Ribonuclease P: an enzyme with an essential RNA component

Proc. Natl. Acad. Sci. USA Vol. 75, No. 8, pp. 3717-3721, August 1978

Biochemistry

Ribonuclease P: An enzyme with an essential RNA component (endoribonuclease/precursor tRNA substrates/RNA subunit)

BENJAMIN C. STARK*, RYSZARD KOLEt, EMMA J. BOWMAN*, AND SIDNEY ALTMAN§ Department of Biology, Yale University, New Haven, Connecticut 06520

Communicated by Joseph G. Gall, June 8,1978

ABSTRACT The activity of ribonuclease P on precursor tRNA substrates from Escherichia coli can be abolished by pretreatment of this enzyme with micrococcal nuclease or pancreatic ribonuclease A, as well as by proteases and by thermal denaturation. Highly purified RNase P exhibits one prominent RNA and one prominent polypeptide com nent when

examined in polyacrylamide gels containing sodum dodecyl sulfate. The buoyant density in CsCl of RNase P, 1.71 g/ml, is characteristic of a protein-RNA complex. The activity of RNase P is inhibited by various RNA molecules. The presence of a discrete RNA component in RNase P appears to be essential for enzymatic function. A model is described for enzyme-substrate recognition in which this RNA component plays an important role. Ribonuclease P (1-3) is necessary for the biosynthesis of the 5' termini of tRNA molecules in Escherichia coli. This enzyme recognizes some aspect of the structural conformation rather than nucleotide sequence at is cleavage sites in tRNA precursor molecules (4-6). Exactly how this recognition occurs is a matter for speculation (7). In this report we show that treatment of highly purified RNase P with either micrococcal nuclease (MN) or pancreatic ribonuclease A abolishes RNase P activity. Our most highly purified RNase P preparations contain one discrete RNA species and one discrete protein species, and several kinds of RNA can inhibit the enzymatic activity. We conclude that the interaction of RNase P with its substrates depends on the presence of RNA in the enzyme complex. METHODS Preparation of RNase P. What follows is an abbreviated description of RNase P purification schemes. Complete details will be published elsewhere. Crude extracts (S30) of E. coil MRE 600 were prepared by grinding of frozen cells with alumina as described (2). Two purification schemes were used, starting with S30. Scheme I. S30 made from 200 g of cells was diluted 1:5 with buffer A [50 mM Tris-HCl, pH 7.5/60 mM NH4Cl/10 mM Mg(OAc)2/6 mM 2-mercaptoethanol] and loaded onto a DEAE-Sephadex A-50 column (12 X 25 cm) equilibrated with buffer A. Bed volume of the column was about 2.5 liters. The column was washed successively with 3 liters of buffer A containing 0.2 M NH4Cl, 0.3 M NH4Cl, and 0.4 M NH4Cl in successive washes. The final wash was with buffer A containing 0.5 M NH4Cl (now called buffer B) and the activity was eluted after about 1 liter of the last wash buffer had flowed through the column. The pooled active fractions wer- precipitated with 0.6 g of (NH4)2SO4 per ml; the precipitate was resuspended and dialyzed against buffer B to give a final volume of about 15 ml. This material was applied to a Sepharose 4B column (2.5 X 90 cm) equilibrated with buffer B and eluted with 300 ml of the The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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same buffer. The RNase P eluted after the rRNA peak and before the 4S RNA peak. The active fractions were pooled and concentrated as described above and then applied to a Sephadex G-200 column (1 X 90 cm) equilibrated and eluted in the same manner as the Sepharose 4B column. The RNase P eluted just after the void volume. This material was again pooled, concentrated, redissolved in 2 M (NH4)2SO4 in buffer B, applied to an n-octyl-Sepharose (a gift of H. Taira, Yale University, New Haven, CT) column (0.5 X 2 cm) that had been equilibrated with 2 M (NH4)2SO4 in buffer B, and then eluted with a linear gradient from 2 M (NH4)2SO4 in buffer B to buffer B. The active fractions eluted at about 0.5 M (NH4)2SO4. Scheme II. S30 made with 600 g of E. coli MRE 600 was further processed to yield a 0.2 M NH4Cl wash of ribosomes. The active RNase P in the salt wash was loaded onto a DEAESephadex column and purified as described (2). RNase P (1.8 mg), purified through the DEAE-Sephadex step, was concentrated by precipitation with (NH4)2SO4, resuspended in 0.5 ml of buffer G [20 mM Tris-HCl, pH 7.6/0.50 M NH4CI/15 mM Mg(OAc)2/6 mM 2-mercaptoethanol/5% sucrose], dialyzed against this buffer, and applied to a column of Sephadex G-200 (0.9 X 55 cm) equilibrated with buffer G. RNase P activity was eluted from this column with buffer G. One-third of the peak fractions (0.4 mg of protein in 4 ml) was pooled and concentrated by dialysis against buffer G containing 15% polyethylene glycol until the final volume was 0.3 ml. This sample was loaded in aliquots onto four 7% polyacrylamide gel columns (6 X 90 mm). The running buffer was 25 mM Tris-HCl/25 mM glycine at pH 9; the gel buffer was the same but at pH 7. The gels were run at 4 mA per column for 80 min at 40, at which time the bromphenol blue marker had run about 70 mm. Ten slices, spaced equidistant apart, were made from the top of the gel to the dye front and eluted, after mincing, with buffer G at 00. Fractions were assayed for activity as described (2, 5). The final NH4Cl concentration in the reaction mixtures was 10 mM. Assay of RNase P Activity. Radioactive substrates for RNase P were prepared from E. coli A49 (RNase P ts) as described (2, 5). These were used in assay mixtures under standard conditions (2, 5) unless otherwise noted. The extent of RNase P reaction was quantitated by cutting appropriate slices out of polyacrylamide gels and assaying the amount of RNA in the slices by monitoring Cerenkov radiation. Abbreviations: NaDodSO4, sodium dodecyl sulfate; MN, micrococcal nuclease; buffer A, 50 mM Tris-HCl, pH 7.5/60 mM NH4Cl/10 mM Mg(OAc)2/6 mM 2-mercaptoethanol; buffer B, 50 mM Tris-HCl, pH 7.5/0.5 M NH4Cl/10 mm Mg(OAc)2/6 mM 2-mercaptoethanol; buffer C, 20 mM Tris-HCl, pH 7.6/0.50 mM NH4Cl/15 mM Mg(OAc)2/6 mM 2-mercaptoethanol/5% sucrose. * Present address: Department of Botany, Washington State University, Pullman, WA 99163. t Permanent address: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland. * Present address: Department of Human Genetics, Yale University, School of Medicine, New Haven, CT 06510. § To whom reprint requests should be addressed.

