Structural Integrity of RNA and Translational Integrity of Ribosomes in

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THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 256, No. 11, Issue o f June 10, pp. 5802-5809, 1981 Printed in L1.S.A.

Structural Integrity ofRNA and Translational Integrityof Ribosomes in Nuclease-treated Cell-free Protein Synthesizing Systems Prepared from Wheat Germ and RabbitReticulocytes* (Received for publication, November 4, 1980)

Theresa D. Kennedy, LindaK. Hanley-Bowdoin, and Byron G . Lane From the Biochemistry Department, University of Toronto, Toronto, Ontario, Canada M5 S 1A8

After treatment at a micrococcal nuclease concentration too low to reduce the endogenous amino acid-incorporating activity of freshly prepared reticulocyte lysate, there islittle, if any, intact 26 S RNA left in the ribosomes of either wheat germ or rabbit reticulocyte cell-free protein synthesizing extracts. The primary scissions, probably at highly exposed sites in the rRNA of plant andanimal ribosomes, produce two fragments which remain complexed until thermal denaturation reveals “hidden breaks.” Molecularweights of the fragments are -0.5 X 10’ and 0.8 X lo6 in the case of wheat, and 0.4 X 10‘ and 1.3 X 10’ in the case of rabbit. There is little perceptible degradation of 5 S, 5.8 S , and 18 S rRNA, or of tRNA in the same extracts. Even though limited degradation of 26 S rRNAby a reticulocyte nuclease has been reportedto severely impair the translational mechanism in reticulocyte ribosomes, micrococcal nuclease-induced degradation of rRNA, whether limited or extensive, does not seriously impair the ability of reticulocyte lysates to discriminate, by selective polypeptide synthesis, between complex populations of cellular mRNA. In an allied study, it is shown that under conditions well suited to recovery of the 5.8 S/26 S rRNA complex, with its naturally occurring hidden break, 5 S/18 S rRNA complexing is not detectable in theRNA of metabolizing embryos, nor in the RNA from untreated or nuclease-treated protein synthesizing extracts from wheat and rabbit. The significance of this finding is briefly elaborated in relation to the suggestion that 5 S rRNA may interact with the m!A-m$A hairpin near the 3’-end of 18 S rRNA.

L-cell rRNA (4), in this laboratory, presaged discovery of a naturally occurring hidden break in the bihelical superstructure of rRNA: the hidden break in 5.8 S/26 S rRNA, first reported in HeLa cells (5), is found in high molecular weight RNA from the large subunit of all eukaryote ribosomes (6-8). Just asthey can sustain structural integration when rRNA molecules are generated by scission of a single transcript (e.g. 5.8 S and 26 S rRNA), noncovalent forces can also promote integration between independently transcribed rRNA molecules (e.g. 5 S and 18 S rRNA). As shown in this laboratory (9, lo), there israpid, selective, and stable complexing between wheat embryo 5 S and 18 S rRNA if they are heated in aqueous solution of moderate ionic strength. Using a similar approach, otherworkers have since shown that aneven more stable “laboratory complex” can be formed between tRNA? and 23 S rRNA from Escherichia coli (11). Both laboratory complexes melt over a narrow range of temperature and they constitute the most stable unions yet shown to be possible between pairs of cellular RNA molecules, beingmore stable than the naturally occurring 5.8 S/26 S rRNA complexes. Largely on the basis of a partial structure for the laboratoryprepared 5 S/l8 SrRNA complex (12), an attractivemolecular basis has been proposed (13) in support of possible (9, 10) involvement of 5 S/18 S rRNA complexing in uniting ribosomal subunits. Signifkantly, however, although much weaker RNA/RNA interactions (e.g.tRNA/mRNA anticodon/codon pairings) can be safely assumed to occur in ribosomes, and some ( e g . 16 S rRNA/mRNA pairings in prokaryotes) (14, 15) have been shown to occur, the stronger interactions manifest in the laboratory (e.g. 5 S/18 S rRNA and tRNAp‘/23 S rRNA) have not been shown to take place in Dekker and Schachman (1) f i s t described how breaks in ribosomes (10, 11). Just asnoncovalent forces between RNA molecules stabilize the covalent structure of a DNA double helix might escape detection, being hidden within a bihelical superstructure that such complexes of proven or potential physiological interest, is stabilized by noncovalent bonds. Detection of unexplained they also sustain structural integrity and translational activity chain termini in bulk wheat embryo rRNA (2, 3) and locali- of ribosomes after extensive nuclease-induced damage of the zation of such termini to the 26 S component’ in the case of RNA in ribosomes of animal, plant, and bacterial origin (1619).Even so, it is noteworthy that although extensive breakage * This work was financially supported by the Medical Research of covalent structure in rRNA also has little effect on the Council of Canada (Grant MRC-MT-1226). The costs of publication structural integrity of rabbit reticulocyte ribosomes (ZO), limof this article were defrayed in part by the payment of page charges. ited nuclease-induced cleavage of rRNA can sharply reduce This article must therefore be hereby marked “advertisement” in their translationalactivity (21)and possibly their translational accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ’ For simplicity, the four rRNA molecules in both wheat and rabbit capacity. Longstanding interest in noncovalent integration of rRNA ribosomes are referred toas 5 S, 5.8 S, 18 S, and 26 S rRNA, overlooking the fact that 26 S rRNA in the large subunit of rabbit molecules and more recent interest (22) in the translational ribosomes has a higher molecular weight (-1.7 X lo6) than its coun- capacity of ribosomes in nuclease-treated (23) cell-free SYSterpart, thehighest molecular weight component in the large subunit tems prompted us to examine the integration and integrity of of wheat ribosomes (-1.3 X 10”).The 5 S, 5.8 S, and 18 S rRNA in RNA in the widely used cell-free protein synthesizing systems both types of ribosome, again basing molecular weights on electrophoretic mobilities, have molecular weights of -0.4 X lo5, 0.5 X lo”, from wheat germ (24, 25) and rabbit reticulocytes (26, 23). The results and implications arising from these and allied and 0.65 X lo6, respectively. 5802

