300. During this incubation, 30 per cent of the mrna was degraded to acid insoluble form. Under these conditions the mrna sedimented exactly as the ribosomes, with more label attached tothe 70 S than to the 100 S ribosomes. This figure is similar to those which we ol ined previously with extracts centrifuged through sucrose gradients at room temperature. It now seems clear that these earlier results4' I reflected mrna degradation and that, normally, addition of mrna to ribosomes increases their sedimentation rate. loos.300r D 90 # 70S i CPM So tprtenthouha ucoe raint S0 10 BS IU2OE S OS CIPM-1-00 Amino acids were incorporated into protein / 1 in a reaction mixture consisting of 0.7 ml 1 50 crude extract, 1 ym ATP, 5,uM PEP, 40,ug.10\ t 60 PKA 0.5auC chlorella hydrolysate diluted protein into a mixture of 10,ug of each of 18 common amino acids, M KCl, M Mg acetate, M tris buffer, ph 7.4, in a total volume, of 1.0 ml. Crude extracts were kept chilled in an ice bath until use. After a 5-min in- TUBE NUMBER cubation at 30 C, the incorporation reaction FIG. 6.-Effect of ribonuclease on nascent was stopped by chilling to 0 C. It was then protein sedimentation. An extract, similar layered on top of a pre-cooled sucrose gradient, to that employed in Figure 5, incorporated centrifuged for 3 1/2 hr at 24,000 rpm and C"-labeled amino acids into protein. Imdrops collected and analysed as in Figure 2. mediately following the 5-min incorporation Control experiments indicate that virtually reaction at 300C, ribonuclease was added at a all the counts in the ribosome region are in final concentration of 25 Mg/ml. After allowprotein and do not represent amino acids at- ing one minute for enzyme action, the treated tached to soluble RNA. About one half the extract was chilled in an ice bath, layered on a counts at the top of the centrifuge tube are in pre-cooled sucrose gradient, and centrifuged newly made soluble protein. The remainder for 3 1/2 hours at 24,000 rpm. Drops were are bound to soluble RNA. collected and analysed as in Figure 2.

5 434 BIOCHEMISTRY: RISEBROUGH, TISSIJRES, AND WATSON PROC. N. A. S. (c) Sedimentation of "active" ribosomes: Approximately two-thirds of the protein synthesized by E coli extracts remains bound, at Mg++ concentrations greater than 10-4 M, to the 70 S and 100 S ribosomes on which it is synthesized.6 Figure 5 shows the sedimentation through a sucrose gradient of a crude extract containing ribosomal bound nascent protein labeled during a 5 minute in vitro incorporation reaction at 300. Most ribosomal bound nascent protein sedimented faster than the majority of the ribosomes. This confirms the conclusions of Tissieres et al.,6 that at any one time only a small fraction of the ribosomes is "active," that is, involved in incorporating amino acids into proteins. The higher sedimentation rate of the "active" ribosomes suggested that they contained mrna. Ribonuclease (12 Mg/ml) was therefore added to a crude extract, after it had incorporated labeled amino acids for 5 minutes at 300 (mrna, in contrast to ribosomal RNA, is very sensitive to ribonuclease4). In Figure 6, it is seen that following the addition of ribonuclease, the "*tive" ribosomes now sediment only slightly faster than ordinary ribosomes. It is thus clear that the faster sedimentation rate of the untreated "active" ribosomes is due to attached mrna. This conclusion is further confirmed by examining extracts which were made to incorporate amino acids in 10-2 M Mg++, and were then dialyzed for three hours at 10-4 M Mg++. At this low Mg++ concentration, most mrna molecules dissociate from ribosomes.3' 4 When these extracts were run in sucrose gradients (Fig. 7), most of the bound nascent protein sedimented at 70 S. A variable fraction sedimented faster, suggesting that all the mrna had not left the ribosomes, a finding previously shown by the experiments of GrotVat.4 The fact that active ribosomes sediment at 70 S when they have lost their mrna suggests that the failure of active ribosomes to break down to their 30 S and 50 S subunits (Tissibres et al.6) is not due 5SO 400-~~~~~~~I 0 -'00 ~~~90 *1 / J 9' a70s :X GPM I60 FIG. 7.