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Stark et al.

Proc. Natl. Acad. Sci. USA 75 (1978)

Ribonuclease Inactivation of RNase P. One hundred microliters of RNase P (11 tig of protein purified through the DEAE-Sephadex step of scheme II was used in the experiments shown in Fig. 2A; 4 ug of protein purified through the nondenaturing gel step of scheme II was used in the experiments shown in Fig. 2B) in buffer G was mixed with 50 Ml of 10 mM CaCl2 and 6 ,ul of MN (1 mg/ml) in 1 mM NaCi. (MN from Sigma Biochemicals, Worthington Biochemicals, Schwarz/ Mann, and an authentic sample received as a kind gift from M. Laskowski, Sr., were all equally effective. Whether or not the stock solutions were made up with bovine serum albumin had no effect on the reaction.) The mixture was incubated for 30 min at 370 and then dialyzed extensively against buffer G at 40 to remove Ca2 . The RNase P was then assayed for activity using the precursor to E. coil tRNATYr as substrate. The final volume (100,Ml) contained 30 mM Tris-HCI (pH 8), 5 mM MgCI2, 0.1 mM EDTA, 0.1 mM 2-mercaptoethanol, and about 104 cpm of substrate with appropriate amounts of treated enzyme. The final concentration of NH4Cl in the assay mixtures was about 50 mM. Incubation was for 10 min at 370; the mixtures were then analyzed in acrylamide gels as described (2,

