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Movement of ribosomes over messenger RNA in polysomes of rel+ and rel−Escherichia coli strains
J. Mol.
Biol.
(1973)
76, 163-179
Movement of Ribosomes over Messenger RNA in Polysomes of reZ+ and ret Escherichia coli Strains AUIX
J. COZZONE~ AND GUNTHER S. STE:NT
Department of Molecular University of California, ( Recehed
Biology and Virus Laboratory Berkeley, Calif. 94720, U.S.A.
30 June 1972, and in revised form 6 November
1972)
The in, vitro movement of ribosomes over messenger RNA was studied in both the presence and the absence of protein synthesis. For this purpose, labeled polysomes were extracted from rel+ and rel- strains of Escherichia coli grown in the presence of radioactive uracil and incubated in a cell-free system containing tRNA, amino acids, soluble enzymes and a source of energy. The gradual conversion of the labeled polysomes into monosomes and ribosomal subunits was followed by subjecting the reaction mixture to sucrose gradient sedimentation after various incubation times and measuring the radioactivity present in the three relevant ribosomal fractions. It was found that when the conditions of incubation allow protein synthesis to occur, polysomes extracted from reZ+ and rel- cells are converted mainly int,o free monosomes, which can be made t,o dissociate into subunits by high-sodium or low-magnesium ion concentrations. Under conditions in which protein synthesis ea,nnot occur because a mutant aminoacyl-tRNA synthetase has been rendered inactive, polysome conversion still occurs, though to a reduced extent. When the products of such residual run-off are examined, however, a difference is manifest between polysomes extracted from rel+ and from rel- strains : whereas the polysomes from the rel- strain are still converted into free monosomes even in the absence of protein synthesis, the polysomes from the reE+ strain are now eonverted mainly into subunits. It can be inferred, therefore, that ribosomes from relbacteria, but not those from rel+ bacteria, cont,inue movement over messenger RNA in the absence of protein synthesis. Studies of mixed extracts from rel- and rel + bacteria have shown that the character of the run-off process does not depend on the source of tRNA and soluble enzymes; the proportions of monosomes and subunits among the run-off products formed in the absence of protein synthesis depend only on the source of the polysomes. It. is suggested that the mutation of the ret! gene alters the functional a,rchiteeture of ribosomes.
1. Introduction Numerous
studies
rel+ and relacyl-tRNA. conditions as in rel(B’riesen, that
strains
have
been made
of Escherichia
In some of this work
of the level
of polysomes
coli deprived it was found
of either that
under
either
of deprivation, the level of polysomes remains bacteria, as compared to the level of polysomes 1968;
Sells & Ennis,
such deprivaeion
+ Present address:
1970).
However,
in most
leaves the level of polysomes Institut
de Chimie Biologique, 163
largely
Universit6
which
an amino
persists
of t,hese two
studies
unaffected de Provence,
different
in rel+ BS well growing 1~4s
unchanged in normally of these
in cella of
acid or an amino-
it was found
in rel-
bacteria,
;Marseille,
France.
164
A. J. COZZONE
AND
6. S. STENT
whereas under deprivation, reE+ bacteria polysomes are rapidly converted into monosomes and ribosomal subunits (Morris & DeMoss, 1966; Weber &, DeMoss, 1966; Ron et al., 1966; Matzura, 1970). The mechanism responsible for the control of the polysome level in such deprived cells is still not clear. It has been proposed that there exists a direct correlation between the level of polysomes and the net synthesis of RNA in rel - and rel+ strains of E. coli (Morris & DeMoss, 1966; Ron et al., 1966). But, contrary to that proposal, Friesen (1968) and Sells & Ennis (1970) have reported that the polysome level of amino acid-deprived cells is not correlated with the rate of residual RNA synthesis. According to Ron (1971), the level of polysomes in rel+ cells deprived of a particular amino acid is a function of the relative abundance of that amino acid in cellular proteins. Recently, it has been shown (Ron, 1971; Cozzone & Donini, 1973) that the polysomes which persist under amino-acyl-tRNA deprivation are dynamic structures in a steady-state condition of disassembly and re-assembly. For, when deprived cells are treated with rifampin, which prevents synthesis of all species of RNA (Hartmann et al., 1967), polysomes cannot re-assemble on nascent mRNA and they quickly disappear. One may assume, therefore, that the higher polysome level observed in deprived rel- cells, as compared to that of rel+ cells, is attributable to a higher rate of polysome re-assembly and/or to a lower rate of dis-assembly, the dis-assembly of polysomes being caused either by mRNA degradation or by ribosome run-off, or by both. In order to resolve, therefore, the previously reported discrepancies concerning the polysome level in rel-- and rel+ -deprived cells, the rates of d&assembly and re-assembly of polysomes must be measured and compared under these conditions . The rate of ribosome run-off can be measured in a cell-free system similar to that described by Matthaei & Nirenberg (1961). As was shown by No11 et al. (1963), when polysomes are incubated in vitro in a reaction mixture containing tRNA, amino acids, soluble enzymes and a source of energy, ribosomes move over the messenger RNA, and the amount of run-off products released from polysomes can be measured as a function of time by sucrose gradient analysis. The absolute value of the rate constant of in vitro ribosome run-off is very likely different from its value measured in an in viva situation. However, one can expect that the relative value of the rate constant obtained by comparing the rates of in vitro ribosome run-off in polysomes of retand reE+ cells is a reflection of its relative value in vivo. In the experiments presented in this paper, the mechanism of ribosome run-off has been studied in vitro. The purpose was to investigate whether or not the relative motion of ribosomes and messenger RNA occurs in rel- and rel + bacteria under aminoacyl-tRNA deprivation, and to estimate the rate of ribosome movement under these conditions. Polysomes isolated from both kinds of cells were therefore incubated under aminoacyl-tRNA deprivation, and their gradual in vitro conversion into run-off products was followed as a function of time.
2. Materials and Methods (a) Bacterial strains and growth conditiona Two pairs of otherwise isogenic sel’ and rel- strains of E. ooli were used. Both pairs are derivatives of strain D2 (Kaplan & Anderson, 1968). Strains 10B6 reZ+ and lOB6 rel-
IN
VITRO
RIBOSOME
If%
RUN-OFF
(Edlm & Stent, 1969) are arginine auxotrophs and have a temperature-sensitive valylt,RNA synthetase. The permissive temperature for these strains is 29°C. The prototropbic pair of strains D2 rel + and D2 rel - was derived from strain D2 by pha,ge Pl transduction (Edlin & Broda, 1968). Cells were grown at 29°C with forced aeration at pH 7.6 in a medium which contained 12 g Tris, 2 g KCl, 2 g NH,Cl, 0.5 g MgCla.6 H@, tha following components per liter: 0.02 g NaaSO,, 14.7 mg CaC1,.2 H,O, I78 mg Na,HPO,.2 H,O, 2 g glucose. This medium was supplemented with 50 pg arginine/ml when necessary. Growth was followed with a Klett-Summerson calorimeter, using a green filter. In experiments requiring long-term labeling of RNA, cells were grown for 2 to 3 generations in the presence of [3H]uracil (4 to 12 pg/ml; 0.4 to 1.0 PCijml) or [14C]uracil (4 to 12 pg/ml; 0.10 to 0.25 &X/ml). (b) Preparation
of supernatant
fractions
Supernatant fractions were prepared from cells of the four strains described above by a method similar to that of Matthaei & Nirenberg (1961). Exponentially growing cells were poured over ice and collected by centrifugation for 8 min at 8000 g. In some experiments, cultures had been shifted to 42.5”C for 5 min before collection. Cells were washed and disrupted by grinding with alumina (2.5 times the weight of washed cells). The soluble fraction wa,s extracted in buffer containing O-01 lx-Tris (pH 7.8), 0.06 in-KCI, 0.01 ivr-magnesium acetate and 0.006 M-mercaptoethanol (5 ml per g of washed cells). Alumina and cellular debris were removed by centrifugation at 20,000 g for 20 min. The supernatant fluid was treated with 5 pg DNase/ml, centrifuged again at 20,000 g for 20 min and finally at 30,000 g for 60 min. The liquid layer, fraction 530, was removed and oentrifuged at 105,000 g for 180 min to sediment the ribosomes. The top two-thirds of the supernatant fraction were removed, divided into portions, quickly frozen in an acetone-dry ice mixture and stored at -20°C until used. Fraction S105 thus prepared contained 19.7 to 25.7 mg protein/ml and 2.3 to 3.5 mg nucleic acid/ml. When fraction 530 was incubated before oentrifugation at 105,000 g, incubation was done for 45 min at 37°C in the presence of 400 rg ATP/ml, 240 pg GTP/ml, 40 pg pyruvate kinase/ml and 1.2 mg phosphoenol pyruvate/ml. Fraction S105 was dialyzed, when indicated, for 16 h against 100 vol. of the buffer with one buffer cha.nge.
(c) Preparation
of crude lysates and isolated
polysomes
The method of preparing crude lysates and isolated polysomes has been described elsewhere (Cozzone & Donini, 1973). Exponentially growing cells were poured over crushed ice chilled at -15°C in order to reduce ribosome run-off (Davis, 1971). All subsequent operations were done at 0 to 4°C. Cells were harvested by eentrifugation and resuspended in a sucrose-buffer solution containing 0.5 M-RNase-free sucrose, 0.016 ivr-Tris .HCl buffer, pH 8.1, and 0.05 M-KCl. Protoplasts were formed by lysozyme action (1 mg/ml) in the presence of 0.2% EDTA (pH 8.0) and lysates were prepared in a buffer solution containing 0.5% Brij 58, 0.5% sodium deoxycholate and 5 rg RNase-free DNase/ml. Lysates were clarified by centrifugation for 12 min at 15,000 g and the supernatant fraction was carefully removed (crude lysate). A crude lysate thus prepared was used either without any further purifloation or was centrifuged through a sucrose gradient to isolate polysomes. In the latter case, the lysate was layered on top of a 15 to 35% or 15 to 40% linear sucrose gradient (11 ml) supported by a 1.5ml cushion of 50% sucrose in 0.01 ivr-Tris (pH 7-S), 0.01 Br-MgCl, and 065 1\1NH&l, and centrifuged for 150 mm at 36,000 revs/min in a Spinco SW36 rotor. The gradient was then pumped through the flow cell of a Gilford spectrophotometer which monitored the optical density at 260 nm, and 30 to 44 fractions were collected (Fig. 1). Only pdysomes larger than tetramers were kept apart, pooled and used in protein synthesis assays and ribosome run-off measurements. A preparation of large polysomes obtained by centrifug&ion of a 1.2..ml lysate prepared from a 250-ml culture contained 0.31 to 0.46 mg polysomes/ml.
166
4.
J-. COZZONE
AND
6.
8. STEXT
O-6
-2 :
04 0 0
02
C
FIG. 1. Zonal sedimentation
I , I
I
5
to
of ribosomes
(d) In vitro
I I
, I
I I!
