J. iMo2. Biol. (1967) 25, 317-330

The Binding of 5s Ribosomal Ribonucleic Acid to Ribosomal Subunits

DONALD G. COMB AND NILIMA SARKAR

Department of Biological Chemistr?) Harvard Medical School

Boston, Massachusetts, U.S.A.

(Heceiaed 8 December 1966, and ifa revised form 23 January 1967)

The 75 s ribosomes of the aquatic fungus, BlastoclacEiella emersonii dissociate at, 1O-4 M-IV& 2+ into 63 s and 45 s subunits. Only the 63 s subunit contains 5 s rRN,4, about one molecule per subunit being present. When these subunits a,rc t,reated with EDTA, the 63 s particle is unfolded to a 47 s particle without exten- sive release of protein but with the release of 5 s rRNA. Using 32P-labeled 6 s rRNA, we have demonstrated that 5 s rRNA will exchange with the 5 s rRNA on the 63 s subunit. Binding studies demonstrate that 2 molecules of 5 s rRNA will bind to ribosomal subunits or to the isolated 47 s largcx subunit. Binding to one of the two sites is displaced by transfer RNAA.

1. Introduction The 70 s ribosomes of protocaryotes and 80 s ribosomes of eucaryotes (Taylor & Storck, 1964) are composed of two unequal subunits. The 70 s particles of Escherichia coli dissociate into 50 s and 30 s subunits at low Mg2+ concentration (Tissieres, Watson, Schlessinger & Hollingworth, 1959). The larger subunit contains 23 s rRNA-t a,nd the smaller subunit’, 16 s rRNA (Kurland, 1960). The picture with ribosomes of eucaryotes is more complex, but in general the 80 s particles dissociate at low Mg2i levels into a large subunit of approximately 60 s and a small subunit of 36 to 45 s (Girard, Lat~ham, Penman & Darnell, 1965; McConkey & Hopkins, 1965; Perry $ Kelly, 1966; Tashiro & Siekevitz, 1965a,b). The large and small subunits contain 28 s and 18 s rRNA, respectively (Girard et al., 1965). The dissociation of liver ribosomes is complicated by the tendency of the large subunit to remain in a more compact form when nascent protein is attached and by the dimerization of small subunits (Tashiro & Siekeveitz, 1965b; Tashiro 8: Morimoto, 1966). There is general agreement that a final dissociated stage is reached by treatment of mammalian ribosomes with EDTA. This yields a large and small subunit with sedimentation coefficients of approximately 50 and 30, respectively.

Less widely appreciated until recently is the fact that the large subunit, derived from ribosomes of either protocaryotes or eucaryotes, contains, in addition to one molecule of 23 s or 28 s rRNA, about one molecule of 5 s rRNA (Rosset, Monier & Julien, 1964; Comb & Katz, 1964, Comb & Zehavi-Willner, 1967). The studies reported below demonstrate that during the EDTA-induced transition of the large subunit from 60 s to 50 s the 5 s rRNA is released. The second aspect of this work demonstrates that the large ribosomal subunit contains a single specific binding site

t Abbreviat,ions used: rRNA, ribosomal RNA; tRNA, transfer RNS.

317

318 D. C. COMB AND N. SARKAR

for 5 s rRNA, and that when the subunits associate to form the monomer, this site is blocked and exchange with added 6 s rRNA does not occur.

2. Materials and Methods Conditions for the cultivation of Bluatocladiellu emersonii, the isolation of ribosomes

and the preparation of isotopically labeled tRNA and 5 s rRNA were as previously described (Comb & Zehavi-Willner, 1967). Ribosome preparations were dialyzed over- night against 0.06 M-KCl-0.01 M-Tris (pH 7*3), low4 M-MgCl, to release bound tRNA. The ribosomal subunits were collected by centrifugation at 105,000 g for 4 hr. The pellet w&8 rinsed with the above solution and then suspended in the same solution and stored at -60°C prior to use.

(a) Binding assays

The assay for detecting the binding of [3aP]tRNA or 32P-labeled 5 s rRNA to ribosomes was similar to that described by Nirenberg & Leder (1964). A typical reaction mixture contained, in 0.05 ml.: 2.5 pmoles of KCl; 2.5 pmoles of Tris (pH 7.3); MgCl, at the desired concentration; 0.5 Aaso unit of ribosomes; and between 0.5 and 2.5 Azeo units of s2P-labeled 5 s rRNA or [3aP]tRNA (1 to 3 x lo6 cts/min/AasO unit). The mixture was incubated at room temperature for 15 min and then diluted to 2 ml. with the same con- centration of KCl-Tris-MgCl, used in the reaotion mixture. The mixture was slowly passed through a Millipore filter (25 mm, 0.45 p) to trap the ribosomes and the filter was washed 5 times with 3-ml. portions of the above buffer. The filters were dissolved in Bray’s solution (Bray, 1960) containing 10% water and counted in a scintillation spectro- meter. The specific activity (cts/min/A 280 unit) of labeled tRNA or 5 s rRNA was deter- mined under the same conditions. At the concentration of ribosomes used in this assay procedure, less than 3% of the ribosome Azeo units passed through the filters.