Ml of buffer G was then added and the sample was filtered

through a Swinnex filter apparatus fitted with a Millipore GAWP 01300 filter (0.45 Mum pore size) to remove the polyacrylamide beads and the pancreatic RNase A. This prevented interference of the pancreatic RNase A in subsequent assays of the RNase P sample. Control reaction mixtures contained 50 MAl of buffer G instead of the pancreatic RNase A suspension; they were incubated and filtered in parallel with the actual reaction mixtures in all experiments. RESULTS Structure of Highly Purified RNase P. RNase P. purified by either scheme I or scheme II, exhibited a simple pattern when analyzed in sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gels (Fig. 1). Scheme I enzyme, purified from a crude extract of E. coli, showed one prominent band staining with Coomassie brilliant blue (C5) and one staining with methylene blue (M2), protein and nucleic acid stains, respectively (Fig. 1A). Estimates of the purification factor (at least 500-fold) of this enzyme were complicated by losses of activity during the steps of scheme I. Scheme II enzyme was somewhat less pure but shows the same protein band predominating and two prominent nucleic acid bands labeled Ml and M2 (Fig. 1 B and C). In separate experiments performed with E. coil labeled with 32P043, we have shown that Ml and M2 isolated from purified RNase P are RNA species with identical RNase Ti fingerprints, indicating that they differ in conformation only. The common RNase TI fingerprint is characteristic of a single RNA species of about 350 nucleotides and is similar to E. coil band IX RNA described by Ikemura and Dahlberg (10). As RNase P was purified through either scheme I or II, the preparation was enriched for band Ml or M2. In addition to the

5).

Polyacrylamide-bound pancreatic RNase A (Sigma Biochemical Corp.) was prepared for use by swelling in water for 2 hr at room temperature with occasional shaking. The swollen beads were washed twice with buffer G and then resuspended in buffer G to give a final concentration of 10 mg of polyacrylamide plus enzyme per ml. RNase P was digested as follows: 50 ,l of polyacrylamide-bound pancreatic RNase A suspension was added to 50 ,l of an RNase P sample in buffer G; this gave about 0.02 Kunitz unit of pancreatic RNase A per Mg of RNA in the RNase P sample. Phenylmethylsulfonylfluoride was added to a final concentration of 10MuM in all reaction mixtures. The sample was incubated with shaking at 370 for 20 min; 150

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FIG. 1. NaDodSO4polyacrylamide gel analysis of purified RNase P. (A) RNase P purified through scheme I was analyzed in a NaDodS0dpolyacrylamide gel as described (8). The gel was stained with Coomassie brillant blue and then overstained with methylene blue (9). (Lanes 1-4) Samples from the n-octyl-Sepharose column (each containing at least 10 ,g of protein) which were dialyzed against distilled water and lyophilized before analysis. RNase P activity eluted from the column in the order of fractions 4, 3, 2, with the peak in 3. (Lane l) Sample with little RNase P activity eluting later than the peak of activity. (Lane 5) Aliquot of the sample loaded on the column. (Lane 6) Standards: Cat, catalase (Mr 58,000); LDH, lactate dehydrogenase (Mr 36,000); Hb, hemoglobin (Mr 16,000). M2, methylene blue-staining material; and C5, Coomassie blue-staining material (see B and C) in lanes 2-4. The high background in lanes 2-4 is due to some breakdown of material staining with methylene blue. (B) RNase P purified through scheme II was analyzed as described in the legend of A, except that individual cylindrical gels were used for each sample. Samples from various stages of purification and those eluted from the final nondenaturing gel step (peak of RNase P activity in fraction 5 with some activity also in fraction 4) were used. (C) Same gel as shown in B, overstained with methylene blue. M1 and M2, regions of fraction 5 that stain prominently with methylene blue. C5 and C6, bands staining with Coomassie blue in fraction 4, only C5 is

visible in fraction 5.

Biochemistry: Stark et al.

Proc. Natl. Acad. Sci. USA 75 (1978)