/ 1
I5 20 Fmctron no
I
I
,
25
30
35
and polysomes
protein
from exponentially
growing
cells.
synthesis
The cell-free system was a modification of that described by Matthaei & Nirenberg (1961). The complete reaction mixture contained the following components per ml: 100 pmol Tris (pH 7*8), 11 pmol magnesium acetate, 50 pmol KCI, 6 pmol mercaptoethanol, 1 pmol ATP (disodium salt), 0.5 prnol GTP (disodium salt), 5 rmol phosphoenolpyruvate (potassium salt), 25 pg pyruvate kinase, 0.05 pmol each of 19 L-amino acids minus proline or valine, or both, 2.5 to 17.5 pCi L-[W]proline and/or 0.4 to I.5 &i L[r4C]valine or 0.5 &i L-[r*C]proline, 0.9 to 1.3 mg 5105 protein, 0.15 to 0.23 mg polysomes. The mixture was incubated for 0 to 75 min at either 29°C or 42*5OC, cooled quickly in an ice bath and trichloroacetio acid was added to a final concentration of 7.5%. In kinetic experiments, 100-p samples were withdrawn from the incubation medium. Acid-treated samples were heated to 92°C for 20 min, filtered on Millipore filters, washed and counted in a Tricarb Packard liquid-scintillation spectrometer using 8 ml of BBOT (2,5-bis-2(5-t-butylbenzoxaloyl)-thiophene)-toluene fluid. (e) In vitro
ribosome
run-off
For the assay of ribosome run-off, the incubation mixture was essentially the same as that used in the protein synthesis assay. Isolated polysomes, or lysates, were prepared from cells grown in the presence of radioactive uracil. After 0 to 60 min of incubation at 42.5”C, the reaction mixture was rapidly cooled in an ice bath, then layered on top of a 15 to 35% or 15 to 40% linear sucrose gradient (8.9 ml) supported by a double cushion of 2.7 ml of 50% sucrose over 0.5 ml of 55% sucrose in 0.01 M-Tris (pH 7.8) 0.01 M-MgCI, and 0*05 M-NH&Cl. After centrifugation for 240 min at 36,000 revs/min in a Spinco SW36 rotor, the distribution of the ultraviolet absorbing material in the gradient was monitored continuously with a, Gilford spectrophotometer, and 26 to 36 fractions were collected. Each fraction was then precipitated with 7.5% cold trichloroacetic acid in the presence of 50 rg of bovine serum albumin, filtered, washed and counted as above. Fig. 2 shows a typical pattern obtained under these experimental conditions. In some experiments, sucrose gradients were prepared with a buffer solution containing either 0.06 %r-NaCl and 0.05 ivr-MgCl, or 0.005 ~-Kc1 and 0.001 M-MgCl,. The total amount of radioactive material present in polysomes, monosomes and ribosomal subunits after centrifugation was taken as 100%. The amount of radioactive material present under each of the two ribosomal regions corresponding to the polysomes (region II) and to the monosomes and subunits (region I) was then measured and expressed
IN
VITRO
RIBOSONE
RUN-OFF
167
6-
5
IO
15
20
25
Fraction no. FTG. 2. Zonal tiedimentation of ribosomal material after incubation of polysomes. La,beled polyaomes were prepared from cells of strain lOB6 rel+ grown for 3 generations in the presence of [Tf]uracil (4 pg/ml; 0.6 pCi/ml) and were incubated in a complete cell-free system. After 20 mm of incubation at 42,5”C, the reaction mixture was analyzed by sucrose gradient sedimentation as described in Materials and Methods. radioactivity of the trichloroacetic Optical density at 260 nm; (--8--O--) t---------j, aaid-preecipitable mat,erial.
as: a percentage of the total counts. In addition, in most run-off experiments, the relative proportions of mcmosomes and subunits in region I were calculated and expressed as a. percentage of the radioactive material present only under region I and taken, in this case, as lCOq/,. (f) Aminoacyl-tRNA synthetase uctivity The valyl-tRNA synthetase activity was measured by the ability of the enzyme to catalyze the aminoacylation of tRNA. The technique used was similar to that described by Anderson & Neidhardt (1972). The reaction mixture contained per ml: 100 pmol Tris (pA 7.2): 15 pmol MgCle, 10 pmol NH,Cl, 100 pg bovine serum albumin, 5 pmol ATP (disodium salt), 1 mg tRNA from E. co& 7 pmol mercaptoethanol, 2.04 to 2.25 mg SlO5 protein, 0.5 &i L-[l%]valine. After 0 to 60 min of incubation at either 29°C or 425°C samples of 100 ~1 were withdrawn from the mixture and 2 ml of 5% trichloroacetic acid were added in the presence of 100 ~1 of a carrier containing, per ml, 320 pmol NaCl, 42 pg yeast RNA and 1.6 mg valine. Precipitates were collected on Millipore filters, washed with 5 rinses of 5% t,richloroacetic acid (3 ml each) and 1 rinse of S7o/o ethanol (5 ml) at - 10°C, The air-dried filters were counted in a scintillation fluid as described above.
Jgaterials were obtained from the following sources : lysozyme, phosphoenolpyruvate (monopotassium salt) and pyruvate kinase (10 mg/ml suspension in ammonium sulfate); Boehringer Mannheim GmbH; ATP (disodium salt), GTP (trisodium salt), UTP (trisodium salt), CTP (disodium salt) and stripped. tRNA, Schwarz Bioresearch Inc. ; DNase
1GY
A. J. COZZONE
AND
G. 9. STENT
(pancreatic, eleotrophoretically pure) and RNase (pancreat,ic, prot,ease-free), Sigma Chemical Co. ; sucrose (RNase-free) and p-chloromercuribenzoic acid (sodium salt), Mann Research Labs. ; sodium deoxycholate, Difco Labs. ; Brij-58 (polyoxyethylene (20) cetyl ether), Atlas Chemical Industries ; spermidine trihydrochloride and puixescine dihydrochloride, Calbiochem; BBOT (2,B-bis-2 (5-t-butylbenzoxaloyI)-thiophene), Nuclea,r
Chicago Corp. Other chemicals used were of reegent grade. L-[4-3H]Proline (20.0 Ci/mmol), Q4C]valine (260 mCi/mmol), mCi/mmol) and [6-3H]uracil (16.8 Ci/mmol) were purchased from Inc.; [2-14C]uracil (52 mCi/mmol) was obtained from New England
L-[14C]proline (260 Schwarz Biorosearch Nuclear Corp.
3. Results (a) In vitro
valyl-tRNA
synthetase activity
When a culture of strain lOB6 rel+ or strain lOB6 sensitive mutant valyl-tRNA synthetase is grown at 29”C, protein synthesis occurs at the same rate as in insensitive strain D2 reE+ or strain D2 rel- . If cells of restrictive temperature of 42@‘C, protein synthesis minutes after the shift (Edlin & Stent, 1969).
rel- carrying a temperaturethe permissive temperature of a culture of the temperaturestrains lOB6 are shifted to the stops abruptly, less than 5
FIG. 3. In vitro valyl-tRNA synthetase activity. The attachment of [14C]valine to tRNA was measured in the S105 supernatant fractions pared from strains D2 Tel+, D2 r&, lOB6 reZ+ and lOB6 rel-. The final concentration in protein was, respectively, 2.26, 2.03, 2,06 and 2.04 mg/ml incubation mixture. Incubation at either 29°C ((a) and (b)) or 42.5”C (( o ) and (d)) and the radioactivity of 100.~1 samples measured &s a function of time after triohloroacetio acid precipitation.
preS105 was was
In a first set of experiments, this temperature effect on in vivo valyl-tRNA syntheactivity was re-examined in vitro. Extracts were prepared from exponentially growing cells of strains D2 and lOB6, and incubated at either 29°C or 425% in the presence of tRNA and radioactive valine. The activity of the valyl-tRNA synthetase was measured by the ability of bhe enzyme to catalyze the aminoacylation of tRNA. Results presented in Figure 3 show that very little, if any, valyl-tRNA synthetase activity is detected in either lOB6 rel+ or lOB6 rel- extracts upon incubation either at 29°C or at 42#‘C. After a 60-minute incubation of a IOB6 rel+ extract at, 29”c, the amount of radioactive valine attached to tRNA is only 2.6% of the amount
tase
IN
VITRO
RIBOSOME
RUN-OFF
rt3i-J
measured in the case of a D2 rel+ extract (Fig. 3(a)), and the radioactivity incorpor ated in a 10436ret- extract is only 0.4% of the radioactivity found in a D2 reE- extra& (Fig. 3(bj). After 60 minutes of incubation at 425”C, similar results are obtained, since the amount of radioactivity incorporated in lOB6 rel+ and 1OB6 rel- extracts is 24o/o and O-4%, respectively, of the amount found in D2 rel+ and D2 rel- extracts (Fig. 3(c) and (d)). Thus, it appears that whereas the valyl-tRNA synthetase is fully active in viwo in cells of strains IOB6 grown at 29”C, it is almost completely inactive in extracts of these cells incubated in vitro at the same temperature. The valyl-tRNA s~thet&se is, CG fortior$ inactive upon in vitro incubation at 425°C. Similar data have been reported in the case of other bacterial strains harboring a temperature-sensitive aminoacyl-tRN,4 synthetase (Eidlio & Neidhardt, 1965; Atherly & Suchanek, 1971; Steinberg 81,Anaguostopoulos, 1971; Anderson & Neidhardt, 1972). As expected, the attachment of valine to tRNA in extracts of strains D2 rel+ and D2 rel- is faster at 425°C than at 2977, as shown by the initial slope of curves in Figure 3, but, the total amount of valine attached to tRNA after 15 to 60 minutes of incubation is independent of the temperature. (b) Cell-free protein-synthesixing
system
Large polysomes prepared from strains D2 rel+ and D2 rel- and from strains lOB6 rel+ and lOB6 rel- were used to direct the in vitro synthesis of proteins in a cellfree system optimized for amino acid incorporation (for composition see Materi.als and Methods). Polysomes from a given strain were incubated in the presence of a supernatant fraction extracted from cells of the same strain (homogeneous system). The incorporation of radioactive proline and valine into trichloroacetie aeidprecipit,able material was measured at 29°C as a function of time in the four diffe~~~~ homogeneous systems : D2 rel+, D2 rel- , lOB6 rel+ and lOB6 rel-. As shown in Figure 4(a) and (b), the incorporating abilities of polysomes from strains D2 rel+ and D2 reZ- are very similar, as shown by the initial slope of the kinetic curves and by the fina! amount of radioactivity incorporated after 50 to 75 minutes. Sirn~la~l~7, the incorporating abilities of polysomes from strains lOB6 reE+ and lOB6 rel- are almost identical; however, both of the latter are only about one-third of those of D2 polysomes. The incorporating abilities of the systems prepared from stringent and relaxed strains lOB6 may, however, still appear surprisingly high, bearing in mind that the mutant valyl-tRNA synthetase present in the supernatant fraction extracted from these strains is almost completely inactive in the in vitro attachment of valine fo tRNA (Fig. 3). In an attempt to reduce this residual incorporating activity, polysomes prepares from strains 10B6 reE+ and lOB6 rel- were incubated in a cell-free system at 42=5”C inst,ead of 29”C, and amino acid incorporation into proteins was measured as before. The amount of radioactive material incorporated by either a D2 rel+ or a D2 retsystem was taken as a control. The results presented in Figure 4(o) and (d) show th after 60 minutes of incubation, the incorporation of proline in the 10136rel+ sy&,e as well as in the lOB6 rel- system is reduced to 13 to 16% of the control value and that, the incorporation of valine is almost completely inhibited, since it is reduced to 25 to 3% of the control value. As in the preceding experiment,, the ~ucorpora~~~g abilities Gf D2 rel’. and D2 rel- polysomes are very similar. 1E
170
A. J. COZZONE
AND
C. S. STENT
_*-* D2 rel-
0
20
40
60
75
20
40
60
75
Time (mm)
FIG. 4. In, vitro incorporstion of proline and valine in a homogeneous system. Polysomes were prepared from the four strains D2 reZ+, D2 reC-, lOB6 reZ+ and 10B6 Tel-, and were incubated in a homogeneous system in the presence of [3H]proline (0.6 nmol/ml; 12.5 &‘i/ml) or [l*C]valine (4 nmol/ml; I.0 @i/ml). Incubation was at either 29’C ((a) and (b)) or 42.5”C. ((c) and (d)). Radioactivity of lOO-~1 samples wss measured as a function of time after trichloroacetic acid precipitation. -0-0--, and -m-m-, proline incorporation; -O-Oand -U-z--, valine incorporation.
St thus appears that in vitro protein synthesis in lOB6 Tel+ and lOB6 rel- systems is more strongly inhibited when incubation is done at 42~5°C rather than at 29”C, even though the heat-sensitive valyl-tRNA synthetase appears equally inactive at both temperatures (Fig. 3). Table 1 reports some other characteristics of the homogeneous incorporating systems prepared from the four strains previously described, and incubated under several different conditions for 60 minutes at 425°C. It can be seen that, in all cases, protein synthesis is much reduced in the absence of ATP but only slightly affected by the absence of GTP. The system is dependent on polysomes, soluble enzymes, meroaptoethanol and magnesium ion concentration, and is inhibited by ribonuclease. Incorporation is reduced in the absence of 20 amino acids but is unaffected by the absence of valine alone. Addition to 1 ml of complete mixture of 0.5 prnol CTP and UTP or 0.1 pmol each of 20 amino acids or 500 pg stripped tRNA, does not significantly affect protein synthesis. Addition of 2 pmol putrescine or 2 pmol spermidine, two polyamines which might have a causal role in the control of RNA synthesis (Edlin & Broda, 1968), does not affect protein synthesis either in stringent or in relaxed extracts.
