Reprinted from the Proceedings of the NaTtonaL ACADEMY OF ScLENCES Vol. 48, No. 8, pp. 1466-1472. August, 1962. THE IDENTIFICATION OF THE RIBOSOMAL RNA CISTRON BY SEQUENCE COMPLEMENTARITY ,* II. SATURATION OF AND COMPETITIVE INTERACTION AT THE RNA CISTRON By 8S. A. YANKOFSKYT AND 8. SpIEGELMAN DEPARTMENT OF MICROBIOLOGY, UNIVERSITY OF ILLINOIS, URBANA, ILLINOIS Communicated by H. S. Gutowsky, May 7, 1962 The presence of a sequence in DNA complementary to homologous ribosomal RNA is indicated by experiments described in a preceding paper.’ The data showed that a hybrid complex resistant to RNAase is formed when mixtures of ribosomal RNA and denatured DNA from the same cell are heated and then subjected to a slow cool.2. When DNA from a heterologous source is substituted, no such com- plex is observed. The specific pairing of ribosomal RNA with a complementary sequence in DNA leads to the following two predictions: (1) The ratio of RNA to DNA in the specific complex should approach a maximum value at levels indicat- ing the involvement of a minor fraction of the DNA; (2) Nonribosomal RNA should not compete for the same site. Experiments relevant to these two issues have been performed. It is the intent of the present paper to describe the results obtained and discuss their implications. The data are consistent with the existence of a specific sequence in DNA capable of complexing with homologous ribosomal RNA and not occupiable by nonribosomal RNA from the same organism. Materials and Methods.—Strain and preparation of materials: FE. coli strains BB and A-155, a uracilless derivative of B were used in the present study. The methods of growth, labeling, counting, CsCl density gradient centrifugation, and the prepa- ration of the following materials are detailed in the earlier papers ;'~* heat-denatured DNA, free of RNAase; P® and H*-labeled 235 RNA free of ‘Gnformational” RNA. The methods of assaying for RN Aase activity and sensitivity of RNA-DNA complexes to nuclease degradation are the same as used in the earlier! study. “Step-down’’ labeling of cells for informational RNA preparations: The procedure used is essentially that of Hayashi and Spiegelman.* Logarithmically growing E. coli A-155 cells in nutrient broth were collected by centrifugation, washed twice Vou. 48, 1962 BIOCHEMISTRY: YANKOFKSY AND SPIEGELMAN 1467 in SC minimal broth, and resuspended in the minimal medium to an O.D.¢60 of 0.360. The cells were aerated for 10 min at 37°C; then 1.48 ng/ml of H*-uridine was added (New England Nuclear Corporation, 3.28 c/mM) and aeration at 37°C continued for 10 min. Isotope incorporation was halted by pouring the cultures into 1/3 their volume of frozen, crushed minimal medium. The cells were collected by centrifugation, then concentrated 20-fold in TM buffer. The RNA was purified and fractionated in linear sucrose gradients by the methods described previously.» 3 The fractions corresponding to 8-128 were pooled, concentrated, and used in the experiments described. Experimental Results.—A. Saturation curve of the ribosomal RNA-DNA complex: The detection of a saturation plateau requires the performance of experiments in- volving the following steps: (1) Mixtures containing fixed amounts of heat-de- natured DNA and varying amounts of labeled ribosomal RNA are exposed to a slow cool from 55°C to 30°C; (2) The resulting products are then subjected to equi- librium density centrifugations in CsCl solutions to separate free RNA from that which is complexed to DNA. The sorts of hybrids observed are exemplified by the two cases in Figure 1. It will be seen that at the lower level of input, most of the RNA in the reaction mixture is complexed to the DNA. The proportion of the input fixed decreases as the concentration of RNA is increased. CsCt EQUILIBRIUM GRADIENTS 50 ay Poo spose roa 7 1000 (0. 3ly) 23S Coli RNA X Coli DNA an , 40b 4 500 Cpm/M! Oo 0 -0-0-0-9 0-6 F-o- of mbt a oO 10 - 20 Fie. 1.—CsCl equilibrium density gradient profiles. Buffer used was TMSV (0.03 M tris, pH 7.3; 0.001 M MgCl; 0.3 M NaCl 0.005 M EDTA. Both contained 71 vg per ml of heat-denatured EZ. coli DNA. Upper contained 0.31 yg and the lower 0.62 ug of P32. 