3719

rylamide gel analysis. RNase P activity was recovered from these gradients in 10-0% yield. When centrifuged in CsCI, the relatively crude DEAE-Sephadex preparation from scheme I overlapped partly with a peak of RNA and protein that may represent ribosome core particles (11) or other RNA-protein complexes. However, the activity in a more purified Sepharose 4B preparation was separated from 90% of the protein layered on the gradient, most of which is at the meniscus (not shown). The apparent molecular weight of RNase P. as determined by gel filtration experiments, was 260,000 (12), but this figure was based on a calibration curve with only protein standards. In fact, the enzyme was eluted from Sepharose 4B or Sephadex G-200 close to the position expected for an RNA molecule of the size of the RNA species that is part of the enzyme complex. It is likely that the enzyme is made up of one RNA molecule and one, or possibly two, protein molecules of molecular weight 20,000. The resolving power of our NaDodSO4/polyacrylamide gels may be inadequate to separate polypeptides with similar molecular weights in the range of interest. Some of our scheme I preparations do exhibit one or two polypeptides bands in addition to C5 in our gel. We cannot rule out the possibility that both the protein and RNA species we identify as components of RNase P are themselves breakdown products of larger protein or RNA species. Inactivation of RNase P by RNases. RNase P purified by either scheme I or II and pretreated with either micrococcal nuclease (MN) or pancreatic RNase A lost the ability to cleave the precursor to E. colh tRNATYr. Experiments with MN are illustrated in Fig. 3 A and B. Control pretreatments were performed in the absence of MN (Fig. 3A) and in the absence of CaCl2 (a necessary cofactor for MN activity) (Fig. 3B). Similar

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FIG. 2. Buoyant density of RNase P in CsCl. RNase P (3 fg) purified through scheme I was layered on a preformed CsCl gradient. The steps of the gradient were 1.6 ml each of 57, 54, and 51% CsCi solutions in buffer A. Centrifugation was for 22 hr at 30 in a Spinco SW65 rotor at 45,000 rpm. Drosophila virilis satellite II [3H]DNA (a gift of C. Yen, Yale University, New Haven, CT) was added with the RNase P sample. X, RNase P activity; *, 3H radioactivity.

NaDodSO4 gel analysis of the purified RNase P showing the presence of an RNA species, results from CsCl buoyant density determinations confirmed that RNA is an integral part of the enzyme. Enzyme purified through scheme I had a buoyant density of 1.71 g/ml in CsCl. Fig. 2 illustrates the buoyant density determination of enzyme purified through the n-octyl-Sepharose step and its separation from a Drosophila virdis DNA standard sample. The RNase P buoyant density corresponds to a species of about 20% protein and 80% RNA (predominantly singlestranded) by mass, as expected from the NaDodSO4/polyac-

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FIG. 3. Inactivation of RNase P with RNase pretreatment. Control reactions were performed without MN in the pretreatment mixture (A) or without CaCl2 (B). In the assay for RNase P activity, about 0.5 Ag of MN was in the final mixtures and about 0.8 ,g of RNase P for the experiments in A and 0.25 .g for the experiments in B. The positions of the intact precursor tRNA'rYr substrate and the two cleavage products are

indicated. Spontaneous breakdown products of the substrate are apparent between the "precursor" and "tRNA" positions. RNase P pretreated with MN had less than 5% the activity of the control reaction RNase P. The extent of inactivation can be varied by changing the conditions as shown. (C) Effect of pancreatic RNase A treatment on RNase P activity. RNase P used in this experiment had been purified by scheme I through the Sepharose 4B step; the RNase A treatment was done with acrylamide-bound RNase A.

3720

Biochemistry:

Stark et al. I

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[RNA), AM FIG. 4. Inhibition of RNase P activity by RNA. About 0.25 ,ug of RNase P (enzyme prepared through various stages of purification of scheme II gave the same result with tRNA; tRNA inhibition was measured with enzyme purified through the DEAE-Sephadex step) was used in the reaction mixtures. Purified E. coli bulk tRNA and 5S rRNA were gifts of B. Clark and R. Garber, respectively. A mixture of 16S and 23S rRNA was prepared by phenol extraction of E. coli MRE 600 ribosomes followed by chromatography of the RNA on Sephadex G-200 to remove low molecular weight species. The extent of RNase P reaction was calculated by comparing the radioactivity in undigested precursor substrate with that found in the bands corresponding to the cleavage products. The molarities of the three inhibitors were calculated from the following figures: one A2so unit equals 40 sg of RNA; average Mr of bulk tRNA is 2.5 X 104, of 5S rRNA is 4 X 104, of the 16S and 23S rRNA mixture is 8.3 X 105. Inhibition by tRNA (@); by 5S rRNA (0); by 16S and 23S rRNA mixture (0). Protein concentrations in all our experiments were determined by the method of Bradford (29).