IN
Vll'RO
RIBOSOME TABLE
i71
RUE-Oh-1
1
Characteristics of polysome-directed cell-free protein synthesis -D2 rel+
Protein D2 rel-
~___~_. Complete --GTP - ATP, ATP regeneration -GTP, ATP, ATP regeneration - S105 supernatant fraction - Polysomes - Mercaptoethanol - 20 a.mino acids - Valine 1jO.5 - GTP,pm01 mercaptoethanol, ~~chloromerouribenzoate/ml + 0.5 pm01 CTP and UTP/ml +O.f pm01 20 amino acids/ml +500 f*g stripped tRNA/ml + 10 pg RN&se/ml + 2 pmol spermidine/ml + 2 pm01 putrescinelml Complete in 5 mm-Mga+
97.6 84.1 15.9 6.3 9.1 7.4
100.0 82.7 16.3 5.1 8.8 6.5
71.3 98.1
68.7 99.2
98.2 98.0 97.9 5.5 37.8
100.4 101.6 6.8 34.3
synthesis (%) lOB6 rel i -___--
lOB6 rel-
12.8 12.1 2.8 1-l 1.4 1.4 2.9 10.9 13.0
16.3 13.3 2.8 1.5 1.2 I.0 3.0 13.5 15.4
0.7 13~4 12.8 12.6 1.2 14.5 11.1 6.7
0~7 15.9 16.4 1.3 18-7 12.7 5.1
Tha incorporation of radioactive proline into proteins was measured, under several different conditions, in the four homogeneous systems prepared from strains D2 reZ+, D2 Tel-, lOB6 rel+ and lOB6 Tel-. Incubations were done at 42.5’C for 60 min. A description of the complete incabation mixture is given in Materials and Methods. Values are expressed as a percentage of the amount of radioactive material incorporated by a complete D2 rel- system, Each value is th.e average of 2 to 7 experiments.
(c) Bibosome run-osf in a homogeneous qptem The in vitro run-off of ribosomes were studied both in the presence and absence of protzein synthesis by measuring the conversion of polysomes into single ribosomes and ribosomal subunits as a function of time. In order to obtain maximum inhibition of protein synthesis in the 10B6 systems, all the experiments described hereafter weye done at 42.5%. Cells of strains D2 rel+, D2 rel-, lOB6 rel+ and lOB6 rel- were grown exponentially in the presence of radioactive uracil, and large polysomal structures were prepared and isolated, in each case, as described in Materials and Methods. Labeled polysomes were then incubated in a complete homogeneous system. Their gradual conversion into monosomes and subunits was followed by measurin.g the radioactivity of each sibosomal fraction after sucrose gradient sedimentation. As shown in Figure 5, the kinetics of appearance of monosomes and subunits in the D2 rel+ and D2 rel- systems are similar. There is proportionality between the rates of protein synthesis (Fig. 4(c) and (d)) and growth of the pool of run-off products, as previously reported by No11 et al. (1963). After 60 minutes of incubation, about 900!, of the polysomes initially present in the reaction medium are converted into monosomes and subunits. In the case of the lOB6 rel+ and lOB6 rel- systems, the kinetics of appearance of monosomes and subunits as a function of time are similar to each other. The initial slope of the kinetic curves is slightly lower than that of the D2 systems. Furthermore, unlike the D2 systems, there is no proportionality between the rates of protein
A. J. COZZONE
172
AND
G. 8. STENT
Time hn)
5. In vitro run-off of ribosomes from isolated polysomes. Polysomes labeled with radioactive uracil were prepared from cells of strains DZ ,rel* , Dd ret-, lOB6 rel* and lOB6 rel-, and were incubated at 42@‘C in a homogeneous system. After 0 to 60 min, the reaction mixture was cooled in an ice bath and analyzed by sucrose gradient sedimentation. The total amount of radioactive material present in polysomes, monosomes and ribosomal subunits was taken as 100%; the amount present in monosomes and subunits (Fig. 2, region I) was expressed as a percentage of the total counts. Each point is the average from 2 to 6 experiments. FIQ.
synthesis and growth of the pool of run-off products after the first 10 to 15 minutes of incubation. Thus, the kinetics of proline incorporation (Fig. 4(c) and (d)) have reached a plateau by this time, while ribosomes continue to run off for 25 to 30 additional minutes. It is to be noted, however, that the fraction of polysomes still remaining intact after 60 minutes of incubation is higher in the lOB6 rel+ system than in the lOB6 rel- system (40% verse 29%). A more detailed characterization of the run-off products was made by determining the proportions of monosomes and ribosomal subunits formed from polysomes after different incubation times (Fig. 6). In the D2 reZ+ and D2 reb- systems, most run-off products consist of monosomes, since only 11 to 21 y0 of the total ribosomal structures are ribosomal subunits. A slightly higher percentage, 21 to 27%, is found in the case of the lOB6 rel- system, whereas as much as 41 to 48% of the products which appear in the lOB6 rel+ system consist of subunits (except for the value measured after 5 min of incubation). In other words, under the conditions of incubation and sucrose gradient analysis described above, in the D2 systems in the presence of protein synthesis, as well as in the lOB6 rel- system in the absence of protein synthesis, the run-off products are mainly monosomes. This is to be contrasted with the lOB6 rel+ system, where in the absence of protein synthesis, a high proportion of ribosomal subunits is formed. Of special interest is the analysis of monosome appearance as a function of time ; it can be calculated from the values given in Figure 5 and Figure 6 that the amount of monosomes formed after 5 minutes of incubation in the D2 rel+, D2 Tel-, and lOB6 rel- systems is, respectively, only 49,45 and 50% of the amount of monosomes formed after 60 minutes of incubation. However, the same calculation for the lOB6 rel+
92.5 (15.9+84.1) 89.3 (20.8+ 79.2) 90.1 (sl.9+18.1) 87.1 (79.6 $ 20.4) 87.9 (80.8+19.2)
86.2 (79.2+ 20.8)
(79.4f20.5) (81.3+ 18.7) (77.51- 22.5) (76.1-I-23.9)
89.2 87.7 SC3 85.6
86.8 (80.7+ 19.3) 28.5 (73.3+ 25,7)
83.4 (81.4+ 18.6) 26.3 (71.6+28*4)
88.4 (17*4+ 82.6) 90.7 (21.6+ 78.4)
90.1 (81.9+ 18.1) 13.0 (59.0$41.0)
Monosomes D2 rel-
12.7 (62.5+37*5)
89.2 (79*4+20*6)
D2 ml +
(%)
10B6 reP
60.0 (54.81-45.2) 61.1 (58~1+41.9) 58.6 (58.3+41.7) 65.7 (56*6+43.4)
59.3 (23.5+ 76.5) 62.3 (20.4+ 79.6)
22.6 (25.4+ 74.6) 61.8 (79.31-20.7)
60.0 (54++45*2) 11.8 (38*4+61.6) 56.6 (62.3+37.7) 19.1 (46*3+53*7)
+ subunits
(20.5-f
79.5)
71.3 (72.7+27.3) 70.4 (73+3+2&2) 68.2 (70,4+ 29.7) 67.1 (71.3+28.7)
71.1 (l&3$-81.7) 73.7 (19*7+80.3)
73.2 (82.1+ 17.9)
23.9
71.3 (72.7+27.3) 12.8 (45.8+54.2) 69.7 (74.4+ 25.6) 20.3 (69.3+ 30.7)
10B6 rel-
Labeled polysomos from the four strains D2 reZ+, 132 reE-, lOB6 re6+ and lOB6 reZ- were incubated l’or 60 mjn at 42.5”C!, under several clifferont conditions, in a homogeneous cell-free system. A description of the complete reaction mixture is given in Materials and Methods. The percentage of run-off products and the proportions of monosomes and subunits among these run-off products (given in parentheses) were calculated and expressed as in Figs 5 and 6.
Complete in 60 mix-Na+ gradients Complete in 1 mwMgZ+ gradients Complete with: non-dialyzed S105 dialyzed S105 pre-incubated and dialyzed 5105 non-dialyzed 8105 from shifted cells
-GTP, ATP, mercaptoethanol, +p-chloromercuribenzoate + 2 pm01 spermidine/mI
Complete - GTP, ATP, ATE regeneration -ATP --S105 supernantant fraction
2
Characteristics of in vitro ribosome run-0JSfin isolated pobysomes
TABLE
174
A. J. COZZONE
AND
oLd 10
20
30 40 Tlme(minl
FIG. 6. Proportion of ribosomal Labeled polysomes were prepared from the four Tel-, and were incubated at 42~5°C for 5 to 60 min The total amount of radioactive material present upon incubation was taken as lOO0/o. The proportion as a percentage of the total oounts.
6.
S. STEST
50
60
subunits in run-off products. strains D2 reZ+, D2 Tel-, lOB6 Tel+ and lOB6 in a complete homogeneous system as in Fig. 5. in monosomes and ribosomal subunits formed of subunits was then calculated and expressed
system leads to the much higher value of 74%. This means that in the lOB6 reE+ system most of the monosomes which are going to be formed have appeared after only 5 minutes of incubation and that run-off products which appear later on are mainly ribosomal subunits. Some other characteristics of the four different systems used for studying the in vitro run-off of ribosomes are presented in Table 2. It can be seen that in all cases, the conversion of polysomes into monosomes and subunits requires the presence of an energy source. Ribosome run-off is strongly reduced in the absence of GTP but only slightly affected by the absence of ATP, as previously reported by Hirashima & Kaji (1972). The presence of an 5105 supernatant fraction, extracted from exponentially growing cells or from cells pre-heated for 5 minutes at 42.5% is essential for polysome conversion. But neither dialysis nor pre-incubation of this fraction before incubation with polysomes seems to affect the degree of conversion. The presence of spermidine leads to a higher proportion of monosomes among the run-off products. The monosomes which are formed upon incubation in a complete system are products of run-off rather than of polysome fragmentation since they can be made to dissociate into subunits by high-sodium or low-magnesium ion concentrations (Ron et al., 1968; Beller & Davis, 1971; Davis, 1971). In the lOB6 cell-free systems used in these experiments, no “non-enzymic translocation” similar to that described by Pestka (1968) and Gavrilova & Spirin (1971) occurs even in the presence of 03 mm-p-chloromercuribenzoate. (d) Ribosome run-ojJ Cn a heterogeneous system In order to assess the relative importance of polysomes and supernatant fraction in determining the character of the run-off products, mixed systems were studied (Table 3).
IA' VITRO
RIBOSOME
176
RUN-OFF
TILED Ribosome run-off in a heterogeneozcscell-free system
Polysomes Supernatant D2 reZ+ D2 rellOB6 reZ+ lOB6 rel-
Monosomes lOB6 reZ+
83.1 (751+24.9) 56.6 (56.9+43.1) 60.0 (544+45.2)t 54.3 (559+44.1) 62.7 (57.2+42+)?
+ subunits
(%) lQB6 rel-
86.6 64.1 73.1 65.0 71.3
(81.1+1&g) (74.0f26.0) (70.7-t-29.3)? (75.8+ 24.2) (72.7-+27Q3)?
Labeled polysomes from a given strain were incubated in a complete cell-free system. in the presence of a supernatant fraction extracted either from the same strain (homogeneous system) or from a different strain (heterogeneous system) as indicated. Ribosome run-off was measured and expressed as in Table 2. The proportions of monosomes and ribosomal subunits are given in parentheses. Incubations were done at 42*5”C for 30 min and, in some instances (t), for 66 min.
