THE JOURNAL OF BIOLOGICAL CHEMLWRY Vol. 254, No. 16. kue of August 25, pp. 7724-7729, 1979 Prmted in U.S.A.

The Ribosomal Protein Binding Site in Saccharomyces cerevisiae Ribosoma15 S RNA A CONSERVED PROTEIN BINDING SITE IN 5 S RNA*

(Received for publication, October 27, 1978, and in revised form, February 8, 1979)

Ross N. Nazar

From the Department of Botany and Genetics, University of Guelph, Guelph, Canada NIG 2Wl

The primary protein binding site of Saccharomyces cerevisiae 5 S RNA was studied by limited ribonuclease digestion of the 5 S RNAoprotein complex and free 5 S RNA. The results indicate that while the digestion prod- ucts were largely equivalent in each case, a portion of the 5 S RNA molecule after limited pancreatic ribonu- clease digestion (F,) remained bound to the ribosomal protein in significant amounts even when the digestion products were separated by gel electrophoresis. Se- quence analyses showed that this RNA was composed of two fragments, a small portion of the 5’-terminal (residues 1 to 12) base-paired with a larger fragment of the 3’-end (residues 79 to 121). A fragment (Fz) contain- ing 5 nucleotides less (residues 1 to 12 and 84 to 121) was also present as a ribonucleoprotein complex in small amounts, but further trimming appeared not to be tolerated. When compared to earlier studies on pro- karyotic 5 S RNA-protein complexes from Escherichia coli (Gray, P. N., Bellemare, G., Monier, R., Garrett, R. A., and Stoffler, G. (1973) J. Mol. Biol. 77,133-152) and Halobacterium cutirubrum (Nazar, R. N., Willick, G. E., Matheson, A. T. (1979) J Biol. Chem. 254, 1506-1512) the results indicate that the primary protein binding site has been conserved in the course of evolution and suggest that all 5 S RNAs share a common binding site. Studies on the reconstitution of fragment F1 and whole 5 S RNA into ribonucleoprotein complexes indicated that the fragment was not able to compete with 5 S RNA for the binding protein suggesting that other re- gions of the 5 S RNA molecule are also required for the initial RNA l protein interaction.

Ribonucleoprotein complexes containing 5 S RNA have been isolated from both eukaryotic (1,Z) and prokaryotic (3- 5) ribosomes. In bacteria, Escherichia coli for example, the complex contains at least two proteins (3-5); depending on the organism or method of preparation a third protein has also been observed (6). In eukaryotes, yeast for example, only one larger protein appears to be associated with the 5 S RNA (1, 2). The primary nucleotide sequences of many ribosomal 5 S RNA’s from both eukaryotic and prokaryotic organisms have been determined (see Ref. 7) and numerous attempts have been made to deduce a secondary structure. Assuming a common role for all 5 S RNA’s in ribosomal structure and function, a number of workers have anticipated common sites

* Part of this work was carried out at the National Research Council of Canada, Ottawa, as study No. 17506. The costs of publi- cation of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertiae- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

in the secondary structure and two of the models have been generalized (8, 9) to include both eukaryotic and prokaryotic species. In contrast, until recently only the structure of the E. coli 5 S RNA-protein complex was extensively examined (3, 10, 11) and presently the studies have only been extended to two other prokaryotes, an extreme halophile, Halobacterium cutirubrum (5, 12) and a moderate thermophile Bacillus stearothermophilus (13).

Both physical and chemical studies suggest that despite significant differences in their primary sequences (14) and in their ionic environments (5), the bacterial complexes share fundamental features in the protein + RNA interaction (12). In each case, circular dichroism measurements indicate that the RNA molecule undergoes conformational changes when in- teracting with binding proteins; as a result, specific nucleotide sites became more susceptible to pancreatic ribonuclease digestion. Furthermore, although there is some disagreement among the studies with respect to the total protein binding site (3, 4, 12) similar regions of the 3’-terminal sequence in each RNA molecule are preferentially resistant to nuclease digestion when complexed with proteins, and similar amounts of ethidium bromide are chased when ethidium bromide- treated 5 S RNA interacts with equivalent proteins (10, 12) EL18 and HL13 from E. coli and H. cutirubrum, respectively. To further evaluate the general features of this RNA. protein interaction, and to attempt to resolve the differences which still remain (3, 4, 12) between models for the prokaryotic complexes, in this study the primary protein binding site in yeast 5 S RNA was examined using limited ribonuclease digestion.