results were obtained when the monomeric precursor to T4 tRNAGIY (a gift of W. H. McClain) or the three dimeric precursors of T4 tRNAs (13) were used as substrates. When the pretreated RNase P was assayed with tRNA precursor as substrate, there was no residual MN activity because CaC12 had been removed by dialysis. The total absence of residual MN activity is shown by the intactness of the precursor tRNA substrate in the reaction mixtures that had no MN at all and those that were exposed to MN. The MN preparations we used do not have proteolytic activity, as judged by their inability to inactivate bacterial muconate lactonizing enzyme, lactate dehydrogenase from beef heart, and bovine pancreatic RNase A (MN also does not inactivate T4 polynucleotide kinase; H. D. Robertson, personal communication), and by the fact that bands staining with Coomassie brilliant blue in NaDodSO4/polyacrylamide gel analysis of crude RNase P remain intact after MN treatment. Furthermore, since pancreatic DNase is unable to inactivate RNase P, the RNase activity of MN must be responsible for the effect we report. Lastly, MN left in the RNase P reaction mixture cannot protect the precursor substrate from attack by RNase P because in the control experiment in which CaCd2 was omitted from the pretreatment reaction, no protection of the substrate was seen. In separate experiments (12) we have also shown that bands stained by methylene blue in NaDodSO4 gel analyses of RNase P treated with MN were either lost entirely

or significantly shifted in mobility when compared with untreated enzyme. We have also used pancreatic RNase A immobilized on polyacrylamide beads to treat RNase P. The beads were removed by filtration from the pretreatment mixture prior to the RNase P assay and no residual RNase A activity was apparent (Fig. 3C). Again, pretreated RNase P became inactive. Pretreatment experiments with RNase A were carried out in the presence and absence of 10 ,M phenylmethylsulfonylfluoride, a serine protease inhibitor (14) that does not affect RNase P activity, with identical results to those shown. Purified E. colt RNase III (15, 16) (a gift of J. J. Dunn) has no effect on RNase P, so the reaction with MN and RNase A may be specific for single-stranded regions in the RNA associated with RNase P. RNase P is resistant to inactivation by several proteases (proteinase K, subtilisin BPN', subtilisin Carlsberg, and subtilopeptidase) at concentrations of 2 mg/ml in buffer G. However, RNase-free pronase (0.2 mg/ml) will inactivate 60% of RNase P activity in buffer G and 100% in the same buffer with only 20 mM NH4C1. RNase P is also more sensitive to thermal denaturation in solutions containing 10 mM NH4Cl rather than in those with 0.19 M NH4Cl. These data, and those from the buoyant density determination, indicate that RNase P is a very compact ribonucleoprotein complex in solutions of high ionic strength but is more accessible to other components in solution when in solutions of relatively low ionic strength. Inhibition of RNase P Activity by RNA Molecules. RNase P activity can be inhibited by tRNA, 5S RNA, or by a mixture of 16S and 23S rRNA (Fig. 4). The degree of inhibition by these molecules, when plotted as a function of the number of moles of RNA molecules used, is the same for each kind of RNA. This result suggests that the number of ends of RNA molecules encountered may play a role in the inhibitory process. tRNA can inhibit RNase P cleavage of both tRNA precursor molecules and bacteriophage 480-encoded M3 RNA (12, 17). Bacteriophage X DNA, at concentrations up to 45 ;g/ml, does not inhibit RNase P action. The data show that various RNA molecules can bind to RNase P but the mode of action has not been determined. Measurements of the Km of RNase P with the precursor to E. colt tRNATYr as substrate give a value of 10-8 M. Estimates of the Ki (assuming either competitive or noncompetitive models) are close to 10- (12), so the interaction of the enzyme with inhibitor molecules, one of which is a product of enzymatic reaction, appears to be much weaker than with the true substrates. Reconstitution of RNase P Activity from Inactivated Enzyme. We have tried to reconstitute RNase P activity from E. colt RNA and enzyme pretreated with MN. Whole cell RNA has yielded some success (about 20% reconstitution of control activity), but the scatter in the experimental points is large so the results are not reliable. The outcome of these experiments is sensitive to the ionic strength of both the reconstitution and enzyme assay medium and the details of MN pretreatment, so the process seems complex. We have been able to recover enzymatic activity from RNase P inactivated with 7 M urea and then dialyzed to remove the urea. Thus, it may be possible to reconstitute enzymatic activity from the enzyme components after separation in and elution from urea-containing polyacrylamide gels or gel filtration columns. DISCUSSION We have shown that the activity of RNase P can be abolished by pretreatment of the enzyme with certain RNases, as well as by protease treatment or thermal denaturation. The RNases