Gradient centrifugations were performed with 28 ml. of a 5 to 20% linear sucrose gradient containing 0.05 M-KCI-0.01 M-Tris (pH 7*3), and MgCI, or EDTA depending on the experiment. The tubes were centrifuged at 4°C in the SW26 rotor with the Spinco model L ultracentrifuge. Approximately 0.9-ml. fractions were collected and the absorb- ancy at 260 mp determined with a Zeiss spectrophotometer. To dissociate B. emeraonii ribosomes into 50 s and 30 s subunits, ribosomes were incubated at 0°C for 15 min with 6 pmoles of EDTA per mg of ribosomes (Tashiro & Siekevitz, 1965b). The subunits were separated by centrifugation through sucrose gradients containing 0.05 M-KCl-0.01 M-Tris (pH 7.3)-0.001 M-EDTA.

RNA was extracted from ribosomes with sodium dodecyl sulfate and water-saturated phenol and chromatographed on methylated albumin columns as described previously (Comb & Zehavi-Willner, 1967).

Protein was estimated by the method of Lowry, Rosebrough, Farr & Randall (1951), using bovine serum albumin as the standard. No correction was made for differences in chromophore content of ribosomal proteins and the albumin standard.

To calculate the amount of B. emereonii [32P]tRNA or 3aP-labeled 5 s rRNA bound to ribosomes, the following molecular weights were used; tRNA, 26,600; 6 s rRNA, 61,000; 18 s rRNA, 0.63 x 10e; 28 s rRNA, l-6 x 106. The chain length of B. emersonii tRNA and 6 s rRNA was determined previously (Comb & Zehavi-Willnsr, 1967). Since B. emersonii ribosomes appear to be typical of other eucaryotes, we have used the values reported by Staehelin, Wettstein, Oura & No11 (1964) for the molecular weights of 18 s and 28 s rRNA. Based on these values, one molecule of 6 s rRNA per ribosome represents 2.2% of the total RNA. A value of 2.8% was determined experimentally from methylated albumin-col- chromatograms of RNA extracted from ribosomes. Since 6 s rRNA is attached to the larger,subunit, it represents 3.2% of the RNA-assuming one molecule per subunit.

(b) Preparation of isotopically labeled 5 s ribosomal RNA In the experiments described below, the binding of 32P- and 14C-labeled 6 s rRNA to

ribosomes at a relatively high input level of 6 s rRNA, is presented. This requires 6 s rRNA of high purity. Unfortunately, we have no quantitative assay for measuring the purity of 6 8 FRNA,

5s RIBOSOMAL RNA BINDING TO RIBOSOMES 3 I 9

Three different preparations of isotopic&y labeled 5 s rRNA were used for the binding experiments. All were isolated in an identical manner and, after methylatad albumin column chromatography, the leading edge of the peak was not pooled since this region would contain the highest contamination with tRNA. The preparations showed a single peak by methylated albumin column chromatography, DEAE-cellulose chromatography at 80% and by countercurrent distribution after 200 transfers (Comb & Zeha,vi-Willner, 1967). The major contaminants would be tRNA and possibly degraded messenger RNA. From in viva labeling of the methyl groups of tRNA, we can state with some nssuranor that cont,amination with tRNA is less than 6%, and probably much less than that since radioactivity due to methylatad bases could not be detected in the final preparat,ion. \Ve know, however, that submethylat,ed tRNA tends to chromatogreph at t,he leading c~lpt’ of 5 s rRNA on methylated albumin columns, and therefore we cannot excah~dc some precursor form of tRNA (low in methylated bases) &s a small contaminant, of our 5 s rRK.4 preparations. Degraded messenger RNA is also a problem; we have no way of tlctermining the amount present. We est,imate that its contamination is not significant, since 5 s rRNA isolated from E. coli by the same procedure shows a very low annetlling efficiency t-o DNA (0.0120/,), indicating that the preparation is relat,ively free of messmgcr RK’A (Zehvi- Willnor & Comb, 1966).