EXPERIMENTAL PROCEDURES

Saccharomyces cerevisiae, strain S266C, grown aerobically in 0.3% Bacto-yeast extract, 0.5% Bacto-peptone, and 2% glucose at 28°C were harvested in late log phase. Nonradioactive 5 S RNA and the 5 S RNAeprotein complex were isolated from 60 S ribosomal subunits prepared essentially by the method of Torano et al. (15). The 5 S RNA. protein complex was released by suspending the subunits in 25 mM EDTA, pH 7.0 (1); much of the r&osomal core was removed by centrifugation at 226,609 x g (Beckman Ti50 rotor at 50,000 rpm) for 18 h and the RNP was further purified by centrifrrgation on a 5 to 20% linear sucrose gradient containing 10 mM Tris-HCl, pH 7.5, for 4 h in a Beckman B-4 zonal rotor at 37,000 rpm. The purified RNP fraction was used directly or dialyzed against an appropriate buffer to remove the sucrose. Unlabeled 5 S RNA was prepared from RNP by applying the fraction (100 to 200 OD 2~) units) to a column of DEAE- cellulose (1.5 X 2.0 cm) (Whatman DE 52) and washing awav the protein with 20 ml of 0.3 M KCl, 6 M urea, i0 mM Tris-HE1 (pR 7.5). The RNA was eluted with 30 ml of 1.0 M KCl. 6 M urea. 10 mM Tris- HCl, pH 7.5, and precipitated with 2 volumes of ethanol.

For 32P-labeled 5 S RNA and complex, cells were labeled in low phosphate medium with 2 to 5 mCi of carrier free [32P]orthophosphate as previously described (16). The 5 S RNA was prepared directly from whole cells by suspending the cell pellet in 35 ml of 0.3% (w/v)

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Protein Binding Site in Yeast 5 S RNA 7725

sodium dodecyl sulfate containing 0.14 M NaCl, 0.05 M sodium acetate, and 0.001 M EDTA (pH 5.1) and extracted with 35 ml of phenol solution at 65°C (17). The RNA was fractionated by polyacrylamide gel electrophoresis on an 8% slab at pH 8.3 and the 5 S rRNA was recovered by homogenizing the gel (18). In some experiments labeled 5 S RNP was released from 60 S subunits with EDTA as described above but the complex was puritied by applying the treated sample directly to an 8% polyacrylamide gel slab as was used for 32P-labeled 5 S RNA. In most experiments the 5 S RNAaprotein complex was labeled in vitro by incubating “P-labeled 5 S RNA of high specific activity with unlabeled complex for 30 min at 0°C or room tempera- ture. The labeled RNA was efficiently incorporated into the complex by equilibrium exchange.

Nucleotide sequences involved in protein binding were probed by limited ribonuclease digestion. 32P-labeled or unlabeled purified 5 S RNA or 5 S RNA. protein complex (25 to 75 pg of RNA) was incubated at 0°C in 100 to 200 /.d of 25 mM EDTA, pH 7.0, for 10 to 20 min with 0.01 to 0.05 ag of pancreatic or T1 ribonuclease. In some experiments, the reaction was stopped with 1 ml of sodium dodecyl sulfate buffer, the RNA fragments were extracted with phenol solution at room temperature as described above and fractionated by electrophoresis on a 12% polyacrylamide slab gel at pH 8.3 as previously described, in other experiments the digests were applied directly to an 8% poly- acrylamide slab gel (18). For nucleotide sequence analyses ‘*P-labeled fragments were eluted from the gels by homogenization in water (18) and completely digested with pancreatic or TI ribonuclease; the digests were separated by one-dimensional electrophoresis on DEAE- paper in 7% formic acid and the nucleotide sequence was determined by comparing the oligonucleotides which were obtained with the known nucleotide sequence of yeast 5 S RNA (14). When necessary the oligonucleotides were further characterized by their nucleotide compositions and products of complete pancreatic or T1 ribonuclease digestion.

