Purification and Properties of a Specific Escherichia coli

Ribonuclease which Cleaves a Tyrosine

Transfer Ribonucleic Acid Precursor

(Received for publication, April 4, 1972)

HUGH D. ROBERTSOK,” SIDNEY ALTMAN,~ ANT) JOHX D. SMITH

From the Medical Research Council Laboratory of Molecular- Biology, Hills Road, Cambridge CR2 2&H, England

SUMMARY

Precursor molecules of Escherichia coli wild type and mu- tant tyrosine tRNA’s contain at both their 5’ and 3’ termini extra nucleotides in addition to those of the mature tRNA molecule. The early steps of processing these precursor molecules must involve specific ribonuclease cleavage. We report the isolation from E. coli extracts of the specific endo- nucleolytic RNase which cleaves only a single phosphodiester bond of the 129 nucleotide tyrosine tRNA precursor mole- cule. This cleavage removes all extra nucleotides present at the 5’ terminus of the precursor as a 41 nucleotide fragment, exposing the 5’ end of the mature tRNA. After sufficient purification, this activity has no effect upon the extra nucleo- tides at the 3’ end of the tRNA precursor. Therefore process- ing of the two ends of this molecule must be carried out by different enzymatic activities.

This novel RNase activity, which we have called RNase P, has been purified by washing ribosomes with 0.2 M NH&l, followed by ammonium sulfate fractionation and chromatog- raphy on DEAE-Sephadex and phosphocellulose. At this stage it shows no evidence of other E. coli RNase activities. RNase P requires both monovalent and divalent cations for optimal activity, and has a pH optimum of 8.0. In the course of purifying RNase P, we have discovered in other subcellular fractions of E. coli RNase activities potentially responsible for additional steps of precursor tRNA processing.

,Z precursor to an &&richia coli tyrosine tRNA species has recently been isolated and characterized (1,2). This 129 nucleo- tide molecule contains 44 extra nucleotides in addition to the 85 normally present in the mature tyrosine tRNA. Of the extra 44, 41, including a ?&terminal nucleoside triphosphate, are lo- cated on the 5’ side of the mature tRNA sequence. It is evident that this precursor molecule must be cleaved in viva in order to give rise to the functional tRNA. Such processing of the tRNA

* Fellow of the Helen Hay Whitney Foundation. Present address, The Rockefeller University, Kew York, New York 10021.

$ Present address, Department of Biology, Yale University, New Haven, Connecticut.

precursor by crude S 30 extracts of E. coli has recently been re- ported by 9ltman and Smith (2), who showed that the extra segments at both the 5’ and 3’ ends of the precursor could be re- moved under proper conditions. One hypothesis to explain these results is that specific endonucleases exist in I?. coli includ- ing one which could cleave the phosphodiester bond immediately adjacent to the normal 5’ end of the tyrosine tRNA sequence, yielding the mature 5’ terminus and a 41 nucleotide fragment. However, such a hypothetical 41 nucleotide fragment was not recovered after digestion with crude extracts, although variable amounts of a smaller fragment containing the 22 to 23 bases nearest the 5’ end of the precursor were observed (2).

Earlier workers have reported a variety of ribonuclease activ- ities of E. coli which may be active in crude extracts (3-8). In order to see whether any of these previously described nucleases are involved in processing the tRNA precursor, as well as t.o characterize further the activity or activities responsible for this processing, we have purified this activity. We have found that more than one ribonuclease activity is involved in the processing, and we describe the extensive purification of one of these activi- ties. This enzyme turns out to be a new ribonuclease specific for the cleavage of only a single phosphodiester bond in the entire tRNA precursor molecule. After sufficient purification, this specific enzyme yields stoichiometric amounts of the expected 41 nucleotide 5’.terminal fragment of the precursor. In the course of purifying this activity, we have also obtained other subcellular fractions which contain activities potentially responsible for other modes of precursor RNA metabolism.

EXPERIMENTAL PROCEDURE

Jlaterials

Bacterial Strains-E. coli MREBOO was kindly provided by Dr. B. F. C. Clark of this laboratory. The RNase I- strain 180, a derivative of A19, was the gift of Dr. D. Morse, Harvard Med- ical School, Boston, Mass. E. coli MB931 was that used by hltman (1) to prepare tyrosine tRNA precursor. E. coli BF266 (9), the gift of Dr. P. Primakoff, Stanford IJniversity, Palo Alto, California, was used interchangeably with strain MB931.

Bacteriophage Strains-hlutant A25 of bacteriophage r&IO

was that reported by Smith et al. (10). This strain carries a mutant in the structural gene for tyrosine tRNA and has been used in the preparat,ion of tyrosine tRNA precursor.

5243

by guest on June 18, 2018http://w

ww

.jbc.org/D

ownloaded from

5244

Enzymes-Electrophoretically purified pxncreatic I )Nase, DPFF, was obtained from Worthington Biochemical Corp., Freehold, N. ,J. RNase Tl was from Sankyo Corp., Tokyo. RNase III was purified from E. coli MREBOO according to the procedure of Robertson et al. (11). Rho factor (12) was the kind gift of Dr. J. Roberts of this laboratory. Rabbit hemoglobin was the gift of Dr. M. B. Mathews of this laboratory, and cata- lase was purchased from Sigma Chemical Co., St. Louis, MO.