(c) Preparation of transfer RKA The same preparation of tRNA was used for all the experiments on the inhibition of

5 s binding to ribosomes. It was isolated from the 105,000 g supernatant solution of cell extracts, and as far as we can tell from chromatographic profiles, it is free of 5 s rRNA. We cannot, however, exclude the possibility of trace contamination. At first, we tended to use much higher levels of tRNA for the inhibition studies than was necessary. We have! not established, as yet, the optimum level of tRNA required for inhibition of 5 s rHNA binding, but have obtained the same results when the excess of t,RNA over 5 s rRKA was anywhere from a 5- to 24-fold.

3. Results (a) Attachment of 5 s rRNA to the 60 s subkt

and its release with EDTA

The aquatic fungus, B. emersonii, has not been widely used in biochemical studies and, therefore, we feel t,he following description of t,he cytoplasmic ribonucleoprotein particles is warranted. Under our standard conditions of growth and cell fractionation, about 16% of the total cytoplasmic ribosomal RNS sediments at 25,000g for 20 minutes with the mitochrondria. This may represent ribosomes attached to membranes or large polysomes. We have not examined this fraction further. The remainder of the cytoplasmic particles, when subjected to sucrose gradient centrifugation, appear as 75 s particles and higher aggregates, presumably polysomes (Fig. l(a)). When the cytoplasmic particles are dialyzed against 10V4 M-Mg2+ buffer overnight, pre- dominantly 63 s and 45 s particles are obtained (Fig. l(b)). In this experiment 32P-labeled ribosomes of B. emersonii were centrifuged in the same gradient with 3H-labeled ribosomes of Escherichia coli so that sedimentation coefficients could be accurately calculated (Martin & Ames, 1961). The observed values were 63 s and 45 s.

The dissociation of ribosomes into 63 s and 45 s particles shown in Fig. l(b) is only observed at very low ribosome concentration. At higher concentrations (10 to 20 -4eo units) a 50 s peak is also observed in the sucrose gradients with a con- comitant decrease in the 45 s peak. This may represent aggregates of the small subunit. We have not characterized the 50 s particles with respect to RNA components.

The sedimentation coefficients of other particles reported were calculated from separate, but otherwise identical, sucrose gradient6 of reference 2. coli particles.

320 D. G. COMB AND N. SARKAlE

10-4 t.mg=+ 47s lO-3 M-EDTA

1 oottom / I , I I I I 1 0 IO 20 0 IO 20 0 IO 20

Fraction no.

(a) (h) (c)

FIG. 1. Sucrose gradient analysis of B. emersonii ribosomes and ribosomal subunits.

(a) Particles isolated from cytoplasmic extracts of B. emersonii by high-speed centrifugation and analyzed at 10ea M-Mg a+. The sample was cent,rifuged for 2.5 hr at 25,000 rev./mill.

(b) Long-term 32P-labeled ribosomes isolated as in (a) but dialyzed overnight at 0°C against 0.06 M-KCl-0.01 M-Tris (pH 7*3)-10-4 M-MgCl, prior to sucrose gradient analysis. 0.1 Azao unit of aaP.labeled ribosomes of B. emersonii were combined with 1.0 A 260 unit of 3H-labeled ribosomes of E. coli B and centrifuged for 16 hr at 17,500 rev./min in a gradient containing low4 M.Mga+ . Separate experiments with higer concentrations of ribosomes indicated that the radioactivity and absorbancy profiles for both types of ribosome preparat.ion were identical. The two peaks for E. coli ribosomes were considered to be 50 s and 30 s and calculations of sedimentation coefficients of B. emersonii ribosomal subunits were baaed on these values. -+-a--, 3aP-labeled ribosomes of B. emersonii; -_O--_O-, “H-labeled ribosomes of E. co.% B.

(c) B. emersonii ribosomes isolated a8 in (b) but treated with 5 pmoles of EDTA at 0°C just prior to centrifugation. The sucrose gradient contained 10e3 M-EDTA in place of Mga+. The sample was centrifuged for 16 hr at 20,000 rev./min.

Treatment of 63 s and 45 s particles with EDTA yields 47 s and 32 s particles in an A,,, ratio of 2.2 to 1, respectively (Fig. l(c)). To maintain uniformity with other reports, the 10M4 M-Mg2’ ribosomal subunits will be referred to as 60 s and 45 s

particles and the EDTA-particles as 50 s and 30 S. In experiments not reported, it

was demonstrated that isolated 60 s particles, when treated with EDTA sediment as 50 s particles plus a small amount of 30 s particles. The 30 s particles account for about 10% of the total A,,, and were probably derived from dimerized 45 s subunits

contaminating the 60 s peak (Tashiro & Morimoto, 1966). The protein content of both 60 s and EDTA-50 s subunits was 39%. Therefore, we feel that within the limits of the calorimetric protein determination employed, the conversion of a 60 s particle to a 50 s particle by EDTA does not involve an extensive release of structural ribo- somal proteins. It is doubtful, however, if this technique would detect the release of one or two of the structural proteins from the large subunit. Tashiro & Siekevitz (1965aJ) suggest that the lower sedimentation coef&ient after EDTA treatment is due to hydration or to change in shape of the particle.