RESULTS

As previously reported with rat liver ribosomes (l), a 5 S RNA. protein complex could be dissociated from yeast ribo- somes or 60 S subunits with 25 mu EDTA (Fig. 1). Formamide has also been used to release this complex from yeast ribo- somes (2) but in our studies the EDTA treatment was found to be more efficient with less dissociation of complex to free 5 S RNA. Further analysis indicated that, as has been reported earlier, this RNP complex, whether purified by gel electro- phoresis or density gradient centrifugation, contained only two components, ribosoma15 S RNA and a ribosomal protein. The ribosomal protein migrated as a single component after electrophoresis on a 10% polyacrylamide sodium dodecyl sul- fate gel (19) or after two-dimensional polyacrylamide gel analysis as described by Kaltschmidt and Wittmann (20). Routinely, about 6 to 8 mg of protein were recovered from 200 Azwn,,, units of complex consistent with a 1:1 binding ratio. Traces of free 5 S RNA were always present in the ribonucle- oprotein preparations (Fig. 1); dissociation to free RNA was minimized by maintaining a relatively high RNP concentra- tion (Xi A260 Dm units/ml).

The protein binding site in yeast 5 S RNA was probed by limited ribonuclease digestion. Two different approaches were used to analyze the digestion products. First, as was used in studies on the H. cutirubrum complex (12), the RNA frag- ments from free and bound 5 S RNA were extracted with sodium dodecyl sulfate-phenol’ and compared after fraction- ation by electrophoresis on 12% polyacrylamide slab gels. Fig. 2 shows the results after digestion with either pancreatic or T, ribonuclease. Surprisingly, -unlike studies on prokaryotic complexes (3, 4, 12) little difference was observed between free and bound RNA. There were no unique bands although there were some quantitative differences with pancreatic ri-

’ This extraction effectively stopped all nuclease digestion; no residual nuclease activity was detected when undigested RNA was added with sodium dodecyl sulfate solution to a typical digestion

mixture and extracted with phenol.

OS Or&

I P i

FIG. 1. Purification of Saccharomyces cerevisiae 5 S RNA* protein complex by preparative gel electrophoresis (left) or density gradient centrifugation (tight). 32P-labeled 60 S subunits were suspended in 25 mM EDTA, pH 7.0, and applied directly to an 8% polyacrylamide slab gel for electrophoresis at pH 8.3; a portion of the autoradiograph is shown here. Unlabeled ribosomes were sus- pended in 25 mM EDTA, centrifuged for 18 h at 226,000 x g, and the supernatant was fractionated on a 5 to 20% linear sucrose gradient; lo-ml fractions were collected; the shaded area indicates the fractions which were pooled as the 5 S RNA. protein complex.

Pancreatic RNase T1 RNase

RNA RNP RNA RNP

Origin

Dye

FIG. 2. Fractionation of limited pancreatic or Ti ribonucle- ase digests of free 5 S RNA or the 5 S RNA*protein complex from Saccharomyces cerevisiae after extraction with a sodium dodecyl sulfate-phenol mixture. The 1.5 A260 “,,, unit samples were digested in 200 ~1 of 25 mM EDTA, pH 7.0, with 0.05 pg of enzyme for 20 min at O”C, extracted with sodium dodecyl sulfate-phenol, and the RNA fragments were fractionated on a 12% polyacrylamide slab gel at pH 8.3 with 35 mA of current for 3.5 h. The gel was stained with methylene blue; the position of the origin and bromophenol blue dye marker is identified.

bonuclease. The results indicate that the presence of protein does not result in any significant preferential resistance or susceptibility to nuclease digestion. Similar conclusions were reached under more severe and milder digestion conditions. Apparently the protein binding site is relatively resistant to nuclease digestion even in free RNA. Because the eukaryotic

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7726 Protein Binding Site in Yeast 5 S RNA

RNP

RNA

Origin

be

Origin

be 5’

FIG. 3. Fractionation of a limited pancreatic ribonuclease digest of “P-labeled 5 S RNA-protein complex from Sac&a- romyces cerevisiae. A 0.6 A260 “,,, unit of complex was digested in 100 ~1 of 25 mu EDTA, pH 7.0, with 0.01 pg of enzyme for 20 min at O’C, and the fragments were fractionated directly on an 8% polyacrylamide slab gel at pH 8.3 with 35 mA of current for 4 h. The autoradiograph (left) compares this digest with an undigested sample; the position of undigested 5 S RNA, 5 S RNA. protein complex, and the bromophenol blue marker dye is indicated. After elution, fragment F1 was denatured with 7 M urea and further fractionated on a 12% polyacrylamide slab gel at pH 8.3 (right).