Clzemicals-All routine chemicals were of reagent grade. Am- monium sulfate was the ultra-pure “Aristar” grade, British I)rug Houses, Poole, England.

Chromatographic ,‘tledin-lI>EAE-Sep~ladex A-50 and Sephades G-25 were obtained from Pharmacia, Ippsala, Sweden. Phos- phocellulose was from Whatman. The DEAE-Sephadex was washed and prepared for use according to Robertson et al. (1 I), whereas the phosphocellulose was washed extensively in 1.0 M Tris-HCl, pH 7.9.

Tyrosine tRNA Precursor-32P-labeled tyrosirle tRNA pre- cursor was prepared and fractionated on polyacrylamide gels as described by Altman (I). The precursor, and other RNA bands on polyacrylamide gels, were eluted as follows. The region de- picting the desired area was cut from an exposed x-ray film, which was used as a template for excising t,he desired regions of the 20 x 40 cm slab gel. The gel band was mechanically homogenized aud eluted in 0.1 M Tris-TICl, pH 9.1, 0.5 RI NaCl, 0.01 M EDTA. Equal volumes (1 to 5 ml) of disrupted polyacrylamide suspended in the above buffer and water-saturated phenol were homogenized mechanically, centrifuged for 10 min at 10,000 x g, and the aqueous layer taken; the other phase was re-extracted with an equal volume of the above buffer by st,irring, and the pooled aqueous phases were filtered through a 0.45.nm Millipore filter. One ABe unit of E. coli tRNA was added per ml of aqueous phase, and the RNA was precipitated with 2.5 volumes of absolute ethanol at -2O”, centrifuged, and the pellet collected and resus- pended in 1 ml of 0.2 M sodium acetate, pH 5.5. Rn’A was pre- cipitated again with 2.5 volumes of absolute ethanol at -20”. After centrifugation, the resulting precipitate was lyophilized and resuspended in 0.1 ml of distilled water. Specific activity of the precursor was 0.5 to 2 X lo6 cpm per pg.

Other Polynucleotides-32P-labeled mixed tRNA was prepared from the same cultures as the tRN.1 precursor and purified in a similar fashion. f2 phage RNA was grown and purified accord- ing to Dahlberg (13). [3H]poly(AIY) copolymer, specific activ- ity 24.3 PC1 per pmole, was that used by Robertson et al. (11). Poly(G) Poly(C), containing [3H]poly(G), was prepared using RNA polymerase with unlabeled poly(C) as template, and had a specific activity of 253 &i per pmole. :Y small RNA species present in @O-infected cells1 was labeled with 32P and purified in a rnanner identical with the precursor. Denatured calf thy mus DNA was the kind gift of I)r. F. Galibert of this laboratory.

Slethods

Standard Assay Conditions for Enzymatic Cleavage OJ” tRn’A Precursor-ITnless otherwise noted, all reactions were carried out in a final volume of 0.1 ml in silicone-treated tubes at 37” for 90 min. Each reaction contained 0.01 M Tris-HCl, pII 8.0; 0.1 M NH4C1; 0.005 M lYIgClz; lop4 M EDT*%; lo+ M 2-mercapto- ethanol; substrate (ordinarily an amount of tyrosine tRiYA pre-

cursor containing 5,000 to 15,000 cpm of “ZP); and ein\me (an amount of t,he fraction to be tested appropriate to give at’ least 25’g cleavage of the precursor during the incubation period). Reactions were terminated by adding & volume of 0.4 M EDT;\, pII 9.4, containing O.OSC,: bromphenol blue. Since more highly purified enzyme fractions contain 5 c/; sucrose, all reactions were normally made 5% in sucrose at this point. Samples were then evaporated to dryness in DCCCUO, resuspended in 25 ~1 of distilled water, and layered on a 10r< polgacrylamide slab gel (20 x 40 x 0.3 cm). Electrophorexis was carried out in a continuous buffer system containing 10.8 g of Tris base, 0.93 g of disodiun- EDTA, 5.5 g of boric arid per lit,er, pH 8.3, for 16 hours at 400 volts and 4”. Positions of the radioactive RNA bands in the gels were determined by radioaut,ography.

In certain experiments it w-as desirable to follow the extensive degradation of RNA substrates. In these, the amount of Rx.1 remaining precipitable in 5?? trichloroacetic acid was determined as described by Robertson et a/. (11).

Fingerprinting analysis of RNA’s was carried out using the methods of Sanger and his collaborators (15, 16) as applied by Goodman et al. (17).