A control experiment showed that when polysomes from the temperature-sensitive strains lOB6 rel+ and lOB6 rel- are incubated in the presence of a supernatant fraction extracted from the temperature-insensitive strains D2 rel+ or D2 rel-, ribosome run-off at 425°C occurs to the same high extent as in a homogeneous D2 rel” or D2 rel- system. When lOB6 rel+ polysomes were incubated in the presence of a lOB6 rel- supernatant fra&ion, or IO%6 rel- polysomes in the presence of a lOB6 rel + supernatant fraction, it was seen that, regardless of the source of the supernatant fraction, polysomes from a given lOB6 strain yield the same amount of run-off products with the same proportions of monosomes and subunits. Whatever the supernatant used in the incubation mixture, conversion of the lOB6 rel- polysomes is always 8 to 13% higher than in the LOB6 rel- polysomes and the proportion of subunits formed is always 1.6 to 2.0 times smaller. (e) Ribosome run-off in crude lysates In all the experiments previously described, polysomes and supernatant fractions were first extracted separately from the cells, then purified and finally incubat,ed together in a reconstituted cell-free system. It is possible that such a treatment may affect the structure and/or some biological properties of the subcellular fractions. In an attempt to examine this possible effect of the purification procedure on the results obtained, ribosome run-off was studied in total crude lysates prepared from the four strains already described (Table 4). Upon incubation for 30 minutes at 42PC in the presence of a supernatant fraction and energy source, ribosome run-off occurred. In D2 lysates 94 to 97%, and in a lOB6 rel- lysate 87.70/,, of the ribosomal material was found as monosomes and subunits after incubation. A lower value; 76.60/b, was found in the case of a IO%6 rel+ lysate. These data are consistent with those obtained in the case of a reconstituted cell-free system (Fig. 5). In other experiments, not reported here, it was found that monosomes released from polysomes upon incubation of crude lysates dissociate into subunit,s after sucrose gradient sedimentation in a buffer containing either
176
A. J. COZZONE
AND
G. X. XTENT
TABLE 4
In vitro ribosome run-off in crude lysates
Non-incubated lysate Lysate incubated alone Lysate + 5105 Lysate + 8105 + energy
D2 rel+
Monosomes D2 rel-
40.6 53-4 66.8 94.5
38-l 54.1 71.0 97.3
+ subunits [%) 10B6 reZ+ lOB6 rel42.4 48.8 59.6 76-6
40.9 47.1 63.2 87.7
Crude lysates were prepared from the four strains D2 reZ+, D2 rel-, lOB6 reZ* and lOB6 relgrown in the presence of radioactive uracil and were incubated for 30 min at 42*5”C as indicated. A lysate from a given strain was incubated, when specified, with an S105 supernatant fraction extracted from cells of the same strain. The amount of radioactivity present in both monosome and subunit fractions was expressed as a percentage of the amount of radioactivity present in the total ribosomal fraction (polysomes+ monosomes+ subunits).
60 mM-sodium chloride and 5 mM-magnesium chloride or 50 mM-potassium chloride and 1 mM-magnesium chloride, which means that those monosomes are really products of run-off rather than of polysome fragmentation.
4. Discussion (a) In vitro protein synthesis In the experiments presented here, ribosome run-off from polysomes was studied in two different kinds of cell-free extracts. In one of these, the temperature-insensitive D2 system containing polysomes and a supernatant fraction prepared from either strain D2 rel+ or strain D2 rel-, protein synthesis occurs. In the other kind of extract, the lOB6 system prepared from either strain lOB6 rel+ or strain lOB6 rel-, which both carry a temperature-sensitive mutant valyl-tRNA synthetase, much less protein synthesis occurs. In fact, since almost no valyl-tRNA synthetase activity is detectable in the supernatant fraction extracted from cells of strains lOB6, it would have been expected that protein synthesis in the lOB6 system would have been completely arrested. However, under valyl-tRNA deprivation at 29”C, the lOB6 system still incorporates 33 to 35’j/, as much proline and 29 to 30% as much valine as the D2 system containing a fully functional valyl-tRNA synthetase. These values are reduced to 13 to 16% and 25 to 3%, respectively, when the reaction mixture is maintained at 42.5%. The unexpectedly high amino acid-incorporating activity of the lOB6 system at 29°C might be due to a residual valyl-tRNA synthetase activity which is not detected by the assay method used. As shown by Anderson & Neidhardt (1972), a very small amount of functional valyl-tRNA synthetase is indeed able to sustain in viva a high rate of protein synthesis. The incorporation of valine at 42.5”C in the lOB6 system, unlike that occurring at 29”C, appears to be completely arrested, since the valyltRNA synthetase is, very likely, totally inactive at this temperature. The fact that, however, some proline incorporation still ocours at 42.5”C might be due to some residual protein synthesis occurring in the absence of any valyl-tRNA synthetase activity.
1N VITRO
RIBOSOME
RUN-QFF
177
(b) In vitro ribosome run-08 It is important for the interpretation of the findings presented here on ribosome run-off in cell-free systems that very little degradation of messenger RNA takes place during incubation of polysomes under these experimental conditions. That this is actually the case has been previously shown; when either isolated polysomes or polysomes in a lysate are incubated in vitro under conditions similar to those used here, no n&ease activity is detected during the conversion of polysomes into run-off products (No11 et cd., 1963 ; Phillips et al., 1969 ; Hirashima $ Kaji, 1972). Furthermore, it has been reported (Kaempfer, 1970; Xubramanian & Davis, 1971) that, in such cell-free systems very little, if any, polysome re-assembly occurs from the run-off products during incubation of polysomes. The absence of significant polysome re-assembly can also be inferred from the results presented here, since of the polysomes present at the start of incubation, more than 90% are converted into monosomes and. subunits after 40 minutes. It seems likely, therefore, that the in vitro polysome disassembly due to run-off observed in the present work occurred under conditions where neither polysome degradation by nucleases nor re-assembly took place to an appreciable extent. Indeed, there are several indications that the ribosomal particles which are formed either in the presence or absence of protein synthesis are really products of run-off rather than of polysome fragmentation. First, the monosomes were found to dissociate into subunits when centrifuged in buffer solutions containing either 60 mM-sodium chloride or 1 mM-magnesium chloride. This supports the inference of their run-off origin, since it has been shown that monosomes obtained by ribosome run-off from polysomes, but not ribosomes &ill attached to mRNA, dissociate in the presence of high-sodium or low-magnesium ion concentrations (Ron et CL, 1968; Belier 8r Davis, 1971; Davis, 1971). Moreover, in an experiment not reported here, polysomes were extracted from cells pulse-labeled with radioactive arginine. After in vitro incubation of these polysomes initially carrying labeled nascent polypeptides, no radioactivity was found in the monosome region by sucrose gradient analysis. This result indicates that monosomes formed upon incubation of polysomes do not bear peptidyl-tRNA and thus are, very likely, not derived from polysome fragmentation. Finally, it may be noted that when polysomes were incubated without an energy source (Tables 2 and 4), very little conversion into monosomes and subunits occurred, indicating that, under these conditions, the polysomal structures are stable. The results presented here that, under aminoacyl-tRNA deprivation, polysomes from reb- and rel+ bacteria show similar rates of decay in vitro thus confirm the previous finding (C&zone & Donini, 1973) that under is viva conditions where neither protein synthesis nor polysome re-assembly is permitted, rel- and TeEicells also show similar rates of polysome decay. Thus, further support has been adduced for the previous inference (Cozzone & Donini, 1973) that the higher polysome level ordinarily maintained by aminoacyl-tRNA-deprived rel- cells as compared to rel+ cells is attributable to a higher rate of polysome re-~sernb~~, while protein synthesis is held in abeyance, rather than to a lower rate of disassembly. The present finding that the products of ribosome run-off in reE+ and rel- in vitro systems during ongoing protein synthesis consist mainly of free monosomes agrees
178
A. J. COZZONE
AND
6.
S. STENT
with previous descriptions of the normal run-off products (Kaempfer, 1970; Subramanian & Davis, 1971; Schlessinger et al., 1967). But that in the absence of protein synthesis rel- polysomes also give rise to the normal monosome yield, whereas rel+ polysomes give rise to a high proportion of subunits among their run-off products, was not foreseen from those earlier studies. This finding might reflect a difference in the post-run-off equilibrium between monosomes and ribosomal subunits. However, the experiments reported in Table 3 rule out the possibility that the high proportion of subunits found in the 10B6 rel+ system could be merely the consequence of a greater ribosome dissociation activity of factor F3(B) present in the supernatant fraction isolated from a rel+ strain, since the same high proportion of subunits is also formed when lOB6 rel+ polysomes are incubated with a lOB6 rel- supernatant fraction. In any event, it can be concluded that this difference in the character of the run-off products of rel+ and rel- polysomes is a consequence of valyl-tRNA deprivation, since under conditions where protein synthesis is permitted, reE+ polysomes yield the normal high proportion of monosomes. Thus, there appears to be a qualitative difference in the mechanisms of ribosome run-off during aminoacyl-tRNA deprivation of rel+ and rel- polysomes. Since, under these conditions, rel- polysomes yield the same high proportion of monosomes as during normal protein synthesis, it would seem likely that here a quasi-normal run-off process continues on these polysomes even though the polypeptide assembly process has been interrupted for lack of aminoaoyltRNA. In particular, one may envisage that upon valyl-tRNA deprivation, translocation of rel- ribosomes would not be arrested upon encounter of a non-translatable valyl codon, and movement over the messenger RNA would continue. In the rel+ system, however, ribosomes reaching a non-translatable valyl codon would not be able to continue their movement along the messenger RNA, Instead, they would be prematurely released from polysomes and accumulate as ribosomal subunits in a form which renders them unable to re-associate while protein synthesis is held in abeyance. Those free monosomes which are mainly formed during the first five minutes of incubation under these conditions would arise from normal ribosome run-off due to some residual protein synthesis (Fig. 4). These proposed differences in the run-off process during valyl-tRNA deprivation between polysomes from rel - and reE+ strains of E. coli imply that these strains differ in the functional architecture of their ribosomes. This inference is supported by the finding that polysomes from a given strain yield the same amount of run-off products with the same proportions of monosomes and subunits regardless of the source of the supernatant fraction used in the incubation mixture (Table 3). Therefore, mutation of the reZ gene (Stent 62Brenner, 1961) seems to alter, directly or indirectly, some functional component, possibly a protein, of the ribosome so that in the rel- mutant strain ribosome translocation can proceed in the absence of protein synthesis.
Thanks are due to Dr Pierluigi Donini for many stimulating discussions and to Elizabeth Mullenbach for excellent technical assistance. We are also indebted to two anonymous This investigation was supported by U.S. referees for helpful criticisms and suggestions. Public Health Service Research grant GM17866 from the Institute of General Medical Sciences. One of us (A. J. C.) was a recipient of a fellowship from N.A.T.O. during part of this project.
