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 same buffer. The RNase P eluted after the rRNA peak and tRNA substrates from Escherichia coli can be abolished by before the 4S RNA peak. The active fractions were pooled and pretreatment of this enzyme with micrococcal nuclease or concentrated as described above and then applied to a Sephadex pancreatic ribonuclease A, as well as by proteases and by ther- mal denaturation. Highly purified RNase P exhibits one prom- G-200 column (1 X 90 cm) equilibrated and eluted in the same inent RNA and one prominent polypeptide com nent when manner as the Sepharose 4B column. The RNase P eluted just examined in polyacrylamide gels containing sodum dodecyl after the void volume. This material was again pooled, con- sulfate. The buoyant density in CsCl of RNase P, 1.71 g/ml, is centrated, redissolved in 2 M (NH4)2SO4 in buffer B, applied characteristic of a protein-RNA complex. The activity of RNase to an n-octyl-Sepharose (a gift of H. Taira, Yale University, P is inhibited by various RNA molecules. The presence of a New Haven, CT) column (0.5 X 2 cm) that had been equili- discrete RNA component in RNase P appears to be essential for brated with 2 M in buffer and then eluted with enzymatic function. A model is described for enzyme-substrate (NH4)2SO4 B, recognition in which this RNA component plays an important a linear gradient from 2 M (NH4)2SO4 in buffer B to buffer B. role. The active fractions eluted at about 0.5 M (NH4)2SO4. Scheme II. S30 made with 600 g of E. coli MRE 600 was Ribonuclease P (1-3) is necessary for the biosynthesis of the 5' further processed to yield a 0.2 M NH4Cl wash of ribosomes. termini of tRNA molecules in Escherichia coli. This enzyme The active RNase P in the salt wash was loaded onto a DEAE- recognizes some aspect of the structural conformation rather Sephadex column and purified as described (2). RNase P (1.8 than nucleotide sequence at is cleavage sites in tRNA precursor mg), purified through the DEAE-Sephadex step, was concen- molecules (4-6). Exactly how this recognition occurs is a matter trated by precipitation with (NH4)2SO4, resuspended in 0.5 ml for speculation (7). In this report we show that treatment of of buffer G [20 mM Tris-HCl, pH 7.6/0.50 M NH4CI/15 mM highly purified RNase P with either micrococcal nuclease (MN) Mg(OAc)2/6 mM 2-mercaptoethanol/5% sucrose], dialyzed or pancreatic ribonuclease A abolishes RNase P activity. Our against this buffer, and applied to a column of Sephadex G-200 most highly purified RNase P preparations contain one discrete (0.9 X 55 cm) equilibrated with buffer G. RNase P activity was RNA species and one discrete protein species, and several kinds eluted from this column with buffer G. One-third of the peak of RNA can inhibit the enzymatic activity. We conclude that fractions (0.4 mg of protein in 4 ml) was pooled and concen- the interaction of RNase P with its substrates depends on the trated by dialysis against buffer G containing 15% polyethylene presence of RNA in the enzyme complex. glycol until the final volume was 0.3 ml. This sample was loaded in aliquots onto four 7% polyacrylamide gel columns (6 X 90 METHODS mm). The running buffer was 25 mM Tris-HCl/25 mM glycine Preparation of RNase P. What follows is an abbreviated at pH 9; the gel buffer was the same but at pH 7. The gels were description of RNase P purification schemes. Complete details run at 4 mA per column for 80 min at 40, at which time the will be published elsewhere. Crude extracts (S30) of E. coil bromphenol blue marker had run about 70 mm. Ten slices, MRE 600 were prepared by grinding of frozen cells with alu- spaced equidistant apart, were made from the top of the gel to mina as described (2). Two purification schemes were used, the dye front and eluted, after mincing, with buffer G at 00. starting with S30. Fractions were assayed for activity as described (2, 5). The final Scheme I. S30 made from 200 g of cells was diluted 1:5 with NH4Cl concentration in the reaction mixtures was 10 mM. buffer A [50 mM Tris-HCl, pH 7.5/60 mM NH4Cl/10 mM Assay of RNase P Activity. Radioactive substrates for RNase Mg(OAc)2/6 mM 2-mercaptoethanol] and loaded onto a P were prepared from E. coli A49 (RNase P ts) as described (2, DEAE-Sephadex A-50 column (12 X 25 cm) equilibrated with 5). These were used in assay mixtures under standard conditions buffer A. Bed volume of the column was about 2.5 liters. The (2, 5) unless otherwise noted. The extent of RNase P reaction column was washed successively with 3 liters of buffer A con- was quantitated by cutting appropriate slices out of polyac- taining 0.2 M NH4Cl, 0.3 M NH4Cl, and 0.4 M NH4Cl in suc- rylamide gels and assaying the amount of RNA in the slices by cessive washes. The final wash was with buffer A containing monitoring Cerenkov radiation. 