5 S RNA. protein complex is relatively stable under low salt conditions, the products of nuclease digestion could also be separated directly as ribonucleoprotein complexes by poly- acrylamide gel electrophoresis. As shown in Fig. 3, when 0.6 AzM),,,,, unit of RNP was digested with 0.01 pg of pancreatic ribonuclease for 20 min at 0°C and applied directly to a cold slab gel, two bands, F1 and Fz, were observed to migrate slower than intact 5 S RNA but considerably faster than the native 5 S RNA-protein complex. Such a mobility could only be consistent with partially digested ribonucleoprotein com- plexes. The remaining bands migrated considerably faster and corresponded to fragments in free 5 S RNA (results not shown). With T1 ribonuclease two similar bands were also observed but in much lower yield and with slightly faster mobilities.

To elucidate the regions of the yeast 5 S RNA molecule which were nuclease-resistant and remained in complex with the 5 S RNA binding protein, the nucleotide sequences of 32P- labeled bands F, and F2 were determined from oligonucleo- tides of complete pancreatic or Tr ribonuclease digestion and the known sequence of yeast 5 S RNA (21). Fig. 4 shows the results of complete pancreatic and T1 ribonuclease digestion after the products were separated by electrophoresis on DEAE-paper in 7% formic acid. Table I summarizes these results and indicates that the two were closely related, F1 contained the sequence of F2 but was extended by an addi- tional five nucleotides. The results further indicated that each band actually contained two fragments from the 3’ and 5 regions of the 5 S RNA molecule which remained base-paired under the electrophoretic conditions that were used. This is consistent with current models of 5 S RNA (8, 9) in which termini are base-paired and was subsequently confirmed by treating band Fr with 7 M urea and rerunning it on a 12% polyacrylamide gel (Fig. 3).

‘; UP

CP 1

ACP

GCP

AUP

GACp

A2Up e

GA3Cp

GUP

C2AUp

C2A2U2

UGP

%Cb

/ AGUp ’

CUGp

UAGp

AGzUP

C3A$JGp + U2Gp

CZA~UGP

G3Up

(PIPPG~UP

(PIPPGP

F1 55 F2 F1 55 F2

Pant RNase 1, RNase

FIG. 4. kactionation of complete pancreatic and T1 ribonu- clease digests of “P-labeled fragments of the 5 S RNA*protein complex from Saccharomyces cerevisioe. Electrophoresis was from bottom to top on DEAE-paper in 7% formic acid. A ‘*P-labeled 5 S RNA control is included for each enzyme and the position of each oligonucleotide product is identified.

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Protein Binding Site in Yeast 5 S RNA

TABLE I

7727

Analysis of fragments obtained by partial digestion with pancreatic RNase After the partial digests were fractionated by gel electrophoresis

(e.g. Fig. 3) bands which migrated slower than free 5 S RNA were underline, one underline, two underlines, and three underlines for

further analyzed by complete digestion with pancreatic or T, ribo- molar yields of 1, 2, 3, and 4, respectively. Molar yields were deter-

nuclease; the products were subsequently separated by one-dimen- mined from the radioactivity and sequences of the digestion products.

sional electrophoresis on DEAE-paper in 7% formic acid (e.g. Fig. 4). The overall sequences were deduced by arranging the end products

The notation for the relative molar yields of the products is no of complete digestion along the known nucleotide sequence of yeast 5 S RNA (21).

Pancreatic RNase Band Digestion Products

T, RNase Digestion Products Sequence

Fl (P)PPG~UP,GJJP~,AGLJP,W,G,C~,GUP, (P)PPGPC,C~A~UGPC,U,GP,C,A,UGPC,

GA,CP,A,UP,GACP,A~~&&P,ACP,CJ, = UAGp,CUGp,U&C,A,U:,C,AUpb,

(p)ppG-G-U-U-G-C-G-G-C-C-A-UP t

A-G-U-G-U-A-G-U-G-G-G-U-G-A-C-

2 AGpQp,GpF) C-A-U-A-C-G-C-G-A-A-A-C-U-C-A-

G-G-U-G-C-U-G-C-A-A-U-C-U

F2 (P)PPG~UP,G,UP~,AG,UP,AGUP,G~C~, (P)PPG&CZA,UGP%~GPC,C,A,UGPC, (p)ppG-G-U-U-G-c-G-G-C-C-A-Up +

GA~CP,A~UP,GAC~,AU~,GCP,AC~,~, = = CUGp,lJ&C,A2UC,,C,AUpb,AGp, A-G-U-G-G-G-U-G-A-C-C-A-U-A-C-

9 W"GP(~) G-C-G-A-A-A-C-U-C-A-G-G-U-G-C-

U-G-C-A-A-U-C-U

a The molar yield of this product was low because a portion remained at the origin (e.g. Fig. 4). * The products of complete pancreatic ribonuclease digestion were AUp and Cp consistent with the sequence C-C-A-Up. ’ The presence of these oligonucleotides was further confirmed by subsequent analysis after pancreatic RNase digestion.