Proc. Natl. Acad. Sci. USA 75 (1978)

Biochemistry: Stark et al. used, MN and RNase A, do not have detectable protease contamination. An indication that RNase P has an essential RNA component is also provided by the copurification of a discrete RNA species with the enzymatic activity through several different kinds of purification schemes. Additionally, the buoyant density in CsCl of the purified enzyme is characteristic of a ribonucleoprotein complex. If the essential RNA component is a characteristic feature of RNase P, an enzyme involved in tRNA biosynthesis, then this feature should be preserved in RNase P found in all organisms. In fact, RNase P extracted from human KB cell extracts can also be inactivated by MN pretreatment (18). We do not know if the RNA component of RNase P is needed for stabilization of the protein moiety or if it plays a more active role in substrate recognition (the nucleolytic activity of the enzyme is reserved for the protein moiety). It is possible, however, to envision a scheme for recognition of tRNA precursor substrates that makes use of nucleotide-nucleotide interactions. The common feature shared by all tRNA moieties of tRNA precursor substrates is the location of invariant nucleotides (19) which supposedly specify the conformation of this part of the substrate molecules. We propose that the RNA component of RNase P interacts in a specific way with the invariant nucleotides found in the tRNA moieties of all precursor tRNA molecules through some of the modes of nucleotidenucleotide interactions involved in maintaining the tertiary structure of tRNA molecules (20, 21). This interaction fixes the position of the enzyme in space and positions the nucleolytic subunit so that it is always at precisely the right position to cleave at the beginning of the 5' terminus of the mature tRNA sequences (7). Studies of mutants of E. coli tRNATYr su3+ and of certain T4 suppressor tRNAs (6) show that any base change that alters the secondary and/or tertiary structure of a tRNA molecule is likely to reduce the rate of RNase P cleavage of the corresponding precursor tRNA (3-5). Mutations or chemical alterations of the C-C-A terminus also affect RNase P cleavage (5, 22), probably by altering enzyme binding near the nucleolytic site. Mutations in the anticodon loop, which do not affect secondary and/or tertiary structure, do not alter RNase P recognition or cleavage of its substrates; thus, this region is left uncovered by the enzyme in our scheme. This scheme is hypothetical: we have no direct evidence that the critical positioning of RNase P on precursor tRNA substrates is determined by RNA-RNA rather than by protein-RNA interactions. In addition to the cleavage of precursor tRNAs, RNase P cleaves other substrates (7, 12, 17, 23). The proposed secondary structures of these other substrates are similar, but differ from the proposed structure of precursor tRNA molecules. The rate of cleavage of these substrates, the precursor to E. coli 4.5 S RNA (23) and bacteriophage 480-induced M.3 RNA (17), by untreated RNase P is about 10%, or lower, of the rate of cleavage of precursor tRNATYr. RNase P pretreated with pancreatic RNase A cannot cleave precursor to 4.5 S RNA. Thus, the RNA moiety of RNase P is required for enzymatic function even in the very slow reaction with nonprecursor tRNA substrate. It seems reasonable and eonomical that an enzyme involved in recognizing a large variety of RNA substrates on the basis of conformation only should take advantage of RNA-RNA interactions in three-dimensional space to achieve the recognition. One obvious and in1portant example of a functional RNAprotein complex is the ribosome. Nucleotide-nucleotide interactions are important in vitro in both mRNA (24) and tRNA (25) binding to ribosomes. Additionally, it appears that 5S rRNA of E. coli stimulates GTPase activity found in a complex with itself and three ribosomal proteins (26). RNA ligase participates in the assembly of bacteriophage T4 tail fibers (27). RNA is also