The studies recorded in Fig. 2 demonstrate that during conversion of the 60 s particle to a 50 s particle, the 5 s rRNA is released. 32P-labeled ribosomes were centrifuged through a sucrose gradient under conditions which yielded either 60 s and 45 s subunits, or 50 s and 30 s subunits. The RNA from the two peak tubes of

0

0

0

0

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Q s

0,

0.

O-

0,

4-

3-

2-

l-

4-

3-

2-

I -

,j s RIBOSOMAL RNA BINDING TO R’IBOSOMES 32 1

(a) 60 s

-6

-4

1 - 21

J I” .J”

fraction no. (2.5 ml.)

Fm. 2. Methylated a,lhumin column ohromatograms of [aaP]RNA extracted from ribosomal subunits of B. erneramii.

The subunits were obtained by sucrose gradient centrifugation, and [3ZP]RNA was extracted from the two peak fractions of aach type of subunit with sodium dodecyl sulfate and phenol. The [32P]RNA was combined with 1 to 2 AzeO units of 5 s rRNA and 10 to 20 A,,, units of high molecular weight rRNA (18 and 28 s) to visualize the absorbancy profiles due to these two RNA components. The column size was 1 cm x 26 cm, and fractions of approximately 2.5-ml. were collected. Other details have been described previously (Comb & Zehavi-Willner, 1967).

Note the scale change for high molecular weight RNA in (a) and (b). The specific activity (cts/min Ass,,) of the ribosomes used for (c) and (d) was much lower than that of (a) and (b). After 1.0 ml. from each fraction was removed for counting, the tubes indicated by the brackets in (b) were pooled and the sample passed directly over a DEAE-cellulose column at 80°C as described in Fig. 3.

322 D. G. COMB AND N. SARKAR

each type of subunit was extracted with sodium dodecyl sulfate and phenol and chromatographed on methylated albumin columns with carrier 5 s rRNA and high molecular weight rRNA. The results in Fig. 2 demonstrate that 5 s rRNA is associated only with the 60 s subunit. The 50 s and 30 s subunits contain no detectable radio- activity due to 5 s rRNA.

The low molecular weight RNA extracted from the 45 s particle which is eluted from methylated albumin columns at the leading edge of 5 s rRNA requires further comment. This component is extremely difficult to distinguish from 5 s rRNA by this chromatographio procedure. Therefore, to distinguish it from 5 s rRNA the material in the tubes under the bracket in Fig. 2(b) was pooled and subjected to chromatography on DEAE-cellulose at 80°C (Comb $ Zehavi-Wilhrer, 1967). Under these conditions, about 90% of the radioactive material is eluted at the same position as tRNA (Fig. 3) suggesting that the chain length of this RNA component is closer to that of tRNA than 5 s rRNA. It represents about 3.3% of the total radioactivity of the 45 s subunit, and assuming a molecular weight close to that of tRNA, there is apparently about one molecule per 45 s subunit. (Since analysis of ribosomes washed in 10m4 M-Mg2+ does not indicate one molecule of this low molecular weight RNA component per monomer, it seems likely that it is present on only those 45 s subunits which do not aggregate or dimerize when relatively high concentrations of subunits are separated in sucrose gradients.) The nature of this RNA component is under investigation at present and will be the subject of future communications from this laboratory.

Fraction no. (3 ml.)

FIG. 3. Comparison of elution position of [3aP]RNA from 45 s subunit with 6 a rRNA by DEAE-cellulose chromatography at 80°C.

The RNA from the tubes under the braoket in Fig. 2(b) wan pooled and passed direotly through a 1 om x 10 cm DEAE-cellulose, Cl- form, column at 80% and the material eluted with a linear salt gradient (Comb UC Zehavi Willner, 1967). In (a) the elution poeition of a mixture of [3HjtRNA and 33P-labeled 6 B rRNA from B. emeraonii is Bhown. The column in (b), which oontained the [3’P]RNA from the 5 8 region of the methylated albumin column of Fig. 2(b), wae run under identical conditions.