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studies of the translational capacity of untreated and nuclease- wheat embryos are viable (32) and roller-milled embryos of treated cell-free protein synthesizing systems are subjects of commercial origin (wheat germ) are not, there is almost no this report. difference between rRNA (33) and mRNA (34) in the NaC1insoluble (0 “C, 24 h) RNA from the two types of embryo. EXPERIMENTALPROCEDURES MateriaZs-Micrococcal nuclease was purchased from Worthing- Whether prepared from whole embryos or from cell-free proton Biochemicals. [35]Methionine(600-1400 Ci/mmol) was purchased tein synthesizing extracts of wheat germ, NaC1-insoluble RNA from Amersham. Sparsomycin was generously supplied by Prof. Ed- is characterized by two principal components when dissolved ward Reich (Rockefeller University) and Prof. I. Goldberg (Harvard in ionically buffered medium (0.1 M NaC1) and subjected to Medical School). electrophoresis in 2.5%polyacrylamide gel: “18S” and “26 S” Preparation and Analysis of RNA-Bulk NaC1-insoluble RNA’ and bulk poly(A)-rich RNA were isolated from dry or imbibing wheat RNA from the small and large ribosomal subunits, respecembryos as described earlier (27, 28). The same methods were used tively (Fig. 1A). When the same RNA is f i s t subjected to to recover NaC1-insoluble and NaC1-solubleRNA after mixing lysates thermal denaturation before electrophoresis, 5.8 S rRNA is and reaction mixtures with 0.06 volumes of 10% sodium dodecyl released from its association with 26 S rRNA and it migrates sulphate and 1 volume of liquified phenol. In this case, the RNA freely as a discrete component (Fig. 1B). The 5.8 S rRNA concentration of the final aqueous phase (-2.5 mg/ml) was greater component is also released as a freely migrating component, than in the final aqueous phasefrom embryo-homogenates (-0.5 mg/ ml) and the NaC1-insoluble RNA was reprecipitated twice more (0 without heating, if NaC1-insoluble RNA is first dissolved in water rather than in an ionically buffered medium (30). “C, 24 h) ata concentration of 1-2 mg of RNA/ml of 2.5 M NaC1. Even when examined in 7.5% polyacrylamide gels in order To prepare 5S/l8 S and 5.8 S/26 S rRNA hybrids, a solution made by mixing wheat germ NaC1-insoluble RNA (0.9ml;10 mg/ml of to sharpenelectrophoretic boundaries of lowmolecular weight water), wheat germ NaC1-soluble RNA (0.44 ml; 10 mg/ml of water), RNA components, no trace of 5 S rRNA can be detected in water (4.06 ml), and 3 M NaCl (0.6 ml) was heated (60 “C) briefly (5 min) before quick cooling to room temperature (10). The resulting solution was then made 2.5 M with respect to NaCl in order to precipitate the hybrids (0 “C, 24 h), leaving unhybridized RNA (largely tRNA) in solution. The precipitated hybrids were dissolved in 0.1 M NaCl and precipitated twice from 2.5 M NaCl (0 “C, 24 h) in order to remove residues of unhybridized RNA. All RNA specimens were dried to salt-free powders by successive washing with 67% EtOH, 95%EtOH, and etherbefore air-drying. The powders were dissolved in sterile water(for aqueous denaturation) or 0.1 M NaCl (for ionic buffering) before electrophoresis of -25 pg in 2.5 or 7.5% polyacrylamide gel (29) as described (30). When dissolved in ionically buffered medium, hidden breaks were detected by heating (-25 pg/40 pl, 2 min, 60 “C) before electrophoresis. The duration of electrophoresis was 1.5 h in 2.5%gels and 2.5 h in 7.5% gels and absorbance (260 nm) was scanned in aGilford 250 spectrophotometer. Cell-free Protein Synthesizing Systems-Rabbit reticulocyte lysate was prepared (26), nuclease-treated (23), and assayed as previously described (22) except that the 2-mercaptoethanol concentration was 0.14 M in the “ion mix.” Wheat germ extract was prepared (24) and nuclease-treated (25) as previously described (10) except that the same buffer (100 mM KAc/3 mM MgAcz/20 mM 4-(2-hydroxyethyl)-lpiperazine sulphate (KOH) (pH 7.5)/1 mM dithiothreitol) was used for homogenizing wheat germ and filtering the supernatant fraction through SephadexG25. For assay, reaction mixtures contained 2 pl of “energy mix” (40 mM creatine phosphate/lO mM ATP/2 mM GTP), 2 pl of “ion mix” (0.5 M KAc/0.2 M 4-(2-hydroxyethyl)-l-piperazine sulphate (KOH) (pH 7.5)/8 mM spermidine/5 mM dithiothreitol), 3 pl of mRNA (0.01 AzwU,0.5 pg), 3 plof [35S]methionine (-30 pCi), and 10 pl of wheat germ extract. Analysis of Proteins-For comparative two-dimensional analysis of radioactive polypeptides (31), 110 ml of“low bis,” pH 9.4, 10% separating gel (22) and 20 ml of low bis, pH 6.8,4.4%stacking gel (22) were needed for second dimensional development in the four-place Pharmacia apparatus (GE-4). The gel is virtually indestructible during processing with dimethyl sulfoxide, 2,5-diphenyloxazole, and drying a t high temperature (-70 “C), and it gives useful widespread distribution of proteins.