-Effect ofdalssgist1-4mfg.8-efc60 o 0 I0 CPMadi Q.240o Fu h a 100S s f ~ ~~~~~~~~~~~~~~~~. ~ M / P ~~~~~~~~~~~.I00-d TUBE NUMBER, TUBE NUMBER FIG. 7.-Effect of dialysis. against 10O4 M FIG. 8.-Effect of addition of mrna upon Mg ++ on nascent protein sedimentation. An sedimentation of ribosomal bound nascent extract incorporated C14-labe'led amino acids protein. A C14 amino acid labeled extract, into proteins under conditions similar to those similar to that of Figure 7, was dialysed for of Figure 5. It was then dialysed for 3 1/2 7 1/2 hours against 5 X 10-3 M tris, ph 7.4, hours at 2VC against 5 X 10-3 M tris, ph 7.4, and 10-4 M Mg++. 1 ml was then brought and 10- M Mg acetate. 1 ml was then cen- to 10-2 M Mg++, layered on a pre-cooled sutrifuged through a sucrose gradient for 31/2 hr crose gradient and centrifuged for 31/4 hours at 24,000 rpm. Drops were then collected at 24,000 rpm. Drops collected and analysed and analysed as in Figure 2. as in Figure 2.

6 VOL. 48, 1962 BIOCHEMISTRY: RISEBROUGH, TISSIPRES, AND WATSON 435 to mrna binding, but instead may relate to the presence of growing polypeptide chains. When extracts dialyzed against 10-4 M Mg++, so that most mrna had left the ribosomes, are brought up again to 10-2 M Mg++, most of the active ribosomes sediment like heavy 100 S and 70 S ribosomes (Fig. 8). This suggests that mrna tends preferentially to combine with "active" ribosomes. Discussion.-In extracts made in 10-4 M Mg++, most mrna is not bound to ribosomes. It sediments as a broad peak, a sign of heterogeneity, with an average sedimentation constants of S.4 Originally, it was thought2' 4 that phenol purified mrna sedimented more slowly (about 8 S) than the unpurified material observed in crude extracts. However, more recent experiments designed to minimize degradation during extract preparation show that the average sedimentation constant of phenol prepared mrna is also S. Since the molecular form of mrna is not known, it is not possible to estimate its molecular weight from the sedimentation behavior. If it sediments, like other RNA, as a random coil, its sedimentation rate suggests9 an average molecular weight of 500,000, with some molecules at least twice this size. Alternatively, if mrna molecules are rigid DNA-like structures, then they must be of even larger sizes. The possibility must also be considered that free mrna molecules are more tightly collapsed than ribosomal or soluble RNA chains. If so, their average molecular weight might be as low as 250,000 (750 nucleotides). All these values are welf within the size necessary to code for a protein molecule of ,000 MW, (assuming a-coding ratio of 3 nucleotides to one amino acid).'0 The presence of attached mrna is clearly responsible for the faster sedimentation of "active" ribosomes which are synthesizing protein. Removal of their bound mrna, either by ribonuclease treatment or by lowering the surrounding Mg++ concentration, changes their sedimentation constant to approximately thiat of, free ribosomes. Active ribosomes are thus also "heavy" ribosomes. Comparison of "heavy" ribosome sedimentation with that of total ribosomes indicates that most mrna is bound to not more than 10-20% of the total ribosomes. This number compares well with the predicted value (6%) based on the presumed amount of mrna (2% of total RNA) and on a probable average molecular weight of 5 X 105. This latter value may be a minimal estimate, both because there are indications7 that 4 per cent may be a more correct value for mrna content, and because many ribosomes may have attached relatively short RNA chains.