3 s RIBOGOMAL RNA BINDING ‘CO RIBOSOMES ‘J.‘.{ . -3

Additional proof that 5 s rRNA is released from EDTA-treated ribosomes was obtained by isolating the 6 s rRNA from the 4 to 5 s region of the sucrose gradient of Fig. l(c) or, as is shown in Fig. 4, from the 105,OOOg supernatant solution nfkr EDTA-treated particles had been removed by centrifugation. The high molecular weight RNA also present with the 5 s rRNA in the 105,OOOg supernatant solution (Fig. 4) probably arises from incompletely sedimented ribosomes. The amount of

5 s rRNA recovered in this case was estimated at about 9576 of that obtained h> direct analysis of ribosomes prior to EDTA treatment.

I I

Fraction no. (3 ml.)

Fxa. 4. Methylated albumin column chromatography of RNA present in the 105,000 g super- natant solution after washed ribosomes had been treated with 5 pmoles of EDTA per mg of ribosomes and pelleted by centrifugation.

The supematant solution was passed directly over a 1 cm x 26 cm methylated albumin column and the RNA eluted with a linear tit gradient. 300 Aaso units of ribosomes, washed in 10V3 M- Mga+, were treated with EDTA in this experiment.

(b) The binding of 32P-labeled 5 i TRNA to ribosomes

Ribosomes isolated from B. emersonii and dialyzed in buffer containing 10 - 4 iv-Mg2 + contain only small amounts of tRNA and about 1 molecule of 5s rRNA per ribosome. Consequently binding experiments with ribosomes prepared in this manner involve binding of tRNA to the subunits, but exchange of 32P-labeled 5 s rRNA with 5 s rRNA present on the 60 s subunit. For complete exchange of labeled 5 s rRNA with that present on the ribosome and for saturation of additional binding sites, a 50- to lOO-fold excess is required. To demonstrate that added 5 s rRNA actually exchanges with that present on the 60 s subunit, the following experiment was done. Long-term 3aP-labeled ribosomes were incubated with either unlabeled tRNA or unlabeled 5 s rRNA at 10e4 M-Mga+ as described under Materials and Methods. The 3aP-labeled ribosomes were then trapped on a Millipore filter and the filtrate, which should contain 32P-labeled 5 s rRNA if exchange occurred, was passed directly over a methylated albumin column. The column was eluted, and the radioactive material which ohromatographed with 5 s rRNA was determined. The results are shown in Table 1. These results clearly demonstrate that unlabeled 5 s rRNA exchanges with 3aP-labeled 5 s rRNA on the ribosome at 10S4 y-MIg2+. They also show that tRNA will not exchange or displace 5 s rRNA from the ribosome to any significant extent. The small exchange that did occur may have been due to trace contamination of our tRNA with 5 s rRNA. In Fig. 5 the binding of 5 s rRNA to

324 D. G. COMB AND N. SARKAR

TABLE 1

Exchange of unlabeled 5 s rRNA with 32P-labeled 5 8 rRNA attached to 32P-lubeled ribosomes

Additions 3ZP-labeled 5 s rRNA

released sb Theoretical (cts/min)

5 s rRNA (2.12 A,,e units) 6105 97.5

tRNA (2.10 Aaso unit’s) 810 14.0

None 0 0

The reaction mixtures (see Materials and Methods) contained lob4 M-MgCl, and 087 Azeo unit of sap-labeled ribosomes (2.84 x lo6 cts/min) and additions as inclicated. After 15 min at room temperature, the ribosomes were trapped on Millipore filters and the e2P-labeled 5 s rRNA in the filtrate determined by methylated albumin column chromatography.

In the experiment with no additions, about 5% of the total radioactive material of the ribosomes passed through the filter, and after methylated albumin column chromatography some of this material, presumably degraded rRNA, was eluted in the tRNA-5 s rRNA region, although no distinct peak was evident. This background radioactivity has been sub&acted from the values reported for tRNA and 5 s rRNA. In the exchange experiment with 5 s rRNA, the radioactive material released by the addition of this component chromatographed with 5 s rRNA on methyl- ated albumin columns. However, when the same material was subjected to DEAE-cellulose at 8O“C, about 20% of the radioactive material was eluted in the tRNA region and the rest chromato- graphed with 5 s rRNA. Therefore, it is possible that 5 s rRNA can also exchange with the low molecular weight RNA associated with the smaller subunit, or that 20% of the exchange observed is due to contamination of our 5 s rRNA preparation with this material.

0.03

t

0.51 A,, unit of ribosomes/

0.01

A,,, units of 32P-labeled 5 s rRNA added

FIG. 5. The binding of 3aP-labeled 5 s rRNA to ribosomal subunits dissociated in 10m4 M-Mge+.