RNP

RNA

Dye

,

FIG. 5. Reassociation of 5 S RNA or nancreatic ribonuclease digestion fragment Fr into RNA*prote& complexes. “P-labeled 5 S RNA (left) or pancreatic ribonuclease digestion fragment F, was incubated with 0.6 A2mnm unit of unlabeled complex in 100 ~1 of 25 rnbr EDTA for 30 min at room temperature and fractionated directly on an 8% polyacrylamide slab gel at pH 8.3. The position of free 5 S RNA, the 5 S RNA-protein complex, and the bromophenol blue marker dye is indicated beside the autoradiograph.

While the present data clearly indicate that at least the primary protein binding site of yeast 5 S RNA is composed of portions of 5’- and 3’-terminal sequences the exact nucleotides or structures which are involved remain unclear. Since the ribonuclease-resistant fragments remained stable in a protein. RNA complex during electrophoresis (Fig. 3), an attempt was made to reconstitute the RNA fragments from band F1 into a ribonucleoprotein complex with the binding protein and to evaluate its ability to compete with native 5 S RNA. As shown in the example in Fig. 5, no competitive reassociation occurred under conditions which permit whole “‘P-labeled 5 S RNA to easily exchange into unlabeled 5 S RNA-protein complex.’ No bands corresponding to the F,-containing ribonucleopro- tein complex were observed; the bands corresponded to the F1 RNA duplex, its 5’- and 3’-end components, and some degra- dation products These results indicate the other regions of the 5 S RNA molecule are required, probably to maintain a unique secondary structure in the nuclease resistant sequence in a form suitable for interaction.

DISCUSSION

Limited pancreatic or T, ribonuclease digests of the yeast 5 S RNA-protein complex and free 5 S RNA indicated that while the digestion products were largely comparable (Fig. 2) a portion of the 5 S RNA molecule (Fl) could remain bound to the ribosomal protein in significant amounts even when fractionated by gel electrophoresis (Fig. 3). Sequence analyses showed that this RNA contained two fragments, a small portion of the 5’terminal (residues 1 to 12) base-paired with a larger portion of the 3’-end (Fig. 6). Because this portion of the 5 S RNA molecule remained bound to the protein com- ponent at least part of it makes up the primary protein binding site. Some additional trimming of residues could be tolerated (F,) but still shorter fragments were not observed in RNP complexes, suggesting that FP was the minimal sequence for

* This reconstitution of 32P-labeled yeast 5 S RNA into a 5 S RNA. protein complex was found to be very RNA specific; when the experiment was repeated with three different ‘*P-labeled noncognate RNAs (yeast 5.8 S RNA, yeast tRNA, or H. cutirubrum 5 S RNA) no ribonucleoprotein complexes were observed. Essentially identical re- sults were obtained by direct reconstitution with purified protein but a great deal of aggregation was also observed owing to the very limited solubility in the purified protein (27).

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7728

Yeast

Protein Binding Site in Yeast 5 S RNA

40 @*UC C ++$U$pP*G U UUGCCA CGAAA GCCCU 3o

E.coli

FIG. 6. A diagrammatic compari- son of the primary protein binding region in Saccharomyces cerevisiae and Escherichia coli 5 S RNA. The secondary structure is estimated accord- ing to Nishikawa and Takemura (9). Data for the E. coli protein binding site is taken from Gray et al. (22). The min- imal RNA sequence which associated with ribosomal protein is shown by Zight shading; the complete region is indi- cated with further bark shading.

protein binding or at least to maintain essential features in the binding site. The inability of F1 to reform a RNA + protein complex or to compete with 5 S RNA for the binding protein, however, indicated that other portions of the 5 S RNA se- quence were required for the initial interaction or at least to maintain the F, sequence in a configuration suitable for pro- tein binding. At present it is not possible to distinguish be- tween these two alternatives.