IN
VITRO
RIBOSOME
RUN-OFF
179
REFERENCES Anderson, J. J. & Neidhardt, F. C. (1972). J. Bact. 109, 307. Atheriy, A. a. & Suchanek, M. C. (1971). J. Bad. 108, 627. Her, R. J. & Davis, B. D. (1971). J. Mol. Biol. 55, 477. zzone, A. & Donini, P. (1973). J. Mol. Biol. 76, 149. Davis, B. D. (1971). Nature, 231, 153. Eidlic, L. & Neidhardt, F. C. (1965). J. Bad. 89, 706. Edlin, G. & Broda, P. (1968). Bact. Rev. 32, 206. Edlin, G. & Stent, 6. S. (1969). Proc. Nat. Acad. Sci., Wash. 62, 475. Friesen, J. D. (1968). J. Mol. Biol. 32, 183. Gavrilova, L. P. & Spirin, A. S. (1971). FEBS Letters, 17, 324. Hartmann, G., Honikel, K. O., Knusel, F. & Neusch, J. (1967). Biochim. Biophys. Ada, 145, 843. Hirashima, A. & Kaji, A. (1972). J. Mol. Bio2. 65, 43. Kaempfer, R. (1970). Xature, 228, 534. Kaplan, 6. & Anderson, D. (1968). J. Bact. 95, 991. Matthaei, J. H. & Nirenberg, M. W. (1961). Proe. Arat. Acad. Scsci., Wash. 47, 1580. Matzura, H. (1970). FEBS Letters, 6, 5. Morris, D. W. & DeMoss, J. A. (1966). Proc. Nat. Acad. Sci., Wash. 56, 262. Nell, H., Staehelin, T. & Wettstein, F. 0. (1963). Nature, 198, 632. Pestka, S. (1968). J. Biol. Chew 243, 2810. Phillips, L. A., Hotham-Iglewski, B. & Franklin, R. RI. (1969). J. Mol. Biol. 45, 23. Ron, E. Z. (1971). J. Bad. 108, 263. Ron, E. Z., Kohler, R. E. & Davis, B. D. (1966). Proc. Nat. Acad. Sci., Wash. 56, 471. Ron, E. Z., Kohler, R. E. & Davis, B. D. (1968). J. Mol. Biol. 36, 83. Schlessinger, D., Mangiarotti, G. & Apirion, D. (1967). Proc. Nat. Acacl. Sci., Wash. 5 1782. Sells, B. H. & Ennis, H. L. (1970). J. Bad. 102, 666. Steinberg, W. & Anagnostopoulos, C. (1971). J. Bad. 105, 6. Stem, G. S. & Brenner, S. (1961). Proc. Nat. Acad. Sci., Wash. 47, 2005. Subramanian, A. R. & Davis, B. D. (1971). Proc. Nat. Accd. SC<., Wash. 68, 2453. Weber, M. J. & DeMoss, J. A. (1966). Proc. Nat. Acad. Sci., Wash. 55, 1224.
Biol.
(1973)
76, 163-179
Movement of Ribosomes over Messenger RNA in Polysomes of reZ+ and ret Escherichia coli Strains AUIX
J. COZZONE~ AND GUNTHER S. STE:NT
Department of Molecular University of California, ( Recehed
Biology and Virus Laboratory Berkeley, Calif. 94720, U.S.A.
30 June 1972, and in revised form 6 November
1972)
The in, vitro movement of ribosomes over messenger RNA was studied in both the presence and the absence of protein synthesis. For this purpose, labeled polysomes were extracted from rel+ and rel- strains of Escherichia coli grown in the presence of radioactive uracil and incubated in a cell-free system containing tRNA, amino acids, soluble enzymes and a source of energy. The gradual conversion of the labeled polysomes into monosomes and ribosomal subunits was followed by subjecting the reaction mixture to sucrose gradient sedimentation after various incubation times and measuring the radioactivity present in the three relevant ribosomal fractions. It was found that when the conditions of incubation allow protein synthesis to occur, polysomes extracted from reZ+ and rel- cells are converted mainly int,o free monosomes, which can be made t,o dissociate into subunits by high-sodium or low-magnesium ion concentrations. Under conditions in which protein synthesis ea,nnot occur because a mutant aminoacyl-tRNA synthetase has been rendered inactive, polysome conversion still occurs, though to a reduced extent. When the products of such residual run-off are examined, however, a difference is manifest between polysomes extracted from rel+ and from rel- strains : whereas the polysomes from the rel- strain are still converted into free monosomes even in the absence of protein synthesis, the polysomes from the reE+ strain are now eonverted mainly into subunits. It can be inferred, therefore, that ribosomes from relbacteria, but not those from rel+ bacteria, cont,inue movement over messenger RNA in the absence of protein synthesis. Studies of mixed extracts from rel- and rel + bacteria have shown that the character of the run-off process does not depend on the source of tRNA and soluble enzymes; the proportions of monosomes and subunits among the run-off products formed in the absence of protein synthesis depend only on the source of the polysomes. It. is suggested that the mutation of the ret! gene alters the functional a,rchiteeture of ribosomes.
1. Introduction Numerous
studies
rel+ and relacyl-tRNA. conditions as in rel(B’riesen, that
strains
have
been made
of Escherichia
In some of this work
of the level
of polysomes
coli deprived it was found
of either that
under
either
of deprivation, the level of polysomes remains bacteria, as compared to the level of polysomes 1968;
Sells & Ennis,
such deprivaeion
+ Present address:
1970).
However,
in most
leaves the level of polysomes Institut
de Chimie Biologique, 163
largely
Universit6
which
an amino
persists
of t,hese two
studies
unaffected de Provence,
different
in rel+ BS well growing 1~4s
unchanged in normally of these
in cella of
acid or an amino-
it was found
in rel-
bacteria,
;Marseille,
France.
164
A. J. COZZONE
AND
6. S. STENT
whereas under deprivation, reE+ bacteria polysomes are rapidly converted into monosomes and ribosomal subunits (Morris & DeMoss, 1966; Weber &, DeMoss, 1966; Ron et al., 1966; Matzura, 1970). The mechanism responsible for the control of the polysome level in such deprived cells is still not clear. It has been proposed that there exists a direct correlation between the level of polysomes and the net synthesis of RNA in rel - and rel+ strains of E. coli (Morris & DeMoss, 1966; Ron et al., 1966). But, contrary to that proposal, Friesen (1968) and Sells & Ennis (1970) have reported that the polysome level of amino acid-deprived cells is not correlated with the rate of residual RNA synthesis. According to Ron (1971), the level of polysomes in rel+ cells deprived of a particular amino acid is a function of the relative abundance of that amino acid in cellular proteins. Recently, it has been shown (Ron, 1971; Cozzone & Donini, 1973) that the polysomes which persist under amino-acyl-tRNA deprivation are dynamic structures in a steady-state condition of disassembly and re-assembly. For, when deprived cells are treated with rifampin, which prevents synthesis of all species of RNA (Hartmann et al., 1967), polysomes cannot re-assemble on nascent mRNA and they quickly disappear. One may assume, therefore, that the higher polysome level observed in deprived rel- cells, as compared to that of rel+ cells, is attributable to a higher rate of polysome re-assembly and/or to a lower rate of dis-assembly, the dis-assembly of polysomes being caused either by mRNA degradation or by ribosome run-off, or by both. In order to resolve, therefore, the previously reported discrepancies concerning the polysome level in rel-- and rel+ -deprived cells, the rates of d&assembly and re-assembly of polysomes must be measured and compared under these conditions . The rate of ribosome run-off can be measured in a cell-free system similar to that described by Matthaei & Nirenberg (1961). As was shown by No11 et al. (1963), when polysomes are incubated in vitro in a reaction mixture containing tRNA, amino acids, soluble enzymes and a source of energy, ribosomes move over the messenger RNA, and the amount of run-off products released from polysomes can be measured as a function of time by sucrose gradient analysis. The absolute value of the rate constant of in vitro ribosome run-off is very likely different from its value measured in an in viva situation. However, one can expect that the relative value of the rate constant obtained by comparing the rates of in vitro ribosome run-off in polysomes of retand reE+ cells is a reflection of its relative value in vivo. In the experiments presented in this paper, the mechanism of ribosome run-off has been studied in vitro. The purpose was to investigate whether or not the relative motion of ribosomes and messenger RNA occurs in rel- and rel + bacteria under aminoacyl-tRNA deprivation, and to estimate the rate of ribosome movement under these conditions. Polysomes isolated from both kinds of cells were therefore incubated under aminoacyl-tRNA deprivation, and their gradual in vitro conversion into run-off products was followed as a function of time.
2. Materials and Methods (a) Bacterial strains and growth conditiona Two pairs of otherwise isogenic sel’ and rel- strains of E. ooli were used. Both pairs are derivatives of strain D2 (Kaplan & Anderson, 1968). Strains 10B6 reZ+ and lOB6 rel-
IN
VITRO
RIBOSOME
If%
RUN-OFF
(Edlm & Stent, 1969) are arginine auxotrophs and have a temperature-sensitive valylt,RNA synthetase. The permissive temperature for these strains is 29°C. The prototropbic pair of strains D2 rel + and D2 rel - was derived from strain D2 by pha,ge Pl transduction (Edlin & Broda, 1968). Cells were grown at 29°C with forced aeration at pH 7.6 in a medium which contained 12 g Tris, 2 g KCl, 2 g NH,Cl, 0.5 g MgCla.6 H@, tha following components per liter: 0.02 g NaaSO,, 14.7 mg CaC1,.2 H,O, I78 mg Na,HPO,.2 H,O, 2 g glucose. This medium was supplemented with 50 pg arginine/ml when necessary. Growth was followed with a Klett-Summerson calorimeter, using a green filter. In experiments requiring long-term labeling of RNA, cells were grown for 2 to 3 generations in the presence of [3H]uracil (4 to 12 pg/ml; 0.4 to 1.0 PCijml) or [14C]uracil (4 to 12 pg/ml; 0.10 to 0.25 &X/ml). (b) Preparation
of supernatant
fractions
Supernatant fractions were prepared from cells of the four strains described above by a method similar to that of Matthaei & Nirenberg (1961). Exponentially growing cells were poured over ice and collected by centrifugation for 8 min at 8000 g. In some experiments, cultures had been shifted to 42.5”C for 5 min before collection. Cells were washed and disrupted by grinding with alumina (2.5 times the weight of washed cells). The soluble fraction wa,s extracted in buffer containing O-01 lx-Tris (pH 7.8), 0.06 in-KCI, 0.01 ivr-magnesium acetate and 0.006 M-mercaptoethanol (5 ml per g of washed cells). Alumina and cellular debris were removed by centrifugation at 20,000 g for 20 min. The supernatant fluid was treated with 5 pg DNase/ml, centrifuged again at 20,000 g for 20 min and finally at 30,000 g for 60 min. The liquid layer, fraction 530, was removed and oentrifuged at 105,000 g for 180 min to sediment the ribosomes. The top two-thirds of the supernatant fraction were removed, divided into portions, quickly frozen in an acetone-dry ice mixture and stored at -20°C until used. Fraction S105 thus prepared contained 19.7 to 25.7 mg protein/ml and 2.3 to 3.5 mg nucleic acid/ml. When fraction 530 was incubated before oentrifugation at 105,000 g, incubation was done for 45 min at 37°C in the presence of 400 rg ATP/ml, 240 pg GTP/ml, 40 pg pyruvate kinase/ml and 1.2 mg phosphoenol pyruvate/ml. Fraction S105 was dialyzed, when indicated, for 16 h against 100 vol. of the buffer with one buffer cha.nge.
(c) Preparation
of crude lysates and isolated
polysomes
The method of preparing crude lysates and isolated polysomes has been described elsewhere (Cozzone & Donini, 1973). Exponentially growing cells were poured over crushed ice chilled at -15°C in order to reduce ribosome run-off (Davis, 1971). All subsequent operations were done at 0 to 4°C. Cells were harvested by eentrifugation and resuspended in a sucrose-buffer solution containing 0.5 M-RNase-free sucrose, 0.016 ivr-Tris .HCl buffer, pH 8.1, and 0.05 M-KCl. Protoplasts were formed by lysozyme action (1 mg/ml) in the presence of 0.2% EDTA (pH 8.0) and lysates were prepared in a buffer solution containing 0.5% Brij 58, 0.5% sodium deoxycholate and 5 rg RNase-free DNase/ml. Lysates were clarified by centrifugation for 12 min at 15,000 g and the supernatant fraction was carefully removed (crude lysate). A crude lysate thus prepared was used either without any further purifloation or was centrifuged through a sucrose gradient to isolate polysomes. In the latter case, the lysate was layered on top of a 15 to 35% or 15 to 40% linear sucrose gradient (11 ml) supported by a 1.5ml cushion of 50% sucrose in 0.01 ivr-Tris (pH 7-S), 0.01 Br-MgCl, and 065 1\1NH&l, and centrifuged for 150 mm at 36,000 revs/min in a Spinco SW36 rotor. The gradient was then pumped through the flow cell of a Gilford spectrophotometer which monitored the optical density at 260 nm, and 30 to 44 fractions were collected (Fig. 1). Only pdysomes larger than tetramers were kept apart, pooled and used in protein synthesis assays and ribosome run-off measurements. A preparation of large polysomes obtained by centrifug&ion of a 1.2..ml lysate prepared from a 250-ml culture contained 0.31 to 0.46 mg polysomes/ml.