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thought to be essential for the structural organizing role of centrioles in eukaryotic cells (28). Thus there is some precedent for believing in the involvement of RNA in intracellular protein complexes that engage in biochemical reactions. We thank L. Atkins and R. M. Gershon for technical assistance and many colleagues for helpful discussions. B.C.S. and E.J.B. were supported by U.S. Public Health Service pre- and post-doctoral training grants, respectively. This work was supported by U.S. Public Health Service Grant GM 19422 to S.A. and was presented in part at the 61st annual meeting of the American Society of Biological Chemists, April 1-8, 1977 (ref. 30). 1. Altman, S. & Smith, J. D. (1971) Nature (London) New Biol. 233, 35-39. 2. Robertson, H. D., Altman, S. & Smith, J. D. (1972) J. Biol. Chem. 247,5243-5251. 3. Altman, S. (1975) Cell 4,21-29. 4. Smith, J. D. (1974) Brookhaven Symp. Biol. 26, 1-11. 5. Altman, S., Bothwell, A. L. M. & Stark, B. C. (1974) Broochaven Symp. Biol. 26, 12-25. 6. Seidman, J. G., Comer, M. M. & McClain, W. H. (1974) J. Mol. Biol. 90, 677'-689. 7. Bothwell, A. L. M., Stark, B. C. & Altman, S. (1976) Proc. Nati. Acad. Sci. USA 73, 1912-1916. 8. Fairbanks, G., Steck, T. L. & Wallach, D. F. H. (1971) Biochemistry 10, 2606-2617. 9. Peacock, A. C. & Dingman, C. W. (1967) Biochemistry 6, 1818-1827. 10. Ikemura, T. & Dahlberg, J. E. (1973) J. Biol. Chem. 248, 5024-5032. 11. Meselson, M. S., Nomura, M., Brenner, S., Davern, C. & Schlessinger, D. (1964) J. Mol. Biol. 9, 696-711. 12. Stark, B. C. (1977) Dissertation (Yale University, New Haven,

CT). 13. Guthrie, C., Seidman, J. G., Altman, S., Barrell, B. G., Smith, J. D. & McClain, W. H. (1973) Nature (London) New Biol. 246, 6-11. 14. Fahrney, D. E. & Gold, A. M. (1963) J. Am. Chem. Soc. 85, 997-1000. 15. Robertson, H. D., Webster, R. E. & Zinder, N. D. (1968) J. Biol. Chem. 243,82-91. 16. Dunn, J. J. (1976) J. Biol. Chem. 251, 3807-3814. 17. Pieczenik, G., Barrell, B. G. & Gefter, M. L. (1972) Arch. Biochem. Biophys. 152, 152-165. 18. Koski, R. (1978) Dissertation (Yale University, New Haven,

CT). 19. Kim, S. H., Quigley, C. J., Suddath, F. L., McPherson, A., Sneden, D., Kim, J. J., Weinzierl, J. & Rich, A. (1973) Science 179, 285-288. 20. Ladner, J. E., Jack, A., Robertus, J. D., Brown, R. S., Rhodes, D., Clark, B. F. C. & Klug, A. (1975) Proc. Natl. Acad. Sci. USA 72,4414-4418. 21. Rich, A. & RajBhandary, U. L. (1976) Annu. Rev. Biochem. 45, 805-860. 22. Seidman, J. G., Schmidt, F. J., Foss, K. & McClain, W. H. (1975) Cell 5,389-400. 23. Bothwell, A. L. M., Garber, R. L. & Altman, S. (1976) J. Biol. Chem. 251, 7709-7716. 24. Steitz, J. A. & Jakes, K. (1975) Proc. NatI. Acad. Sci. USA 72, 4734-4738. 25. Erdmann, V. A. (1976) Prog. Nucleic Acid Res. Mol. Biol. 18, 45-90. 26. Gaunt-Kl6pfer, M. & Erdmann, V. A. (1975) Biochim. Biophys. Acta 390, 226-230. 27. Snopek, T. J., Wood, W. B., Conley, M. P., Chen, P. & Cozzarelli, N. R. (1977) Proc. Natl. Acad. Sci. USA 74,3355-3359. 28. Heidemann, S. R., Sander, G. & Kirschner, H. W. (1977) Cell 10, 337-350. 29. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. 30. Stark, B. C., Bowman, E. J. & Altman, S. (1977) Fed. Proc. Fed. Am. Soc. Exp. Biol. 36,659.