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RESULTS

Examination of 5 S/18 S and 5.8 S/26 S rRNA Complexing in NaC1-insoluble RNA-Whereas laboratory-prepared The abbreviations used are: NaC1-insoluble RNA, RNA which is insoluble in aqueous 2.5 M NaCl solution a t 0 “C (includes 5.8 S, 18 S, and 26 S rRNA, mRNA and SnRNA); NaC1-soluble RNA, RNA which is soluble in aqueous 2.5 M NaCl solution a t 0 “C (includes transfer RNAand 5 S rRNA). Unless otherwise stated, NaC1-insoluble RNA was thrice precipitated (2.5 M NaC1, 0 “C) at -2 mg of RNA/ ml, in order to remove traces of NaC1-soluble RNA species carried over in the interstices of pellets; pellets usually contain -10% and less than 1%of the total tRNA and 5 S rRNA after the fist and second precipitations, respectively, following centrifugation of suspensions for 15 min, 0 “C, 27,000 X g; AzwU,quantity of material in 1 ml of a solution having an absorbance of 1 at 260 nm in a 1-cm path length quartz cell.

I

5

Distanco (cm) FIG. 1. Electrophoretic comparison of NaC1-insolubleRNA from untreated and nuclease-treated wheat germ extracts. Origins of gels (2.5%)are at theleft. RNA specimens were recovered from untreated (A, B ) and nuclease-treated (C, D ) extracts, and after dissolution in ionically buffered medium, they were subjected to electrophoresis without (A, C ) or with (B, D ) a heat-treatment to reveal hidden breaks. The conditions of nuclease treatment of the wheat germ extract (-4 mg of ribosomes/ml) were as recommended by Pelham and Jackson (23): 75 units of micrococcal nuclease/ml of extract, 15 min, -20 “C.