166
4.
J-. COZZONE
AND
6.
8. STEXT
O-6
-2 :
04 0 0
02
C
FIG. 1. Zonal sedimentation
I , I
I
5
to
of ribosomes
(d) In vitro
I I
, I
I I!
/ 1
I5 20 Fmctron no
I
I
,
25
30
35
and polysomes
protein
from exponentially
growing
cells.
synthesis
The cell-free system was a modification of that described by Matthaei & Nirenberg (1961). The complete reaction mixture contained the following components per ml: 100 pmol Tris (pH 7*8), 11 pmol magnesium acetate, 50 pmol KCI, 6 pmol mercaptoethanol, 1 pmol ATP (disodium salt), 0.5 prnol GTP (disodium salt), 5 rmol phosphoenolpyruvate (potassium salt), 25 pg pyruvate kinase, 0.05 pmol each of 19 L-amino acids minus proline or valine, or both, 2.5 to 17.5 pCi L-[W]proline and/or 0.4 to I.5 &i L[r4C]valine or 0.5 &i L-[r*C]proline, 0.9 to 1.3 mg 5105 protein, 0.15 to 0.23 mg polysomes. The mixture was incubated for 0 to 75 min at either 29°C or 42*5OC, cooled quickly in an ice bath and trichloroacetio acid was added to a final concentration of 7.5%. In kinetic experiments, 100-p samples were withdrawn from the incubation medium. Acid-treated samples were heated to 92°C for 20 min, filtered on Millipore filters, washed and counted in a Tricarb Packard liquid-scintillation spectrometer using 8 ml of BBOT (2,5-bis-2(5-t-butylbenzoxaloyl)-thiophene)-toluene fluid. (e) In vitro
ribosome
run-off
For the assay of ribosome run-off, the incubation mixture was essentially the same as that used in the protein synthesis assay. Isolated polysomes, or lysates, were prepared from cells grown in the presence of radioactive uracil. After 0 to 60 min of incubation at 42.5”C, the reaction mixture was rapidly cooled in an ice bath, then layered on top of a 15 to 35% or 15 to 40% linear sucrose gradient (8.9 ml) supported by a double cushion of 2.7 ml of 50% sucrose over 0.5 ml of 55% sucrose in 0.01 M-Tris (pH 7.8) 0.01 M-MgCI, and 0*05 M-NH&Cl. After centrifugation for 240 min at 36,000 revs/min in a Spinco SW36 rotor, the distribution of the ultraviolet absorbing material in the gradient was monitored continuously with a, Gilford spectrophotometer, and 26 to 36 fractions were collected. Each fraction was then precipitated with 7.5% cold trichloroacetic acid in the presence of 50 rg of bovine serum albumin, filtered, washed and counted as above. Fig. 2 shows a typical pattern obtained under these experimental conditions. In some experiments, sucrose gradients were prepared with a buffer solution containing either 0.06 %r-NaCl and 0.05 ivr-MgCl, or 0.005 ~-Kc1 and 0.001 M-MgCl,. The total amount of radioactive material present in polysomes, monosomes and ribosomal subunits after centrifugation was taken as 100%. The amount of radioactive material present under each of the two ribosomal regions corresponding to the polysomes (region II) and to the monosomes and subunits (region I) was then measured and expressed
IN
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RUN-OFF
167
6-
5
IO
15
20
25
Fraction no. FTG. 2. Zonal tiedimentation of ribosomal material after incubation of polysomes. La,beled polyaomes were prepared from cells of strain lOB6 rel+ grown for 3 generations in the presence of [Tf]uracil (4 pg/ml; 0.6 pCi/ml) and were incubated in a complete cell-free system. After 20 mm of incubation at 42,5”C, the reaction mixture was analyzed by sucrose gradient sedimentation as described in Materials and Methods. radioactivity of the trichloroacetic Optical density at 260 nm; (--8--O--) t---------j, aaid-preecipitable mat,erial.
as: a percentage of the total counts. In addition, in most run-off experiments, the relative proportions of mcmosomes and subunits in region I were calculated and expressed as a. percentage of the radioactive material present only under region I and taken, in this case, as lCOq/,. (f) Aminoacyl-tRNA synthetase uctivity The valyl-tRNA synthetase activity was measured by the ability of the enzyme to catalyze the aminoacylation of tRNA. The technique used was similar to that described by Anderson & Neidhardt (1972). The reaction mixture contained per ml: 100 pmol Tris (pA 7.2): 15 pmol MgCle, 10 pmol NH,Cl, 100 pg bovine serum albumin, 5 pmol ATP (disodium salt), 1 mg tRNA from E. co& 7 pmol mercaptoethanol, 2.04 to 2.25 mg SlO5 protein, 0.5 &i L-[l%]valine. After 0 to 60 min of incubation at either 29°C or 425°C samples of 100 ~1 were withdrawn from the mixture and 2 ml of 5% trichloroacetic acid were added in the presence of 100 ~1 of a carrier containing, per ml, 320 pmol NaCl, 42 pg yeast RNA and 1.6 mg valine. Precipitates were collected on Millipore filters, washed with 5 rinses of 5% t,richloroacetic acid (3 ml each) and 1 rinse of S7o/o ethanol (5 ml) at - 10°C, The air-dried filters were counted in a scintillation fluid as described above.
Jgaterials were obtained from the following sources : lysozyme, phosphoenolpyruvate (monopotassium salt) and pyruvate kinase (10 mg/ml suspension in ammonium sulfate); Boehringer Mannheim GmbH; ATP (disodium salt), GTP (trisodium salt), UTP (trisodium salt), CTP (disodium salt) and stripped. tRNA, Schwarz Bioresearch Inc. ; DNase
1GY
A. J. COZZONE
AND
G. 9. STENT
(pancreatic, eleotrophoretically pure) and RNase (pancreat,ic, prot,ease-free), Sigma Chemical Co. ; sucrose (RNase-free) and p-chloromercuribenzoic acid (sodium salt), Mann Research Labs. ; sodium deoxycholate, Difco Labs. ; Brij-58 (polyoxyethylene (20) cetyl ether), Atlas Chemical Industries ; spermidine trihydrochloride and puixescine dihydrochloride, Calbiochem; BBOT (2,B-bis-2 (5-t-butylbenzoxaloyI)-thiophene), Nuclea,r
Chicago Corp. Other chemicals used were of reegent grade. L-[4-3H]Proline (20.0 Ci/mmol), Q4C]valine (260 mCi/mmol), mCi/mmol) and [6-3H]uracil (16.8 Ci/mmol) were purchased from Inc.; [2-14C]uracil (52 mCi/mmol) was obtained from New England
L-[14C]proline (260 Schwarz Biorosearch Nuclear Corp.
3. Results (a) In vitro
valyl-tRNA
synthetase activity
When a culture of strain lOB6 rel+ or strain lOB6 sensitive mutant valyl-tRNA synthetase is grown at 29”C, protein synthesis occurs at the same rate as in insensitive strain D2 reE+ or strain D2 rel- . If cells of restrictive temperature of 42@‘C, protein synthesis minutes after the shift (Edlin & Stent, 1969).
rel- carrying a temperaturethe permissive temperature of a culture of the temperaturestrains lOB6 are shifted to the stops abruptly, less than 5
FIG. 3. In vitro valyl-tRNA synthetase activity. The attachment of [14C]valine to tRNA was measured in the S105 supernatant fractions pared from strains D2 Tel+, D2 r&, lOB6 reZ+ and lOB6 rel-. The final concentration in protein was, respectively, 2.26, 2.03, 2,06 and 2.04 mg/ml incubation mixture. Incubation at either 29°C ((a) and (b)) or 42.5”C (( o ) and (d)) and the radioactivity of 100.~1 samples measured &s a function of time after triohloroacetio acid precipitation.
preS105 was was
In a first set of experiments, this temperature effect on in vivo valyl-tRNA syntheactivity was re-examined in vitro. Extracts were prepared from exponentially growing cells of strains D2 and lOB6, and incubated at either 29°C or 425% in the presence of tRNA and radioactive valine. The activity of the valyl-tRNA synthetase was measured by the ability of bhe enzyme to catalyze the aminoacylation of tRNA. Results presented in Figure 3 show that very little, if any, valyl-tRNA synthetase activity is detected in either lOB6 rel+ or lOB6 rel- extracts upon incubation either at 29°C or at 42#‘C. After a 60-minute incubation of a IOB6 rel+ extract at, 29”c, the amount of radioactive valine attached to tRNA is only 2.6% of the amount
tase
IN
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RIBOSOME
RUN-OFF
rt3i-J
measured in the case of a D2 rel+ extract (Fig. 3(a)), and the radioactivity incorpor ated in a 10436ret- extract is only 0.4% of the radioactivity found in a D2 reE- extra& (Fig. 3(bj). After 60 minutes of incubation at 425”C, similar results are obtained, since the amount of radioactivity incorporated in lOB6 rel+ and 1OB6 rel- extracts is 24o/o and O-4%, respectively, of the amount found in D2 rel+ and D2 rel- extracts (Fig. 3(c) and (d)). Thus, it appears that whereas the valyl-tRNA synthetase is fully active in viwo in cells of strains IOB6 grown at 29”C, it is almost completely inactive in extracts of these cells incubated in vitro at the same temperature. The valyl-tRNA s~thet&se is, CG fortior$ inactive upon in vitro incubation at 425°C. Similar data have been reported in the case of other bacterial strains harboring a temperature-sensitive aminoacyl-tRN,4 synthetase (Eidlio & Neidhardt, 1965; Atherly & Suchanek, 1971; Steinberg 81,Anaguostopoulos, 1971; Anderson & Neidhardt, 1972). As expected, the attachment of valine to tRNA in extracts of strains D2 rel+ and D2 rel- is faster at 425°C than at 2977, as shown by the initial slope of curves in Figure 3, but, the total amount of valine attached to tRNA after 15 to 60 minutes of incubation is independent of the temperature. (b) Cell-free protein-synthesixing
system
Large polysomes prepared from strains D2 rel+ and D2 rel- and from strains lOB6 rel+ and lOB6 rel- were used to direct the in vitro synthesis of proteins in a cellfree system optimized for amino acid incorporation (for composition see Materi.als and Methods). Polysomes from a given strain were incubated in the presence of a supernatant fraction extracted from cells of the same strain (homogeneous system). The incorporation of radioactive proline and valine into trichloroacetie aeidprecipit,able material was measured at 29°C as a function of time in the four diffe~~~~ homogeneous systems : D2 rel+, D2 rel- , lOB6 rel+ and lOB6 rel-. As shown in Figure 4(a) and (b), the incorporating abilities of polysomes from strains D2 rel+ and D2 reZ- are very similar, as shown by the initial slope of the kinetic curves and by the fina! amount of radioactivity incorporated after 50 to 75 minutes. Sirn~la~l~7, the incorporating abilities of polysomes from strains lOB6 reE+ and lOB6 rel- are almost identical; however, both of the latter are only about one-third of those of D2 polysomes. The incorporating abilities of the systems prepared from stringent and relaxed strains lOB6 may, however, still appear surprisingly high, bearing in mind that the mutant valyl-tRNA synthetase present in the supernatant fraction extracted from these strains is almost completely inactive in the in vitro attachment of valine fo tRNA (Fig. 3). In an attempt to reduce this residual incorporating activity, polysomes prepares from strains 10B6 reE+ and lOB6 rel- were incubated in a cell-free system at 42=5”C inst,ead of 29”C, and amino acid incorporation into proteins was measured as before. The amount of radioactive material incorporated by either a D2 rel+ or a D2 retsystem was taken as a control. The results presented in Figure 4(o) and (d) show th after 60 minutes of incubation, the incorporation of proline in the 10136rel+ sy&,e as well as in the lOB6 rel- system is reduced to 13 to 16% of the control value and that, the incorporation of valine is almost completely inhibited, since it is reduced to 25 to 3% of the control value. As in the preceding experiment,, the ~ucorpora~~~g abilities Gf D2 rel’. and D2 rel- polysomes are very similar. 1E
170
A. J. COZZONE
AND
C. S. STENT
_*-* D2 rel-
0
20
40
60
75
20
40
60
75
Time (mm)
FIG. 4. In, vitro incorporstion of proline and valine in a homogeneous system. Polysomes were prepared from the four strains D2 reZ+, D2 reC-, lOB6 reZ+ and 10B6 Tel-, and were incubated in a homogeneous system in the presence of [3H]proline (0.6 nmol/ml; 12.5 &‘i/ml) or [l*C]valine (4 nmol/ml; I.0 @i/ml). Incubation was at either 29’C ((a) and (b)) or 42.5”C. ((c) and (d)). Radioactivity of lOO-~1 samples wss measured as a function of time after trichloroacetic acid precipitation. -0-0--, and -m-m-, proline incorporation; -O-Oand -U-z--, valine incorporation.