238 RNA (6.7 & 104 cpm/ug) of BE. coli. Reaction mixtures slow (ca. 13 hr) eooled from 35°C to 33°C, Mixtures were then brought to a density of 1.73 with CsCl and a total volume of 3ml, They were centrifuged for 52 hr at 33,000 rpm and 25°C. 1468 BIOCHEMISTRY: YANKOFSKY AND SPIEGELMAN Proc. N. A. 5. PERCENT RNAase RESISTANT RNA T T T T wool IN DNA DENSITY REGION 4 x L ae Xe y O.3ly 0.62y 5 |.86y 7 4.96y Free RNA Control 4 on 1 1 1 1 a L 4 1 1 o 10 20 30 40 50 MINUTES Fig. 2.—RNAase resistance of RNA-DNA complexes. 109 ug/ml of heat-denatured E. colt DNA were slow-cooled in TMSV with the in- dicated concentrations of FE. coli 238-P?-RNA. (6.7 X 10* epm/yg). After separation of hybrids by centrifugation as in Figure 1, sensi- tivity to RNAase was examined as described previously.? 0.005 t t 1 t r 2 eS 23S E.Coli RNA X Coti DNA oO luo 004 L 4 a uw x Total RNA Z o L 4 D0 003 SY x a 2 wa 7 a“ a 0.002 F 4 4 z ! a“ a : x ~ ‘ “7 a L “ Zz ©00l F es ir x 4 RNAase Resisant RNA 1 1 1 1 t ° ol 02 03 04 05 INPUT RNA/DNA Fra. 3.—Saturation curves of total and RN Aase-resistant counts in DNA. region of CsCl equilibrium gradients. Details of incubations and centrifugation are as noted in Figures 1 and 2. to RNAase. From our previous experience! with ribosomal RNA, it might be expected that nonspecific binding involving incidental coincidences over small regions will inevitably complicate such experiments. Ad- ventitious complexing of this sort could become quantitatively serious at high levels of RNA input. For- tunately, as has been shown,! sen- sitivity to RNAase permits a ready distinction between nonspecific and specific complexes. This same de- vice was used in the present study. Fractions in the hybrid regions of the density gradients were pooled, dialyzed, and exposed to RNAase.! The results obtained for four levels of RNA are detailed in Figure 2. In each case, free P*?-labeled RNA was included as an internal control in the reaction mixture. The be- havior of only one control is de- picted since they were all identical. It will be noted that the free RNA is almost completely solubilized in 5 min, whereas the H-labeled RNA from the hybrid regions exhibit RN Aase-resistant residues of vary- ing percentages. The data of Figure 2 are in agreement with what would have been expected from in- creasing amounts of nonspecific com- plexing at higher concentration levels of RNA. The greater the in- put of RNA, the larger is the propor- tion of the counts in the hybrid re- gion which are found to be sensitive A composite summary of these and similar experiments are plotted in Figure 3. We have here a comparison of the total RNA found in the DNA density region with that which resists degradation by RNAase. The behavior of these two are strik- ingly different. The total RNA shows no signs of saturation, whereas the resistant residue clearly approaches a plateau, corresponding approximately to an RNA: DNA ratio of 0.0015. The existence of this maximal level is consistent with the view that the DNA contains a restricted region capable of specifically complexing with ribosomal RNA. Von, 48, 1962 BIOCHEMISTRY: YANKOFSKY AND SPIEGELMAN 1469 B. Competitive and noncompetitive interaction in the course of hybrid formation: Competition experiments can also be used to reveal the existence of a localized specific interaction between ribosomal RNA and its homologous DNA. In the present system this can be realized by using two identifying isotopic labels on dif- ferent RNA preparations. If the two RNA molecules are competing for the same site and the total concentration is at or near the saturation level, one label will displace the other as its proportion is increased in the reaction mixture. If they do not compete for the same site, fixation to the DNA of one will be essentially in- different to the presence of the other. To examine questions of this nature, the following types of experiments were performed with #. coli RNA and DNA. Mixtures containing fixed levels of P#- 238 RNA, heat-denatured DNA, and varying amounts of H*-23S RNA were incu- bated and then centrifuged in CsCl. The amounts of P#2 and H*-labeled RNA fixed in the DNA and resistant to enzyme were then determined. Jor purposes of com- parison, similar experiments were carried out with H*-informational RNA (8- 128) replacing the H*-238 RNA in the mixtures. The informational variety was chosen since it was known to complex well with DNA and thus provides a test for the specificity of the ribosomal combination with DNA. Figure 4 gives two examples of the hybrid regions observed in CsCl gradients. HYBRID REGION, H3- INF RNA, P32-235 RNA 0.0. 