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NaC1-insoluble RNA from dry wheat embryos, in which all of the ribosomes are in the form of monoribosomes (10). The “laboratory prepared” 5 S/l8 S rRNA complex is more thermostable than thenatural 5.8 S/26 S rRNA complex, but only the latter complex is recovered by the procedures used to isolate NaC1-insolubleRNA, suggesting that any 5 S/18 S rRNA complexing inthe ribosomes of dry embryos is transient and/or reversibly dependent on factors not affecting stability of 5.8 S/26 S rRNA complexing (10). In view of possible mRNA-dependence of 5 S/l8 S rRNA complexing in ribosomes (35),it was of interest to search for such complexes in NaC1-insoluble RNA from imbibing embryos, in which a large proportion (-50%) of the ribosomes is complexed with mRNA, in polyribosomes. As shown in Fig. 2, there is heat-dependent release of 5.8 S but not 5 S rRNA when NaC1-insolubleRNA from imbibing wheat embryos (1024 h postimbibition) is subjected to electrophoresis in 7.5% polyacrylamide gels (Fig. 2 A ) . If mixed hybrids of 5 S/18 S and 5.8 S/26 S rRNA are subjected to thesame isolation and analytical procedures, there is heat-dependent release of 5 S, as well as 5.8 S rRNA from NaC1-insoluble RNA in the control experiment (Fig. 2B). Although formed with lower efficiency and characterized by a broader melting range, a 5 S/18 S rRNA complex can be prepared when the 5 S and 18 S rRNA molecules from higher animal cells are briefly heated at 60 “C in 0.3 M NaCl (35). It is of interest that NaC1-insoluble RNA from untreated or nuclease-treated rabbit reticulocyte cell-free protein synthesizing systems is also devoid of 5 S rRNA, there being heat-dependent release of 5.8 S rRNA, but not 5 S rRNA, when rabbit reticulocyte NaC1-insoluble RNA is subjected to electrophoresis in 7.5% polyacrylamide gel. Because sparsomycin prevents chain elongation but does not interfere with formation of 40 S initiation complexes, or with attachment of large subunitstothese complexes, it seemed that the drug might stabilize any transient 5 S/18 S rRNA association occurring after formation of a 40 S initiation complex, and before chain elongation. If wheat embryos are

imbibed in amedium containing 200 PM sparsomycin, there is -50% inhibition of protein synthesis as measured by pukelabeling between 10- and 11-h postimbibition of dry embryos, but NaC1-insoluble RNA from such embryos is again devoid of detectable 5 S rRNA. Similarly, if the wheat germ cell-free protein synthesizing system is programmed by poly(A)-rich RNA from dry embryos, either in the presence or absence of 200 PM sparsomycin (36), NaC1-insoluble RNA is again devoid of detectable 5 S rRNA, although in the presence of sparsomycin there is strong inhibition (-85%) of protein synthesis. Integrity of RNA from Cell-free Protein Synthesizing Systems-As with RNA from the wheat germ cell-free system (Fig. I), NaC1-insoluble RNA from the rabbit reticulocyte cellfree system consists of two conspicuous “18 s’’ and “26 s’’ components before denaturation (Fig. 3A) and of three components, including 5.8 S rRNA, after denaturation (Fig. 3 B ) .

!6r

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Disram. (em)

Distow(cm) FIG. 3. Electrophoreticcomparison oftheproperties of FIG. 2. Electrophoretic comparisonof low molecular weight NaC1-insoluble RNA from untreated and nuclease-treated rabcomponents released by heat treatment before and NaClafter bit reticulocytelysates. Origins of gels (2.5%)are at the left. The insoluble RNA from imbibing wheat embryos is hybridized RNA specimens were recovered from untreated ( A ,B ) and nucleasewith NaC1-soluble RNA from wheat embryos. Originsof gels treated (C,D)extracts, and after limited aqueous denaturation, were (7.5%) are at the left. Thrice-precipitated NaC1-insoluble RNA from subjected to electrophoresis without (A, C ) or with (B,D) subsequent imbibing wheat embryos, before ( A )and after ( B )hybridization with thermal denaturation. The conditions of nuclease treatment of the NaC1-soluble RNA, was dissolved in ionically buffered medium and rabbit reticulocyte lysate (-4 mg of ribosomes/ml) were as follows: subjected to brief heat treatment before electrophoresis. 150 units of micrococcal nuclease/ml of extract, 15 min, -20 “C.