St thus appears that in vitro protein synthesis in lOB6 Tel+ and lOB6 rel- systems is more strongly inhibited when incubation is done at 42~5°C rather than at 29”C, even though the heat-sensitive valyl-tRNA synthetase appears equally inactive at both temperatures (Fig. 3). Table 1 reports some other characteristics of the homogeneous incorporating systems prepared from the four strains previously described, and incubated under several different conditions for 60 minutes at 425°C. It can be seen that, in all cases, protein synthesis is much reduced in the absence of ATP but only slightly affected by the absence of GTP. The system is dependent on polysomes, soluble enzymes, meroaptoethanol and magnesium ion concentration, and is inhibited by ribonuclease. Incorporation is reduced in the absence of 20 amino acids but is unaffected by the absence of valine alone. Addition to 1 ml of complete mixture of 0.5 prnol CTP and UTP or 0.1 pmol each of 20 amino acids or 500 pg stripped tRNA, does not significantly affect protein synthesis. Addition of 2 pmol putrescine or 2 pmol spermidine, two polyamines which might have a causal role in the control of RNA synthesis (Edlin & Broda, 1968), does not affect protein synthesis either in stringent or in relaxed extracts.
IN
Vll'RO
RIBOSOME TABLE
i71
RUE-Oh-1
1
Characteristics of polysome-directed cell-free protein synthesis -D2 rel+
Protein D2 rel-
~___~_. Complete --GTP - ATP, ATP regeneration -GTP, ATP, ATP regeneration - S105 supernatant fraction - Polysomes - Mercaptoethanol - 20 a.mino acids - Valine 1jO.5 - GTP,pm01 mercaptoethanol, ~~chloromerouribenzoate/ml + 0.5 pm01 CTP and UTP/ml +O.f pm01 20 amino acids/ml +500 f*g stripped tRNA/ml + 10 pg RN&se/ml + 2 pmol spermidine/ml + 2 pm01 putrescinelml Complete in 5 mm-Mga+
97.6 84.1 15.9 6.3 9.1 7.4
100.0 82.7 16.3 5.1 8.8 6.5
71.3 98.1
68.7 99.2
98.2 98.0 97.9 5.5 37.8
100.4 101.6 6.8 34.3
synthesis (%) lOB6 rel i -___--
lOB6 rel-
12.8 12.1 2.8 1-l 1.4 1.4 2.9 10.9 13.0
16.3 13.3 2.8 1.5 1.2 I.0 3.0 13.5 15.4
0.7 13~4 12.8 12.6 1.2 14.5 11.1 6.7
0~7 15.9 16.4 1.3 18-7 12.7 5.1
Tha incorporation of radioactive proline into proteins was measured, under several different conditions, in the four homogeneous systems prepared from strains D2 reZ+, D2 Tel-, lOB6 rel+ and lOB6 Tel-. Incubations were done at 42.5’C for 60 min. A description of the complete incabation mixture is given in Materials and Methods. Values are expressed as a percentage of the amount of radioactive material incorporated by a complete D2 rel- system, Each value is th.e average of 2 to 7 experiments.
(c) Bibosome run-osf in a homogeneous qptem The in vitro run-off of ribosomes were studied both in the presence and absence of protzein synthesis by measuring the conversion of polysomes into single ribosomes and ribosomal subunits as a function of time. In order to obtain maximum inhibition of protein synthesis in the 10B6 systems, all the experiments described hereafter weye done at 42.5%. Cells of strains D2 rel+, D2 rel-, lOB6 rel+ and lOB6 rel- were grown exponentially in the presence of radioactive uracil, and large polysomal structures were prepared and isolated, in each case, as described in Materials and Methods. Labeled polysomes were then incubated in a complete homogeneous system. Their gradual conversion into monosomes and subunits was followed by measurin.g the radioactivity of each sibosomal fraction after sucrose gradient sedimentation. As shown in Figure 5, the kinetics of appearance of monosomes and subunits in the D2 rel+ and D2 rel- systems are similar. There is proportionality between the rates of protein synthesis (Fig. 4(c) and (d)) and growth of the pool of run-off products, as previously reported by No11 et al. (1963). After 60 minutes of incubation, about 900!, of the polysomes initially present in the reaction medium are converted into monosomes and subunits. In the case of the lOB6 rel+ and lOB6 rel- systems, the kinetics of appearance of monosomes and subunits as a function of time are similar to each other. The initial slope of the kinetic curves is slightly lower than that of the D2 systems. Furthermore, unlike the D2 systems, there is no proportionality between the rates of protein
A. J. COZZONE
172
AND
G. 8. STENT
Time hn)
5. In vitro run-off of ribosomes from isolated polysomes. Polysomes labeled with radioactive uracil were prepared from cells of strains DZ ,rel* , Dd ret-, lOB6 rel* and lOB6 rel-, and were incubated at 42@‘C in a homogeneous system. After 0 to 60 min, the reaction mixture was cooled in an ice bath and analyzed by sucrose gradient sedimentation. The total amount of radioactive material present in polysomes, monosomes and ribosomal subunits was taken as 100%; the amount present in monosomes and subunits (Fig. 2, region I) was expressed as a percentage of the total counts. Each point is the average from 2 to 6 experiments. FIQ.
synthesis and growth of the pool of run-off products after the first 10 to 15 minutes of incubation. Thus, the kinetics of proline incorporation (Fig. 4(c) and (d)) have reached a plateau by this time, while ribosomes continue to run off for 25 to 30 additional minutes. It is to be noted, however, that the fraction of polysomes still remaining intact after 60 minutes of incubation is higher in the lOB6 rel+ system than in the lOB6 rel- system (40% verse 29%). A more detailed characterization of the run-off products was made by determining the proportions of monosomes and ribosomal subunits formed from polysomes after different incubation times (Fig. 6). In the D2 reZ+ and D2 reb- systems, most run-off products consist of monosomes, since only 11 to 21 y0 of the total ribosomal structures are ribosomal subunits. A slightly higher percentage, 21 to 27%, is found in the case of the lOB6 rel- system, whereas as much as 41 to 48% of the products which appear in the lOB6 rel+ system consist of subunits (except for the value measured after 5 min of incubation). In other words, under the conditions of incubation and sucrose gradient analysis described above, in the D2 systems in the presence of protein synthesis, as well as in the lOB6 rel- system in the absence of protein synthesis, the run-off products are mainly monosomes. This is to be contrasted with the lOB6 rel+ system, where in the absence of protein synthesis, a high proportion of ribosomal subunits is formed. Of special interest is the analysis of monosome appearance as a function of time ; it can be calculated from the values given in Figure 5 and Figure 6 that the amount of monosomes formed after 5 minutes of incubation in the D2 rel+, D2 Tel-, and lOB6 rel- systems is, respectively, only 49,45 and 50% of the amount of monosomes formed after 60 minutes of incubation. However, the same calculation for the lOB6 rel+
92.5 (15.9+84.1) 89.3 (20.8+ 79.2) 90.1 (sl.9+18.1) 87.1 (79.6 $ 20.4) 87.9 (80.8+19.2)
86.2 (79.2+ 20.8)
(79.4f20.5) (81.3+ 18.7) (77.51- 22.5) (76.1-I-23.9)
89.2 87.7 SC3 85.6
86.8 (80.7+ 19.3) 28.5 (73.3+ 25,7)
83.4 (81.4+ 18.6) 26.3 (71.6+28*4)
88.4 (17*4+ 82.6) 90.7 (21.6+ 78.4)
90.1 (81.9+ 18.1) 13.0 (59.0$41.0)
Monosomes D2 rel-
12.7 (62.5+37*5)
89.2 (79*4+20*6)
D2 ml +
(%)
10B6 reP
60.0 (54.81-45.2) 61.1 (58~1+41.9) 58.6 (58.3+41.7) 65.7 (56*6+43.4)
59.3 (23.5+ 76.5) 62.3 (20.4+ 79.6)
22.6 (25.4+ 74.6) 61.8 (79.31-20.7)
60.0 (54++45*2) 11.8 (38*4+61.6) 56.6 (62.3+37.7) 19.1 (46*3+53*7)
+ subunits
(20.5-f
79.5)
71.3 (72.7+27.3) 70.4 (73+3+2&2) 68.2 (70,4+ 29.7) 67.1 (71.3+28.7)
71.1 (l&3$-81.7) 73.7 (19*7+80.3)
73.2 (82.1+ 17.9)
23.9
71.3 (72.7+27.3) 12.8 (45.8+54.2) 69.7 (74.4+ 25.6) 20.3 (69.3+ 30.7)
10B6 rel-
Labeled polysomos from the four strains D2 reZ+, 132 reE-, lOB6 re6+ and lOB6 reZ- were incubated l’or 60 mjn at 42.5”C!, under several clifferont conditions, in a homogeneous cell-free system. A description of the complete reaction mixture is given in Materials and Methods. The percentage of run-off products and the proportions of monosomes and subunits among these run-off products (given in parentheses) were calculated and expressed as in Figs 5 and 6.
Complete in 60 mix-Na+ gradients Complete in 1 mwMgZ+ gradients Complete with: non-dialyzed S105 dialyzed S105 pre-incubated and dialyzed 5105 non-dialyzed 8105 from shifted cells
-GTP, ATP, mercaptoethanol, +p-chloromercuribenzoate + 2 pm01 spermidine/mI
Complete - GTP, ATP, ATE regeneration -ATP --S105 supernantant fraction
2
Characteristics of in vitro ribosome run-0JSfin isolated pobysomes
TABLE
174
A. J. COZZONE
AND
oLd 10
20
30 40 Tlme(minl
FIG. 6. Proportion of ribosomal Labeled polysomes were prepared from the four Tel-, and were incubated at 42~5°C for 5 to 60 min The total amount of radioactive material present upon incubation was taken as lOO0/o. The proportion as a percentage of the total oounts.
6.
S. STEST
50
60
subunits in run-off products. strains D2 reZ+, D2 Tel-, lOB6 Tel+ and lOB6 in a complete homogeneous system as in Fig. 5. in monosomes and ribosomal subunits formed of subunits was then calculated and expressed
system leads to the much higher value of 74%. This means that in the lOB6 reE+ system most of the monosomes which are going to be formed have appeared after only 5 minutes of incubation and that run-off products which appear later on are mainly ribosomal subunits. Some other characteristics of the four different systems used for studying the in vitro run-off of ribosomes are presented in Table 2. It can be seen that in all cases, the conversion of polysomes into monosomes and subunits requires the presence of an energy source. Ribosome run-off is strongly reduced in the absence of GTP but only slightly affected by the absence of ATP, as previously reported by Hirashima & Kaji (1972). The presence of an 5105 supernatant fraction, extracted from exponentially growing cells or from cells pre-heated for 5 minutes at 42.5% is essential for polysome conversion. But neither dialysis nor pre-incubation of this fraction before incubation with polysomes seems to affect the degree of conversion. The presence of spermidine leads to a higher proportion of monosomes among the run-off products. The monosomes which are formed upon incubation in a complete system are products of run-off rather than of polysome fragmentation since they can be made to dissociate into subunits by high-sodium or low-magnesium ion concentrations (Ron et al., 1968; Beller & Davis, 1971; Davis, 1971). In the lOB6 cell-free systems used in these experiments, no “non-enzymic translocation” similar to that described by Pestka (1968) and Gavrilova & Spirin (1971) occurs even in the presence of 03 mm-p-chloromercuribenzoate. (d) Ribosome run-ojJ Cn a heterogeneous system In order to assess the relative importance of polysomes and supernatant fraction in determining the character of the run-off products, mixed systems were studied (Table 3).