- At -any given moment mrna chains are being made and broken down, and our extracts may contain many molecules "caught" in the process of degradation. In this case, at least 12 per cent of the 70 S ribosomes (or 24% if the ribosomes are in the 100 S form) would have mrna attached. The fraction of ribosomes which contain nascent protein is more accurately known. The fact that "active" ribosomes do not immediately break apart to their 30 S and 50 S subunits when suspended ini 10-4 M Mg++ allowed a direct determination' of their maximum percentage (5-10%) in the total ribosome population. It is thus possible that the proportions of heavy and active ribosomes are identical and that in our in vitro systems all ribosomes containing mrna participate in protein synthesis. Our results suggest that all "heavy" ribosomes do not sediment at similar rates. Some of the variability may be caused by a tendency for mrna containing ribo-

7 436 BIOCHEMIST'RY: RISEBROUGH, TISSIERES, AND WATSON PROC. N. A. S. somes to aggregate. Another part may reflect size differences in their mrna components. Still another cause may be due to differences in heavy ribosome shape. Attention should be drawn to the fact (Fig. 4) that when heavy ribosomes are briefly incubated at 300, they then sediment at exactly the same rate as free ribosomes. This occurs even though the average sedimentation constant of the remaining undegraded ribosome-bound mrna, when released from ribosomes, remains almost the same. This would imply that shape factors are important in governing heavy ribosome sedimentation. There is a strong tendency for mrna to combine preferentially with 100 S ribosomes. Likewise, most nascent protein sediments as heavy 100 S ribosomes. This hints that 100 S ribosomes, not 70 S ribosomes, may be the principal sites of protein synthesis. However, since 100 S and 70 S ribosomes are easily interconvertible,11' 1 there now does not exist a method for deciding whether only one or both these ribosomes are the functional particles. Summary.-Attachment of messenger-rna to 100 S and 70 S ribosomes results in "heavy" 100 S (70 S) ribosomes which sediment faster than free ribosomes. Many, if not all, "heavy" ribosomes are also "active"; that is, capable of functioning as sites for protein synthesis. Only a small fraction (5-10%) of total ribosomes are "heavy" ("active"). Thus, it appears that, at any given moment, only a correspondingly small ribosome fraction functions in protein synthesis. The continued support of the National Science Foundation (Molecular Biology Division) and the National Institutes of Health is gratefully a&nowledged. We have greatly benefited from numerous discussions with Dr. W. Gilbert and from the technical assistance of Mrs. A. Nevins and Mrs. V. Tissieres. * The following abbreviations are used. RNA (ribonucleic acid); mrna (messenger-rna); D)NA (deoxyribonuieleic acid); DNase (deoxyribonuclease); TCA (trichloracetic acid); PEP (phosphoenol pyruvate); PK pyruvate kinase); and ATP (adenosine 5-triphosphoric acid.) l Jacob, F., and J. Monod., J. Mol. Biol., 3, 302 (1961). 2 Nomura, M., B. D). Hall, and S. S. Spiegelman, J. Mol. Biol., 2, 306 (1960). Brenner, S., F. Jacob, and M. Meselson, Nature, 190, 576 (1961). 4Gros, F., W. Gilbert, H. Hiatt, C. Kurland, R. W. Risebrough, and J. D. Watson, Nature, 190, 581 (1961). Gros, F. H. Hiatt, and K. Asano., unpublished experiments (1961). fitissibreps A., D. Schlessinger, and Francoise Gros, these PROCEEDINGS, 46, 1450 (1960). 7Cohen, S.:S., H. 1). Barner, and J. Lichtenstein, J. Biol. Chem., 236, 1448 (1961). 8 Gros, F., W. Gilbert, H. H. Hiatt, G. F. Attardi, P. F. Spahr, and J. D. Watson, in Cold Spring Harbor Symposia on Quantitative Biology, vol. 26 (1962), in press. 9 Hall, B. D., and P. M. Doty, J. Mol. Biol., 1, 111 (1959). 10 Crick, F. H. C., L. Barnett, S. Brenner, and R. J. Watts-Tobin, Nature, (1962, in press). 11 Tissibres, A., J. D. Watson, D. Schlessinger, and B. R. Hollingworth, J. Mol. Biol., 1, 222 (1959). 12 Roeten, E. T., B. H. Hoyer, and D. B. Ritter, Microsomal Particles and Protein Synthesis (New York: Pergamon Press, 1958), p. 18.