Because 5 S RNAs are integral parts of ribosomes and appear to share common features in their secondary struc- tures, it is attractive to postulate that they also share common features in their interactions with ribosomal proteins. Earlier studies on protein binding sites in E. coli 5 S RNA (22) and recent studies on RNA. protein interactions in the H. cutiru- brum 5 S RNA *protein complex (5, 12) both suggest that comparable sequences in the 3’-end of the RNA molecule make up the primary binding site. When the protected se- quences in the E. coli 5 S RNA. protein complex are compared to the F, band from yeast RNP (Fig. 6), the conclusions are strikingly similar. In both studies, however, it is not clear whether the 5’-terminal fragment is essential for protein bind- ing or simply present because the high degree of base-pairing made this structure very resistant to nuclease digestion. Fur- thermore, in their studies on the E. coli 5 S RNA-protein complex (13,23), Zimmermann and Erdmann did not find any protection of the 5’-end and, the 5’-end, which is apparently not as extensively paired in H. cutirubrum 5 S RNA (12), is not included in the halophihc protein binding site. Therefore, all the studies suggest that at least part of the primary protein binding site resides in a specific comparable region of the 3’- terminal sequence of 5 S RNA; there appears to be a universal binding region in 5 S RNAs.

In search of structural features which may better define this primary binding site, the equivalent region was examined in all known 5 S RNA sequences; two noteworthy features were observed. As pointed out by Nishikawa and Takemura (8) and by Hori (24) in addition to the 5’-, 3’-terminal pairing, a hairpin loop structure can always be formed from residues 78 through 99, a loop which in prokaryotes is sometimes referred

to as the prokaryotic loop (9). Furthermore, as recently pointed out by Nichols and Wijesinghe (25) in wheat embryo and rye 5 S RNA, some 5 S RNAs contain two symmetrical sequences in this region. Palindromes have been noted previ- ously in DNA sequences which interact with specific proteins (see Ref. 26). Whether either of these features is significant in protein binding must await further experimentation; never- theless, it may be useful to direct special attention to them.

The models in Fig. 6 differ somewhat with studies by Zimmermann and Erdmann on reconstituted 5 S RNA.pro- tein complexes from E. coli and B. stearothermophilus (13, 23). Both studies with the E. coli 5 S RNA. protein complex (3, 23) agree that EL18 and EL25 bind to residues 69 to 116, but differ significantly with respect to the third protein, EL5. Gray et al. (22) did not observe a resistance to nuclease digestion in residues 18 to 57 with EL5 as reported by Zim- mermann and Erdmann (23). Similarly, this region of the molecule has been omitted in other models including yeast. Experimentally, several differences exist in the various stud- ies; a filter assay was used for RNP instead of gel electropho- resis, T1 ribonuclease was used for limited digests instead of pancreatic ribonuclease, and one study included 23 S rRNA while the other did not.

Studies on other 5 S RNA.protein complexes may offer some explanation for these differences. As was pointed out with the H. cutirubrum 5 S RNA. protein complex (12) almost the entire RNA molecule was largely resistant to T1 digestion when it was bound to protein as reported by Zimmermann and Erdmann (23) for the E. coli complex. In contrast, a significant portion was digested away with pancreatic ribo- nuclease as reported by Gray et al. (22) for the E. coli complex. That study concluded that a greater region of the sequence is indirectly stabilized by protein binding but that the primary binding site is largely restricted to the smaller pancreatic ribonuclease-resistant fragments. The present study on the yeast complex tends to support this view. The fact that F, and Fz remained complexed with protein during electropho- resis is the strongest evidence that this nucleotide sequence makes up the primary protein binding site. However, since F1

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Protein Binding Site in Yeast 5 S RNA 7729

and Fz cannot compete with 5 S RNA for the binding protein, it is also clear that other portions of the 5 S RNA molecule play a structural role. This is consistent with the general resistance to T1 ribonuclease in H. cutirubrum and perhaps also explains some of the differences observed by Zimmer- mann and Erdmann (13,23). All the studies, therefore, suggest that the primary protein binding site in 5 S RNAs resides in the 3’-terminal region of the molecule, but there is also signif- icant evidence that other sequences may be involved at least indirectly.

The fact that there appears to be a conserved primary protein binding site in 5 S RNA despite significant differences in the number of protein components raises an important question about the co-evolution of the 5 S RNA binding proteins. The total molecular weight of the protein compo- nents is approximately equal in each complex and preliminary comparisons of their primary amino acid sequences suggest that the yeast protein may result from a fusion of the prokar- yotic protein sequences (27). A conserved primary protein binding site in yeast with only one larger protein component further supports this hypothesis.

Aclznowledgment-I wish to acknowledge the excellent technical assistance of C. F. RoIIin.

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R N NazarRNA. A conserved protein binding site in 5 S RNA.

The ribosomal protein binding site in Saccharomyces cerevisiae ribosomal 5 S

1979, 254:7724-7729.J. Biol. Chem. 

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