IA' VITRO
RIBOSOME
176
RUN-OFF
TILED Ribosome run-off in a heterogeneozcscell-free system
Polysomes Supernatant D2 reZ+ D2 rellOB6 reZ+ lOB6 rel-
Monosomes lOB6 reZ+
83.1 (751+24.9) 56.6 (56.9+43.1) 60.0 (544+45.2)t 54.3 (559+44.1) 62.7 (57.2+42+)?
+ subunits
(%) lQB6 rel-
86.6 64.1 73.1 65.0 71.3
(81.1+1&g) (74.0f26.0) (70.7-t-29.3)? (75.8+ 24.2) (72.7-+27Q3)?
Labeled polysomes from a given strain were incubated in a complete cell-free system. in the presence of a supernatant fraction extracted either from the same strain (homogeneous system) or from a different strain (heterogeneous system) as indicated. Ribosome run-off was measured and expressed as in Table 2. The proportions of monosomes and ribosomal subunits are given in parentheses. Incubations were done at 42*5”C for 30 min and, in some instances (t), for 66 min.
A control experiment showed that when polysomes from the temperature-sensitive strains lOB6 rel+ and lOB6 rel- are incubated in the presence of a supernatant fraction extracted from the temperature-insensitive strains D2 rel+ or D2 rel-, ribosome run-off at 425°C occurs to the same high extent as in a homogeneous D2 rel” or D2 rel- system. When lOB6 rel+ polysomes were incubated in the presence of a lOB6 rel- supernatant fra&ion, or IO%6 rel- polysomes in the presence of a lOB6 rel + supernatant fraction, it was seen that, regardless of the source of the supernatant fraction, polysomes from a given lOB6 strain yield the same amount of run-off products with the same proportions of monosomes and subunits. Whatever the supernatant used in the incubation mixture, conversion of the lOB6 rel- polysomes is always 8 to 13% higher than in the LOB6 rel- polysomes and the proportion of subunits formed is always 1.6 to 2.0 times smaller. (e) Ribosome run-off in crude lysates In all the experiments previously described, polysomes and supernatant fractions were first extracted separately from the cells, then purified and finally incubat,ed together in a reconstituted cell-free system. It is possible that such a treatment may affect the structure and/or some biological properties of the subcellular fractions. In an attempt to examine this possible effect of the purification procedure on the results obtained, ribosome run-off was studied in total crude lysates prepared from the four strains already described (Table 4). Upon incubation for 30 minutes at 42PC in the presence of a supernatant fraction and energy source, ribosome run-off occurred. In D2 lysates 94 to 97%, and in a lOB6 rel- lysate 87.70/,, of the ribosomal material was found as monosomes and subunits after incubation. A lower value; 76.60/b, was found in the case of a IO%6 rel+ lysate. These data are consistent with those obtained in the case of a reconstituted cell-free system (Fig. 5). In other experiments, not reported here, it was found that monosomes released from polysomes upon incubation of crude lysates dissociate into subunit,s after sucrose gradient sedimentation in a buffer containing either
176
A. J. COZZONE
AND
G. X. XTENT
TABLE 4
In vitro ribosome run-off in crude lysates
Non-incubated lysate Lysate incubated alone Lysate + 5105 Lysate + 8105 + energy
D2 rel+
Monosomes D2 rel-
40.6 53-4 66.8 94.5
38-l 54.1 71.0 97.3
+ subunits [%) 10B6 reZ+ lOB6 rel42.4 48.8 59.6 76-6
40.9 47.1 63.2 87.7
Crude lysates were prepared from the four strains D2 reZ+, D2 rel-, lOB6 reZ* and lOB6 relgrown in the presence of radioactive uracil and were incubated for 30 min at 42*5”C as indicated. A lysate from a given strain was incubated, when specified, with an S105 supernatant fraction extracted from cells of the same strain. The amount of radioactivity present in both monosome and subunit fractions was expressed as a percentage of the amount of radioactivity present in the total ribosomal fraction (polysomes+ monosomes+ subunits).
60 mM-sodium chloride and 5 mM-magnesium chloride or 50 mM-potassium chloride and 1 mM-magnesium chloride, which means that those monosomes are really products of run-off rather than of polysome fragmentation.
4. Discussion (a) In vitro protein synthesis In the experiments presented here, ribosome run-off from polysomes was studied in two different kinds of cell-free extracts. In one of these, the temperature-insensitive D2 system containing polysomes and a supernatant fraction prepared from either strain D2 rel+ or strain D2 rel-, protein synthesis occurs. In the other kind of extract, the lOB6 system prepared from either strain lOB6 rel+ or strain lOB6 rel-, which both carry a temperature-sensitive mutant valyl-tRNA synthetase, much less protein synthesis occurs. In fact, since almost no valyl-tRNA synthetase activity is detectable in the supernatant fraction extracted from cells of strains lOB6, it would have been expected that protein synthesis in the lOB6 system would have been completely arrested. However, under valyl-tRNA deprivation at 29”C, the lOB6 system still incorporates 33 to 35’j/, as much proline and 29 to 30% as much valine as the D2 system containing a fully functional valyl-tRNA synthetase. These values are reduced to 13 to 16% and 25 to 3%, respectively, when the reaction mixture is maintained at 42.5%. The unexpectedly high amino acid-incorporating activity of the lOB6 system at 29°C might be due to a residual valyl-tRNA synthetase activity which is not detected by the assay method used. As shown by Anderson & Neidhardt (1972), a very small amount of functional valyl-tRNA synthetase is indeed able to sustain in viva a high rate of protein synthesis. The incorporation of valine at 42.5”C in the lOB6 system, unlike that occurring at 29”C, appears to be completely arrested, since the valyltRNA synthetase is, very likely, totally inactive at this temperature. The fact that, however, some proline incorporation still ocours at 42.5”C might be due to some residual protein synthesis occurring in the absence of any valyl-tRNA synthetase activity.
1N VITRO
RIBOSOME
RUN-QFF
177
(b) In vitro ribosome run-08 It is important for the interpretation of the findings presented here on ribosome run-off in cell-free systems that very little degradation of messenger RNA takes place during incubation of polysomes under these experimental conditions. That this is actually the case has been previously shown; when either isolated polysomes or polysomes in a lysate are incubated in vitro under conditions similar to those used here, no n&ease activity is detected during the conversion of polysomes into run-off products (No11 et cd., 1963 ; Phillips et al., 1969 ; Hirashima $ Kaji, 1972). Furthermore, it has been reported (Kaempfer, 1970; Xubramanian & Davis, 1971) that, in such cell-free systems very little, if any, polysome re-assembly occurs from the run-off products during incubation of polysomes. The absence of significant polysome re-assembly can also be inferred from the results presented here, since of the polysomes present at the start of incubation, more than 90% are converted into monosomes and. subunits after 40 minutes. It seems likely, therefore, that the in vitro polysome disassembly due to run-off observed in the present work occurred under conditions where neither polysome degradation by nucleases nor re-assembly took place to an appreciable extent. Indeed, there are several indications that the ribosomal particles which are formed either in the presence or absence of protein synthesis are really products of run-off rather than of polysome fragmentation. First, the monosomes were found to dissociate into subunits when centrifuged in buffer solutions containing either 60 mM-sodium chloride or 1 mM-magnesium chloride. This supports the inference of their run-off origin, since it has been shown that monosomes obtained by ribosome run-off from polysomes, but not ribosomes &ill attached to mRNA, dissociate in the presence of high-sodium or low-magnesium ion concentrations (Ron et CL, 1968; Belier 8r Davis, 1971; Davis, 1971). Moreover, in an experiment not reported here, polysomes were extracted from cells pulse-labeled with radioactive arginine. After in vitro incubation of these polysomes initially carrying labeled nascent polypeptides, no radioactivity was found in the monosome region by sucrose gradient analysis. This result indicates that monosomes formed upon incubation of polysomes do not bear peptidyl-tRNA and thus are, very likely, not derived from polysome fragmentation. Finally, it may be noted that when polysomes were incubated without an energy source (Tables 2 and 4), very little conversion into monosomes and subunits occurred, indicating that, under these conditions, the polysomal structures are stable. The results presented here that, under aminoacyl-tRNA deprivation, polysomes from reb- and rel+ bacteria show similar rates of decay in vitro thus confirm the previous finding (C&zone & Donini, 1973) that under is viva conditions where neither protein synthesis nor polysome re-assembly is permitted, rel- and TeEicells also show similar rates of polysome decay. Thus, further support has been adduced for the previous inference (Cozzone & Donini, 1973) that the higher polysome level ordinarily maintained by aminoacyl-tRNA-deprived rel- cells as compared to rel+ cells is attributable to a higher rate of polysome re-~sernb~~, while protein synthesis is held in abeyance, rather than to a lower rate of disassembly. The present finding that the products of ribosome run-off in reE+ and rel- in vitro systems during ongoing protein synthesis consist mainly of free monosomes agrees
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S. STENT
with previous descriptions of the normal run-off products (Kaempfer, 1970; Subramanian & Davis, 1971; Schlessinger et al., 1967). But that in the absence of protein synthesis rel- polysomes also give rise to the normal monosome yield, whereas rel+ polysomes give rise to a high proportion of subunits among their run-off products, was not foreseen from those earlier studies. This finding might reflect a difference in the post-run-off equilibrium between monosomes and ribosomal subunits. However, the experiments reported in Table 3 rule out the possibility that the high proportion of subunits found in the 10B6 rel+ system could be merely the consequence of a greater ribosome dissociation activity of factor F3(B) present in the supernatant fraction isolated from a rel+ strain, since the same high proportion of subunits is also formed when lOB6 rel+ polysomes are incubated with a lOB6 rel- supernatant fraction. In any event, it can be concluded that this difference in the character of the run-off products of rel+ and rel- polysomes is a consequence of valyl-tRNA deprivation, since under conditions where protein synthesis is permitted, reE+ polysomes yield the normal high proportion of monosomes. Thus, there appears to be a qualitative difference in the mechanisms of ribosome run-off during aminoacyl-tRNA deprivation of rel+ and rel- polysomes. Since, under these conditions, rel- polysomes yield the same high proportion of monosomes as during normal protein synthesis, it would seem likely that here a quasi-normal run-off process continues on these polysomes even though the polypeptide assembly process has been interrupted for lack of aminoaoyltRNA. In particular, one may envisage that upon valyl-tRNA deprivation, translocation of rel- ribosomes would not be arrested upon encounter of a non-translatable valyl codon, and movement over the messenger RNA would continue. In the rel+ system, however, ribosomes reaching a non-translatable valyl codon would not be able to continue their movement along the messenger RNA, Instead, they would be prematurely released from polysomes and accumulate as ribosomal subunits in a form which renders them unable to re-associate while protein synthesis is held in abeyance. Those free monosomes which are mainly formed during the first five minutes of incubation under these conditions would arise from normal ribosome run-off due to some residual protein synthesis (Fig. 4). These proposed differences in the run-off process during valyl-tRNA deprivation between polysomes from rel - and reE+ strains of E. coli imply that these strains differ in the functional architecture of their ribosomes. This inference is supported by the finding that polysomes from a given strain yield the same amount of run-off products with the same proportions of monosomes and subunits regardless of the source of the supernatant fraction used in the incubation mixture (Table 3). Therefore, mutation of the reZ gene (Stent 62Brenner, 1961) seems to alter, directly or indirectly, some functional component, possibly a protein, of the ribosome so that in the rel- mutant strain ribosome translocation can proceed in the absence of protein synthesis.
Thanks are due to Dr Pierluigi Donini for many stimulating discussions and to Elizabeth Mullenbach for excellent technical assistance. We are also indebted to two anonymous This investigation was supported by U.S. referees for helpful criticisms and suggestions. Public Health Service Research grant GM17866 from the Institute of General Medical Sciences. One of us (A. J. C.) was a recipient of a fellowship from N.A.T.O. during part of this project.
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