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somewhat, possibly because of ambient temperature variation (20-22 " C ) . If unprotected by architectural constraintsof the ribosome, as when bulk NaC1-insoluble RNA is treated with an even lower concentration of micrococcal nuclease (30 units/ml), 18 S and 26 S rRNA are extensively degraded (Fig. 4). As shown in Fig. 5,there is a broad range of nuclease concentration (30150 units/ml) over which enzymic action on the ribosome is selective; only at high nuclease concentration (750 units/ml) is there extensive degradation of the RNA in ribosomes (Fig. 5F). Bulk NaC1-insoluble RNA has the same electrophoretic properties whether recovered from freshly prepared reticulocyte lysate having a high ribosome concentration (-4 mg/ml) (Figs. 3, A and B, and 5) or from "old" lysate (stored at -70 "C for 2 years) having a relatively low ribosome concentration (-1 mg/ml) (Fig. 6, A and B ) . As might be anticipated, when treated at the same micrococcal nuclease concentration (150 units/ml), there is much greater degradation of rRNA at the higher nuclease/ribosome ratio (cf Figs. 6D and 30). This is a practical consideration of some importance since the riboA some concentration in reticulocyte lysates can vary appreciably. The usual conditions of nuclease-treatment (23, 25) do 0.5not cause appreciable degradation of the principal components of bulk NaC1-soluble RNA, tRNA and 5 S rRNA, although notably, the tRNA/5 S rRNA proportion in the reticulocyte extract is -2-fold greater than in the plant extract. Effect o f Nuclease-treatment on Cell-free Protein Synthe260 sizing Capacity-As previously shown (22), the two-dimen6 sional electrophoretic distribution of proteins made in reI t I 0.5sponse to a supplement of mRNA fromdry wheat embryos is different from that of proteins made when mRNA from imbibing wheat embryos is added to cell-free protein synthesizing reaction mixtures containing nuclease-treated reticulocyte lysate. Because there is virtually complete scission of ribosomal 26 S RNA at a micrococcal nuclease concentration (30 units/ml) too low to reduce the endogenous amino acid-incorporating activity of freshly prepared reticulocyte lysate (Fig. Distance (ern) 5A), it is important to determine if this degradation of riboFIG. 4. Electrophoretic comparison of the properties of the somal RNA has an effect on the specificity of responses to degradation productsformed when NaC1-insolubleRNA from different supplements of mRNA. By using reticulocyte lysates wheat germ (A) and rabbitreticulocyte (B) lysates are treated having a relatively high concentration of ribosomes (-4 mg/ with micrococcal nuclease. Origins of gels (2.5%)are at the left. The positions, of markers are indicated by vertical lines in B : 26 S ml), as in Figs. 3 and 5 and extending the time of fluorographic rRNA (left), 18 S rRNA (middle), and 5.8 S rRNA (right). The development in detecting the products made in reaction mixconditions of nuclease-treatment of the RNA specimens (-2 mg of tures containing untreated lysate, it is possible to observe the RNA/ml) were as follows: 30 units of micrococcal nuclease/ml of polypeptide distributions resulting from additions of exogeRNA solution, 15 min, -20 "C. nous wheat mRNA to reaction mixtures containing untreated

When wheat germ extract is treated with micrococcal nuclease under prescribed conditions (23, 25), there is selective scission of ribosomal 26 S RNA. The cleavage is not manifest until after thermal denaturation of bulk NaC1-insoluble RNA (Figs. 1, C and D). One fragment migrates behind (Mr= -0.8 X 10") and another migrates ahead (Mr= -0.5 X 10") of 18 S rRNA. Similarly, when reticulocyte lysate is treated with micrococcal nuclease under prescribed conditions (23), there is also selective scission of ribosomal 26 S RNA. The cleavage is partially manifest after limited aqueous denaturation (Fig. 3C), but fully shown by thermal denaturation (Fig. 30) of NaC1-insoluble RNA. One fragment migrates behind (M, = -1.3 x 10") and another migrates ahead (M, = -0.4 X lo6) of 18 S rRNA. Mock incubation in the presence of Ca2+ andin the absence of nuclease does not lead to the appearance of new peaks, although in replicate experiments, in the presence of nuclease, the amounts of26 S rRNA left intact vary