The reaction mixtures contained lOA4 re-Mge+ and were otherwise as described under Materials amd Methods. The specific activity of the 5 s rRNA was 130,000 cts/min/d,e, unit. In those reaction mixtures containing 0.61 Aas,, unit of ribosomes, 0.023 Aaso unit of 6 s rRNA bound is equivalent to about two molecules per 75 s ribosome. In these and other binding experiments, we assume that the total molecular weight of the RNA associated with B. emeraonii ribosomes is 2.3 x 1Oe and that one molecule of 5 s rRNA (mol. wt 51,000) per ribosome is 2.2% of the total. A background of about 60 cts/min was obtained with the highest concentration of eaP-labeled 5 s rRNA used when riboeomes were omitted from the reaction mixture.

5 s RIBOSOMAL RNA BINDING TO RIB0801\iES 92,3

ribosomal subunits as a function of both 32P-labeled 5 s rRNA and ribosomc con- centration is shown. Under these conditions, saturation of binding sites generally occurs when two to three molecules of 5 s rRNA have been bound.

The effect of Mg2+ concentration on the binding of 5 s rRNA to ribosomes is striking. Figure 6 shows t,hat at 10m2 M-Mg2+, where ribosomes exist in the 75 s monomeric form, very little 5 s rRNA binds to (exchanges with) t,he particles. On t,he other hand, [32P]tRNA readily attaches to the ribosomes and approaches satura- tion at slightly more than one molecule per ribosome. At 10m4 M-Mg2+, the reverse situation prevails; 5 s rRNA binding reaches saturation at about two molecules per ribosome, whereas tRNA binds rather poorly and would require higher concentrations t’han those used in these experiments to reach saturation. Although Cannon, Krug $ Gilbert (1963) did not detect binding of tRNA to ribosomes at 10m4 M-Mg2+. t)hcx assay procedure employed was quite different and much less sensitive than t’hat of t,ht> present studies. We have adso used a much higher input of tRN,4 per ribosome and this may, in pa.rt, account for the different results obtained.

AzbO units of C’2PIRNA added

FIG. 6. Effect of Mg2+ concent.rat.ion on the binding of 32P-1abeletl .i s ILISA and [32PJtRS;\ to B. emersonii ribosomes.

The react,ion mixtures contained the lMg2+ concentration indicated and were otherwise as stated under Materials and Methods. The specific activit,y of t.he tRNA was always close to t,hat, of 5 s rRXA, since bot,h were isolated from t,he same 32P-labeled cells. For calculating t,he amount of t,RNA bound per ribosome, we have made the same assumptions a.s for 5 s rR,NA, namel!.. one molecule per ribosome represents I.l”/& of the total RNA.

(c) Effect of unlabeled tRNA on 3YP-labeled 5~ rRNA bindi~ng to ribosonws

Direct analysis of ribosomes has indicated that about one molecule of 5 s rRNA is bound per monomer. The experiments reported above show, however, that about two molecules of 5 s rRNA can bind in the in citro experiments. Since Cannon et al., (1963) have demonstrated a single binding or exchange site on the large subunit for tRNA, it seemed possible that 5 s rRNA may be binding to this site as well as ittl own specific site. Figure 7 demonstrates that the addition of a large excesti of unlabeled tRNA to the assay mixtures inhibits the binding of 32P-labeled 5 s rRNA to one of the two &es.

326 D. G. COMB AND N. SARKAR

A 260 units of 32P-labeled 5 s rRNP

per incubation mixture

FIQ. 7. Effect of unlabeled tRNA on the binding of 3aP4abeled 5 s rRNA t,o ribosomes at lo-’ ar-MgZ+.

The reaction mixtures (Materials and Methods) contained lo-* M-Mg2 +. The reaction mixtures represented by the lower curve (-•--) contained, in addition, 8 A,,, units of unlabeled tRNA per I.0 Azao unit of saPTlabeled 6 s rRNA used in the maction mixture. On a molar basis, this represents a l&fold excess of tRNA.

(d) The binding of 5 s rRNA to the 50 s ribosonucl subunit

In these experiments ribosomes were treated with EDTA to dissociate the ribosomes into 50 s and 30 s subunits. The subunits were separated by sucrose gradient centri- fugation and the 50 s particles isolated by high-speed centrifugation and used in the binding experiments recorded in Fig. 8(a). When only 50 s particles are present in the reaction mixtures, a plateau in the binding curve at 10e4 M-Mga+ is reached at about two molecules of 5 s rRNA per 50 s subunit. In the presence of excess unlabeled tRNA, only one molecule of 5 s rRNA binds to the 50 s subunit.