260 CPM/ml Fig. 4.-Hybrid regions in CsCl gradients of mixtures contain- ing ribosomal and informational RNA. Both incubation mix- tures contain 63 ug/ml of heat-denatured E. coli DNA and 2.66 ug/ml of E. coli P2238 RNA. (A) contained in addition 2.9 pg of H*-informational RNA and (B) 5.7 ug of H*-informational RNA per ml. Details of incubations and centrifugations are as in Figures 1 and 2. Both types of nucleic acids have hybridized with, however, an interesting and con- sistent difference. The H*labeled informational RNA appears to be symmetrically distributed among the denatured strands of the DNA. This is clearly not the case with the ribosomal complex which is distinctly displaced toward the heavy side of the mean density of denatured DNA. The likely significance of this will be briefly noted in the Discussion. Again, to avoid confusion with irrelevant complexes, the fractions in the hybrid regions were pooled and the RNAase-resistant residue of complexed radioactivity determined. Figure 5 summarizes the resulting data. There is clear evidence (Fig. 5A) of displacement of the P*-labeled ribosomal RNA as more H3-labeled RNA of the same kind is incorporated into the complex. Further, a saturation 1470 BIOCHEMISTRY: YANKOFSKY AND SPIEGELMAN Proc. N. A. 8. 0.5 T (A) P32-235 Ribosomal RNA (Constant conc.) O4F (B) 2 + H7-23S Ribosomal RNA +H% - Inf RNA oO ~ S 03F S ™~ a x O02 HF Ww qf 2 ae Ol aa \ Le ~~e. —— ee oe ee @e 1 i | i ! i I | \ | I I 2 3 4 5 6 | 2 3 4 5 6 H3-RNA ADDED (pg) s 5 06t- ~ O° 6 05+ 3B 23S Ribosomal! (P32) RNA Ww oo4a- +H7-inft RNA re 2 03h x 23S Ribosomal RNA a ef (P32 + HF) e —_@ oO e Of \ | ! ! 1 i | ! 1 | 2 3 4 5 6 7 8 9 TOTAL RNA INPUT ( jg} Fig. 5.—Tests for competitive interactions in mixtures of ribosomal and informational RNA molecules. (A) All mixtures contained 63 ug/ml of heat- denatured £. coli DNA. and 2.66 ug/ml of FE. colt P3?-238 ribosomal RNA. H3- 238 ribosomal RNA of E. cold varied as shown. (B) DNA and P3?-RNA same as (A) except that H*informational RNA was added in the amounts indicated. (C) The same preparations as(A) and (B). The plot here is total (P#? + H’) RN Aase stable counts per 100 ng of DNA as a function of total input of RNA. All incubations during slow cool and equilibrium centrifugations were carried out as described in Figure 1. plateau in the H*-RNA complexed is observed within the concentration range tested. On the other hand, when the H*-label is on informational RNA, its hybridization with the DNA has no effect on the ability of the DNA to combine with the P32 labeled ribosomal material (Fig. 5B). In addition, no evidence of saturation is observed with the informational variety within the concentration range tested. This difference in saturation behavior is more clearly illustrated in Figure 5C in which the total (P*? + H*) RNA fixed per 100 y of DNA is plotted against the total RNA in the mixture. When the ribosomal varieties alone are present, the sum of Vou. 48, 1962 BIOCHEMISTRY: YANKOFSKY AND SPIEGELMAN 147] the two labels fixed approaches a plateau—a phenomenon completely absent in the mixture of informational and ribosomal RNA. These experiments provide additional evidence supporting the contention that ribosomal RNA specifically complexes with a restricted region not occupiable by nonribosomal RNA from the same organism. Discussion.