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FIG. 5. Electrophoretic comparison of NaC1-insolubleRNA specimens from reticulocyte lysates treatedat different micrococcal nuclease concentrations. Origins ofgels (2.5%)are at the left. Under conditions othewise the same as described by Pelham and Jackson (23), the micrococcal nuclease concentrations were 30 ( A ) ,60 ( B ) ,90 (C), 120 ( D ) ,150 ( E ) ,and 750 units/ml (Fj.

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Structural and Translational Integrity of Nuclease-treated Ribosomes B

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grammed by wheat mRNA, in reaction mixtures containing untreated reticulocyte lysate, are very similar to the corresponding distributions obtained when the same saturating amounts (20pg of mRNA/ml reaction mixture)of the different types of RNA are used to (fully) restore (to thatobserved in reaction mixtures containing untreated lysate) the level of amino acid incorporation in reaction mixtures containing nuclease-treated lysate. For detecting the wheat-polypeptides made in such reaction mixtures, the period of fluorographic development needed in the caseof reaction mixtures containing untreated lysate is -5-fold greater than is neededfor equivalent fluorographic developmentin the case of reaction mixtures containing nuclease-treated lysate. Although a quantitative effect of massive amounts of globin mRNA (in reaction mixtures containing untreated lysate) on the proportional synthesisof different wheat proteins can be expected (37), visual examination of fluorograms reveals virtually the same differences between protein distributions directed by mRNA from dry and imbibing embryos whether reaction mixtures contain untreated (present study) or nuclease-treated lysate (22). Isolated differences can sometimes be observed when different reticulocyte lysates programed are by the same specimen of mRNA, as mentioned earlier (34), but the value of the lysate for discriminating between different translational capacities of bulk mRNA specimens is not affected in any important way by nuclease treatment (23). Exogenous supplementsof wheat mRNA restore only -75% of the [''5S]methionine-incorporatingactivity of untreated lysates when reticulocyte lysates are treated at an unusually high concentrationof micrococcal nuclease (Fig. 5F), whereas more usual conditions of nuclease-treatment (23) allow 100130% restoration of thesameactivity, noting thatwheat proteins contain about twice as much methionine as does globin. In any event, even at the high micrococcal nuclease

H+ OHMstonc. (em) FIG. 6. Electrophoreticcomparison of theproperties of NaC1-insoluble RNA from untreated and nuclease-treated rabbit reticulocyte lysates. Origins of gels (2.5%)are at the left. The RNA specimens were recovered from untreated (A,B)and nucleasetreated ( C , D)extracts,andafter dissolution in ionicallybuffered medium, they were subjected to electrophoresis without (A, C) or with (B,D ) thermal denaturation. The conditions of nuclease treatment of the rabbit reticulocyte lysate(-1 mg of ribosomes/ml) were as described in Fig. 3. Ribosome concentrations of extracts were roughly approximated as being double the mass quantities of NaClinsoluble RNA recovered by aqueous phenolic extraction of 1 rnl of lysate.

H+

t

reticulocyte lysate (and its accompanying complement of globin mRNA)." When mRNA from dry imbibing or wheat embryos is added to reaction mixtures containing untreated reticulocyte lysate, there is little change (&lo%)in the high level of amino acid FIG. 7. Two-dimensional electrophoretogramscomparing incorporation obtained without an exogenous supplement of mRNA (-10 pCi of acid-insoluble '"S-radioactivity in a 25-p1 the products made when wheat embryo NaC1-insoluble RNA is used to direct cell-free protein synthesis in nuclease-treated reaction mixture (22) containing 10 pl of reticulocyte lysate wheat germ and rabbit reticulocyte extracts. The direction of and -75 pCi of ["S]methionine (specific activity, -lo00 Ci/ isoelectric focusing in the first dimension is indicated by the disposimmol)). The polypeptidedistributions for products pro- tion of thesymbols OH- and H' (pH 5-7) andthe direction of ''Globin. synthesized in large amounts in response to the large amount of glbbin mRNA in reaction mixtures containing untreated lvsate., is not retained during isoelectric focussing (pH 5-7) and does not interfere with distributions of wheat polypeptides ( e g . Fig. 7). ~

_

I

~

molecular weight sieving in the second dimension is top to bottom. The numbered spots are found whencell-free synthesis is directed by mRNA from dry or imbibing wheat embryos; the lettered spots are found in conspicuously proportions when greater cell-free is synthesis directed by RNA from imbibing embryos, as in this case (see Ref. 22).