0.5 M NH4Cl (now called buffer B) and the activity was eluted Abbreviations: NaDodSO4, sodium dodecyl sulfate; MN, micrococcal after about 1 liter of the last wash buffer had flowed through nuclease; buffer A, 50 mM Tris-HCl, pH 7.5/60 mM NH4Cl/10 mM the column. The pooled active fractions wer- precipitated with Mg(OAc)2/6 mM 2-mercaptoethanol; buffer B, 50 mM Tris-HCl, pH 0.6 g of (NH4)2SO4 per ml; the precipitate was resuspended and 7.5/0.5 M NH4Cl/10 mm Mg(OAc)2/6 mM 2-mercaptoethanol; dialyzed against buffer B to give a final volume of about 15 ml. buffer C, 20 mM Tris-HCl, pH 7.6/0.50 mM NH4Cl/15 mM This material was applied to a Sepharose 4B column (2.5 X 90 Mg(OAc)2/6 mM 2-mercaptoethanol/5% sucrose. * Present address: Department of Botany, Washington State University, cm) equilibrated with buffer B and eluted with 300 ml of the Pullman, WA 99163. t Permanent address: Institute of Biochemistry and Biophysics, The costs of publication of this article were defrayed in part by the Polish Academy of Sciences, Warsaw, Poland. payment of page charges. This article must therefore be hereby marked * Present address: Department of Human Genetics, Yale University, "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate School of Medicine, New Haven, CT 06510. this fact. § To whom reprint requests should be addressed. 3717 Downloaded by guest on September 23, 2021 3718 Biochemistry: Stark et al. Proc. Natl. Acad. Sci. USA 75 (1978) Ribonuclease Inactivation of RNase P. One hundred mi- Ml of buffer G was then added and the sample was filtered croliters of RNase P (11 tig of protein purified through the through a Swinnex filter apparatus fitted with a Millipore DEAE-Sephadex step of scheme II was used in the experiments GAWP 01300 filter (0.45 Mum pore size) to remove the polyac- shown in Fig. 2A; 4 ug of protein purified through the nonde- rylamide beads and the pancreatic RNase A. This prevented naturing gel step of scheme II was used in the experiments interference of the pancreatic RNase A in subsequent assays shown in Fig. 2B) in buffer G was mixed with 50 Ml of 10 mM of the RNase P sample. Control reaction mixtures contained 50 CaCl2 and 6 ,ul of MN (1 mg/ml) in 1 mM NaCi. (MN from MAl of buffer G instead of the pancreatic RNase A suspension; Sigma Biochemicals, Worthington Biochemicals, Schwarz/ they were incubated and filtered in parallel with the actual Mann, and an authentic sample received as a kind gift from M. reaction mixtures in all experiments. Laskowski, Sr., were all equally effective. Whether or not the stock solutions were made up with bovine serum albumin had RESULTS no effect on the reaction.) The mixture was incubated for 30 min at 370 and then dialyzed extensively against buffer G at Structure of Highly Purified RNase P. RNase P. purified 40 to remove Ca2 . The RNase P was then assayed for activity by either scheme I or scheme II, exhibited a simple pattern using the precursor to E. coil tRNATYr as substrate. The final when analyzed in sodium dodecyl sulfate (NaDodSO4)/poly- volume (100,Ml) contained 30 mM Tris-HCI (pH 8), 5 mM acrylamide gels (Fig. 1). Scheme I enzyme, purified from a MgCI2, 0.1 mM EDTA, 0.1 mM 2-mercaptoethanol, and about crude extract of E. coli, showed one prominent band staining 104 cpm of substrate with appropriate amounts of treated en- with Coomassie brilliant blue (C5) and one staining with zyme. The final concentration of NH4Cl in the assay mixtures methylene blue (M2), protein and nucleic acid stains, respec- was about 50 mM. Incubation was for 10 min at 370; the mix- tively (Fig. 1A). Estimates of the purification factor (at least tures were then analyzed in acrylamide gels as described (2, 500-fold) of this enzyme were complicated by losses of activity 5).