—The use of double labeling combined with equilibrium centrifuga- tions in density gradients and enzyme sensitivity permitted the accumulation of data which leads to the conclusion that DNA does contain a localized sequence com- plementary to ribosomal RNA. The nature of the data obtained in the present and previous investigation! may be summarized asfollows: (1) The RNA complexed in a hybridization test is ribosomal as demonstrated by the base composition of the hybridized material. (2) Complex formation stable to RNAase occurs only with homologous DNA. (3) Saturation of homologous DNA with ribosomal RNA in a complex stable to RNAase occurs when approximately 0.2 per cent of the DNA is occupied. (4) Competition for a specific site was established by using two identical ribosomal RNA preparations distinguishable by isotopic labeling. Under the same conditions, nonribosomal RNA from the same organism does not compete. It will not escape the attention of the reader that certain problems of obvious in- terest have not received explicit mention. We may briefly cite a few. First, all the experiments described have employed only the 288 ribosomal component. This raises the obvious question of sequence similarity with the 16S variety. Further, no details are reported on heterologous tests among the bacteria, a point of obvious interest in view of the similarity of the ribosomal RNA base composi- tions. These, and related problems have been the subject of continuing investiga- tions by the methods developed in the present investigation. The results will be reported in separate communications. We may perhaps briefly allude to one point centering around the significance of the fact that ribosomal RNA saturates at a level corresponding to approximately 0.2 per cent of the DNA in £. colt. In view of the complicated operations required to obtain this number, its exact value must not be taken too seriously, and certainly not until similar determinations are reported with other organisms. However, values similar to this have been repeatedly obtained in independent experiments, and it would seem that it is certainly correct as to order of magnitude. This, however, is about 10 times greater than one would calculate from the DNA content per nu- cleus‘ and the assumption that there is only one complementary sequence per ge- nome. We conclude, therefore, that there must be repeating units of this kind. They could be similar in sequence, or identical, depending on whether the popula- tion of ribosomal RNA molecules are identical or different. There is one highly suggestive observation which leads us to conclude that, whichever is the case, these repeating units are not scattered throughout the genome, but contiguous. A persistent pecularitiy observed with homologous ribosomal RNA-DNA hybrids (Fig. 1 and P*? curves of Figs. 4A and 4B) is the pronounced displacement of the complex towards the heavy side of the mean density of the heat-denatured DNA. This displacement is not observed with the heterologous complexes.!| Further- more, it is not seen with informational RNA-DNA hybrids at this level of RNA in- put (H*-curves of Figs. 44 and 4B). The simplest interpretation for this shift is to assume that the repeating units complementary to ribosomal RNA are contiguous 1472 BIOCHEMISTRY: YANKOFSKY AND SPIEGELMAN Proc. N. ALS. and that any DNA strand which has one such sequence is likely to have another. Consequently, this selected set of strands complex with several ribosomal RNA molecules, resulting in the observed increase in density. Summary.—The experiments reported offer further evidence for the presence in DNA of a sequence complementary to ribosomal RNA. Saturation experiments suggest that the particular region involved corresponds to between 0.1 and 0.2 per cent of the total genome. Competition for a restricted DNA region can be exhibited by the use of variously labeled homologous ribosomal RNA. Nonribo- somal RNA from the same organism does not compete for the same site. The amount of ribosomal RNA complexed per unit of DNA at saturation suggests a number of repeating, similar, or identical sequences. Further, the density shift of the hybrids suggests that these units are not scattered but contiguous in the DNA structire. * This investigation was aided by grants in aid from the U.S. Public Health Service, National Science Foundation, and the Office of Naval Research. { Predoctoral fellow trainee in Molecular Genetics (USPH 2G-319). 1 Yankofsky, 8., and 8. Spiegelman, these PRocEEDINGS, 48, 1069 (1962). 2 Hall, B. D., and 8. Spiegelman, these Proceepines, 47, 137 (1961). 3 Hayashi, M., and S. Spiegelman, these Procrepinas, 47, 1564 (1961). 4 Barner, H., and 8. 8. Cohen, J. Bactertel., 72, 115 (1956).