In addition, isolated 50 s subunits bind somewhat more than one molecule of b s rRNA in 10ea M-Mg2+ (Fig. 8(b)). Compare the binding in this experiment with that shown in Fig. 6 with 75 s monomers at lOma Icl-Mga + . In the latter case, very little binding to monomer occurred. The addition of unlabeled tRNA only partially inhibits the binding of 5 s rRNA to the 50 s subunit at 10W2 En-Mga + . Interpretation of the results obtained with 5 s binding to the 50 s subunit at 10U2 M-Mga+ and the effect of tRNA on the binding will require more extensive investigations, but the results do suggest that 5 s rRNA binds primarily to its own specific site at high

Mb2 a+ levels and only poorly to the tRNA binding site, if indeed separate sites are present on the 50 s subunit.

(e) The binding of 5s rRNA to the 309 subunit

The 30 s subunits obtained by EDTA treatment are unstable and when isolated and recentrifuged through a sucrose gradient most of the material sediments with a much lower dedimentation coefficient. Consequently, we have not considered it, meaningful to study binding to this particle until it can be stabilked, However, the

6 s RIBOSOMAL RNA BINDING TO RIBOSOMES 327

(b) W2 M--M~~’ 1

A,, units of “C-labeled 5 s rRNA added

FIG. 8. The binding of ‘V-labeled 6 s rRNA to isolated 60 s subunits in (a) IO-’ aa-@“+ and (b) 10-s M-Mga+ and the effect of unlabeled tRNA.

The 50 s subunits were isolated from sucrose gradients of EDTA-treated ribosomes by diluting the sucrose fractions threefold with 0.05 M-KCl-0.01 M-Tris (pH 7.3) and centrifuging for 0 hr at 105,000 g, The 60 s subunit pellet was resuspended in 0.05 M-KCl-0.01 M-Tris (pH 7.3) and 10e4 M-MgCl, and stored at -6O’C. In reaction mixtures containing unlabeled tRNA, 2.5 Aas0 units of tRNA were added for each Ass,, unit of 14C-labeled 6 s rRNA. The speci6c activity of the 14C-labeled 6 s rRNA was 12,600 cts/min/&ss units.

Fraction no.

FIQ. 9. The binding of 3aP-labeled 5 s rRNA to 30 s subunits and the effect of unlabeled tRNA.

Ribosomes dissociated in 10e4 M-Mga + were treated with EDTA and the subunits separated by sucrose gradient centrifugation. A portion (0.1 ml.) from alternate tubes throughout the gradient was assayed for binding of 3aP-labeled 6 s rRNA. The reaction mixtures contained the following, in 0.12 ml.: 6 pmoles of Tris (pH 7.3); 6 pmoles of KCl; O-024 pmole of MgCl,; 0.95 Asso unit of 3aP-labeled 5 s rRNA (1.34 x 10s cts/min/&s,J; 0.1 ml. of sucrose gradient fraction and, where indicated, unlabeled tRNA (12 Aoso units/ApaO unit of saPlabeled 6 s rRNA). The subunits were trapped on Millipore filters, washed and counted as described (see Materials and Methods). The radioactivity associated with each reaction mixture is plotted. Without subunits, a back- ground of about 20 ots/min was obtained.

22

328 D. G. COMB AND N. SARKAR

experiment recorded in Fig. 9 suggests that 5 s rRNA can bind to freshly isolated 30 s particles and that binding is not inhibited by tRNA. In this experiment, ribo- somal subunits prepared in 10m4 M-Mg2+ buffer were treated with EDTA and then centrifuged through a sucrose gradient to yield the absorbancy profile shown in Fig. 9. Portions of various fraction (0.1 ml.) were then assayed for their ability to bind 32P-labeled 5 s rRNA in the presence or absence of unlabeled tRNA. The results demonstrate that both 50 a and 30 s subunits are able to bind 5 s rRNA. Unlabeled tRNA inhibits about 50% of the binding to the 50 s subunit, but it has no effect on the binding to the 30 s subunit.

The observation that 5 s rRNA binds to the 30 s subunit should be viewed with caution, since excess 5 s rRNA was used for binding to this subunit and it is possible that contaminants in the 5 s rRNA, particularly the low molecular weight RNA present on the 45 s subunit, are responsible for the binding observed.

4. Discussion

The present studies have demonstrated that during the dissociation of B. emersonii ribosomes the following subribosomal particles are formed:

lo-‘M-M@+ 75 8. ‘+ lo-aidaga+

45 s %A 30 s

We have not demonstrated the conversion of 45 s particles to 30 s particles by EDTA, but studies with HeLa cell ribosomal subunits indicates this to be the case (Girard, et al., 1965). As shown in the present studies, as well as those of others (Tashiro $ Siekevitz, 1965b), the conversion of the 60 s particle to the 50 s particle does not result in extensive release of structural ribosomal protein, but probably involves a change in shape or unfolding of the particle. The evidence that 5 s rRNA is released from the 60 s particle during this conversion is unequivocal and is based on the following observations. (a) The 60 s particles contain 5 s rRNA, whereas the 45 s subunits do not. (b) No detectable 5 s rRNA is present on either the 50 s or 30 s subunit derived by EDTA treatment. (c) After EDTA treatment of ribosomes, 5 s rRNA can be recovered from the 4 to 5 s region of sucrose gradients or from the 105,000 g supernatant solution after removal of 50 and 30 s subunits by centrifuga- tion; in both cases, the amount recovered is comparable to that present on the particles before EDTA treatment, The 45 s subunit does contain an RNA component which chromatographs at the leading edge of 5 s rRNA on a methylated albumin column, but this component is eluted with tRNA on DEAE-cellulose chromatography at 80°C. It is possible that this RNA species is the submethylated tRNA previously reported to be attached to B. emersonii ribosomes (Comb, 1964; Sarkar & Comb, 1966). An RNA component with similar chromatographic properties is present on E. coli 30 s ribosomal subunits (Zehavi-Willner & Comb, unpublished results).

Recent studies by Tashiro & Morimoto (1966) indicate that subunits isolated by EDTA treatment fail to re-associate into the monomer form of one large and one small subunit. In view of the above results, it would be of interest to determine if

5 6 RIBOSOMAL RNA BINDING TO BIBOSOMES 420

5 s rRNA is required for re-association of subunits. Experiments in this direction are

being pursued. The 5 s rRNA binding experiments have disclosed several important features of

this reaction. First, it is clear that 5 s rRNA binds (exchanges) poorly to mixtures of subunits in 10m2~-Mg2+, where presumably 75 s monomers exist. At lo-* w- Mg2+, where the monomers have dissociated into 60 s and 45 s subunits and possibly dimers of the small subunit, about two molecules of 32P-labeled 5 s rRNA bind to these particles. One molecule of 32P-labeled 5 s rRNA can be displaced with unlabeled tRNA. An experiment not reported in this paper suggests that the 60 s component accounts for most of the binding observed with mixtures of subunits. Studies with isolated 50 s subunits demonstrate that at low magnesium ion concentrations, these particles bind about two molecules of 5 s rRNA, and that binding to one of the two sites can be inhibited by excess tRNA. Finally, 5 s rRNA binds primarily to its own site on isolated 50 s subunits in 10m2 M-Mg2+, since excess tRNA had little effect’ on the binding.

The simplest interpretation of these observations is that the large ribosomal sub- unit contains one specific binding site for 5 s rRNA. When the large subunit associates with the small subunit to form the 75 s monomer, the 5 s rRNA attached to the large subunit is not available for exchange with added 5 s rRNA. Since 5 s rRNA can bind specifically to the large subunit in 10m2 M-Mg2+, it would appear that the lack of binding or exchange of 5 s rRNA which occurs when both subunits are present at low2 rd-i%g2+ is due to blocking of the 5 s rRNA site by the association of subunits rather than a change in conformation of the large subunit. Although preliminary studies showed that 5 s rRNA could also bind specifically (not inhibited by tRNA) to the 30 s subunit, we have not yet determined the extent of binding to this subunit. This leads to the interesting possibility that 5 s rRNA may be involved in binding the two subunits together to form the monomer. This hypothesis is open to experi- mental testing, but ao far the instability of the EDTA-treated particles has com- plicated these investigations.

The present studies demonstrate that with either isolated 50 s subunits or a mixture of 60 s and 45 s subunits, unlabeled tRNA prevents the binding of 32P-labeled 5 s rRNA to one of the two binding sites on the ribosome. We prefer to interpret these results with caution, since we have not demonstrated that tRNA bound to the ribo- some is specifically displaced by the addition of 5 s rRNA. It is possible that the attachment of tRNA to the ribosome modifies one of two binding sites for 5 s rRNA without directly competing for a 5 s rRNA binding site.

In conclusion, these studies have demonstrated that 5 s rRNA is released from the large ribosomal subunit during its conversion to the 50 s particle by EDTA. Two sites are present on the large subunit which will bind 5 s rRNA. The binding of 5 s

rRNA to one of these sites can be inhibited by tRNA, whereas t,he ot’her binding site appears to be specific for 5 s rRNA.

We wish to acknowledge the expert technical assistance of Mr Jose DeVallet and MI Douglas Keagle.

This study was aided by grant no. GM 12632 from the National Institutes of Health of the United States Public Health Service, and grant no. GB 2349 from the National Science Foundation. One of us (D. G. C.) is a Research Career Development Awardee of the United States Public Health Service, I-K3-GM 6385 and the other (N. S.) is a Fellow of the National Cystic Fibrosis Research Foundation.

330 D. a. COMB AND N. SARKAR

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