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protein (17, 20), or their ability to promotepolypeptide synthesis in response to supplementsof mRNA (19) or synthetic polynucleotides (18, 20). On theotherhand, verylimited degradation of ribosomal 26 S RNA, under theinfluence of a membrane-associated nuclease, has been concluded to be rewhen sponsible fora sharp loss of protein synthesizing activity rabbit reticulocyte ribosomes are treated with the nuclease (21). Although discrete andlimited fragmentation of the high molecular weight RNA component in the large subunit of animal, plant, and bacterial ribosomes has been the subjectof a diverseliterature (39-41), therehave been noreported studies of the effect that nuclease treatment might have on therabbitreticulocyte,wheatgerm,andother nucleasetreated cell-free systems now widely used, inconjunction with two-dimensional electrophoresis (31,42-49), to assess cell-free translational capacities of bulk cellular mRNA specimens. After discovery that intact 26 S RNA is virtually absent in the ribosomesof nuclease-treated cell-free systems (Figs. 1,3, 5, 6), it seemed important to examine the effect that such scission might have on the way ribosomes respond to different supplements of mRNA. Inspite of somequantitative differenceswithdifferent lysates, nuclease treatment has little effect on the responses obtained when bulk mRNA from dry and imbibing wheat embryos are used to probe the abilities of the wheat and rabbit systemsto distinguish between different populations of mRNA molecules. As noted by others (50), premature chain terminations in the wheat germ system limit its effectiveness FIG.8. One-dimensional electrophoretogram comparing the in relation to other cell-free systems, especially if products are products made during “early” and “late” imbibition of dry wheat embryoswith those made in cell-free protein synthesiz- only analyzed by one-dimensional electrophoresis in SDSby mRNA from dry andimbibing (22 polyacrylamide gel. As could be anticipated, the limitations ing systems programmed complexity of the mRNA h) embryos. The conditions of electrophoresis in SDS-polyacryl- become more severe with increased amide (12.5%)were as described (60,61), theorigins being at the top. specimens used to direct cell-free systems. For example, as The positions of standard marker proteins are shown in the first slot shown in Fig. 8, there is discriminating response to mRNA ( A )of the slab-gel: in order of ascending mobility, they are phospho- from dry and imbibing wheat embryos in the reticulocyte = 94,000), bovine serum albumin (M, = 67,000), ovalrylase A (M, system buta relatively undifferentiated response to these two = 43,000), carbonic anhydrase (M, = 30,000), and cytobumin (M, if products areonly = 12,400). The other tracks in the gel contain polypep- types of mRNA in the wheat germ system chrome c (M, tide products made in B, early imbibing embryos (“pulse-labeled” analyzed by one-dimensional electrophoresis. between 0- and 1-h postimbibition of dry embryos), C, wheat germ I t is doubtful if premature chain terminations are responextract programmedby mRNA fromdry embryos, D, reticulocyte sible for some of the more obvious differences between twolysate programmed by mRNA from dry embryos, E , late imbibing dimensional distributions of products made in the wheat and embryos (“pulse-labeled” between 21- and 22-h postimbibition of dry embryos), F, wheat germ extract programmed with mRNA from rabbit systems (Fig. 7). For instance, although proteinsa and in the reticulocyte thanin the imbibing (22 h) embryos, and G, reticulocyte lysate programmed by b are made in greater amounts wheat germ system (Fig. 7), some higher molecular weight mRNA from imbibing (22 h) embryos. proteins (e.g. 4 and 34) are made in similar or even greater quantity in the wheat germ extract. A diffuse background, concentration (Fig. 5F), or at high nuclease/ribosome ratio is to program wheat (Fig. 6D), distributions of proteins made in response to mRNA sometimes seen when wheat mRNAused from dry and imbibing embryos are very similar to the cor- extracts (Fig. 7), is not observed when the same extract is responding distributions obtained under more usual condi- programmed by bulk mRNA from ratliver,4 possibly because tions of nuclease treatment, although at the lower ribosome rat liver mRNA directs synthesis which is channeled more concentration (Fig. 6D), there are isolated increments in the fully into a smaller number of prominent polypeptide prodproportional synthesis of some proteins suchas b in Fig. 7. In ucts. Although our efforts have thusfailed far to adduceevidence general, differences betweenproteindistributionswhen a given specimen of mRNA is added to reticulocyte and wheat of 5 S/18 S rRNA complexing in the ribosomes of dry (10) and imbibing embryos, orin the ribosomes of wheat germ and germ extracts(Fig. 7) are greater than when the same mRNA is added to independently prepared reticulocyte lysates. As rabbitreticulocyteproteinsynthesizingsystems(present circumscribed the shown in Fig. 8, the reticulocyte lysate is notably superior to study),theresults of thestudieshave the wheat germ systemfor differentiating between the trans- properties of any suchcomplex. Either inmonoribosome- (10) lational capacities of mRNA from dry and imbibing wheat or polyribosome- (present study) containing cellular or cellembryos when characterizationof products is limited to one- free systems, any5 S/18 S rRNA interaction(9) does not have dimensional electrophoresisin sodium dodecyl sulphate-poly- the sort of stable existencewhich characterizes the5.8 S/26 S rRNA union and those RNA/RNA interactions which sustain acrylamide gel. structural and translational integrityof ribosomes even after DISCUSSION extensive nuclease-induced damage of RNAstructure. As Hunt (51) has noted, transient ribosomal interactions, as in Grossdegradation of theRNA in bacterial,plant,and animalribosomes diminishes, but does not abolish, either R. Sharma, R. K. Murray, and B. G . Lane unpublished observatheir endogenous capacity to incorporate amino acids into tions.

A

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Structural and Translational Integrity of Nuclease-treated Ribosomes

40 S/mRNA/tRNAMet interactions, are strong and specific,

IO. Oakden, K. M., Azad, A. A. & Lane, B. G. (1977) Can. J. Biochem.

yet the next step in protein synthesis requires that such associations be quickly dissipated as new and equally specific associations are established many times during the synthesis of each protein molecule. The elusiveness of 5 S/18 S rRNA complexing in the ribosome might be viewed in this context, but additionally and perhaps alternatively, a physiological role for the sort of specific and strong cell-free interaction shown between sequences in 5 S and 18 S rRNA might be sought at the level of the genome, where transcription of 5 S and pre- (18 5 / 5 3 S/26 S ) rRNA might be modulated, even coordinated through such an agency (cfi Ref. 52). It is tempting to speculate that similar molecular weights for the smaller fragments (0.4-0.5 X 10‘) derived by limited nuclease treatment of animal, plant (present study), andbacterial (41) ribosomes reflect the existence of a labile, exposed site common to all ribosomes. In this same context of universally occurring sites in the ribosomes of all cells, and allied with any discussion of the integrity and stability of rRNA, it is relevant to recall an unusual property which directly led to our discovery of the “universal” mfiA-mgA sequence (53): its remarkable stability toward hydrolysis of its internucleoside phosphodiester bridge. This peculiar property is a reflection of enhanced base/base stacking, already strong and responsible for exceptional stability in the parent diadenylate sequence (54-56). Augmented stability arising by tetramethylation of the diadenylate sequence may serve to stabilize a site of acute tortional stress between the limbs of the mfiA-mfiAhairpin if, as suggested in connection with 5 S118 S rRNA interaction (13), there is repeated opening and closing of the hairpin during association and dissociation of ribosomal subunits. This property wouldexplainwhy a universally conserved sequence is highly favourable, though not indispensable for biological function (57). A similar view emerged in the course of our studies of the biogenesis of G-C-mzG-C sequences in transfer RNA (58, 59), in which case it was concluded that emergent properties associated with an interstem G-C-G-C sequence may already be operative in the tRNA molecules of prokaryotes, which lack mgG, the same properties being usefully modulated by introduction of methyl substituents at the corresponding sites in higher organisms. Whereas exceptional stability led to discovery of a sequence now known to surmount a universal hairpin loop near the 3‘-end of 18 S (16 S ) rRNA in the ribosomes of all cells, it remains to be seen if exceptional lability in the mid-region of 26 S (23 S) rRNA in the ribosomes of animal, plant, and bacterial cells may point to another universal site, this one in thelarge rather than the small ribosomal subunit.

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