Preparation 0s Subcellular Fractions-All operations were car ried out at 4”. Five grams of B. coli AIRE or 180 were ground with 10 g of levigated alumina with a previously chilled ( -20”) mortar and pestle until a 1)aste was formed. Five milliliters of Buffer il (0.05 M ‘I’&HCl, pH 7.5, 0.06 M NH,Cl, 0.01 M MgClz, 0.006 M 2-mercaptoethanol) were added, and 10 pg per ml of pancreatic DNaae were added to the resultant slurry. After 30 min at 4”, the mixture was centrifuged for 10 min at 8,000 rpm in 12-ml glass centrifuge tubes in the SS34 rotor of the Sorvall RCS-B centrifuge. The supernatant was then centri- fuged for 40 min at 15,500 rpm as above; the resulting 30,000 x g supernatant is called S 30 and was that in which precursor cleaving activity was previously detected (2). R.ibosomes were prepared from this S 30 suprrnatant by centrifugation for 4 hours at 45,000 rpm in the type 65 rotor of the Reckman model L ultracentrifuge. The upper two-thirds of the resulting S 100 supernatant was removed with a Pasteur pipette, and the rest was discarded. The ribosomal pellet was rinsed with 2 ml of l3uffer A, which was discarded, and the pellet was then redis- solved in 2 ml of fresh Isuffer A. After removal of an aliquot of resuspended ribosomes for assays, the ribosomes were washed with the desired concentration of NH,Cl as follows. An appro- priate arnount of 4 M NH&I in Buffer A was added, aiid the mixture was transferred to a Beckman cellulose nitrate ceiitrifuge tube (3 X 2 inch). This tube was attached to the cup of a varin- ble-speed Vortex mixer (Lab-Line Instruments) with vinyl tape and allowed t,o agitate gently overnight at 4”. The volume leas increased to 5 ml with Buffer A containing the appropriate NH,CI concentration, and the mixture was centrifuged for 4 hours in the SW39 or SW50 rotor of the Beckman model L ultracentrifuge at 37,000 rpm. The upper two-thirds of the resulting superna tant was removed and retained, while the rest was discarded. The ribosomes were again resuspended in Buffer A, an aliquot, removed, and a further washing st,ep initiated. Protein con centrations were determined by the procedure of Lowry et al. (18).

Determination of Radioactivity--Samples dried on glass fiber filters or on paper were assayed for radioactivity using the to-

1 CT. Pieczenik, B. G. Rarrell, and M. L. (iefter (see R.c>fercncc uene-based scintillat,ion fluid of Robertson et al. (11). Quaiitita 14). tive analysis of the kinetics of tRKA precursor reactions \~a::

by guest on June 18, 2018http://w

ww

.jbc.org/D

ownloaded from

5245

performed after cutting out the appropriate bands from the gel as described above. The radioactivity in each sample was as- sayed by placing it in an empty vial and measuring the Cerenkov radiation in a scintillation spectrometer. The efficiency of de- tectiou of z21’ under optimal settings was 950/, using scintillation fluid and 2Ocl, for Cerenkov radiation.

Sucrose Density Gradieni Centrifugation-Sucrose density gradients (7 to 25c/;,) were prepared in a buffer containing 0.01 M Tris-HCl, pH 8.0, 0.005 M RlgC12, 1OV M EDTA, and 10m4 M

2-mercaptoethanol. Centrifugation was carried out for 10 hours in the SW50 rotor of the Beckman L265B ultracentrifuge at a t)emperature of 5”.

RESULTS

Partial Purification of a Specific Precursor-cleaving Activity- We atternpted to devise a purification procedure which avoided harsh treatment of subcellular components for as long as possible. In particular we attempted not to disrupt ribosomes. This ap- Irroach was used successfully by Robertson et al. in their purifica- tion of E. coli RNase III (11) and was also an alternative purifi- cation of JG:. coli RKase IT suggested by Spahr (19). An S 30 extract from 5 g of E. coli lUREGO was prepared as described un- der “Experimental Procedure,” and the ribosomes were isolated and washed as described under “Experimental Procedure.” The resuspended ribosornes and the supernatants, after washing at various NH&l coucentrations, were assayed for their ability to cleave t,he tRNh precursor.

Fig. 1 shows the effect of various subcellular fractions upon the tyrosine tRNA precursor. The large change in mobility of the precursor upon specific cleavage is almost entirely due to the re- moval of the 41 rrucleotides located to the 5’ side of the tRNA sequence in the precursor, since the 5’-terminal region accounts for 41 of the 44 extra uucleotides in the precursor. A comparison of Lanes I and 14 shows that the 8 30 extract contains an activity which cleaves the precursor to yield a major product with the mobility of tRNA, as noted by Altman and Srnith (2). ‘I‘hi~ activity remains with the ribosomes after washing and cerr- t,rifugatioii iu Buffer -1 containing 0.06 M N&Cl. Furthermore, about half of the activity is separated from the ribosomes after waslling and centrifugation in buffer containing 0.2 M ?JH&l (I,an~ 4). The rest of this specific RNase appears to remain as- aoriated with the ribosomes. However, when the ribosomes are washed and centrifuged in Buffer A cont,aining 0.5 M NH&l, a previously latent nonspecific ribonuclease activity is released. This activity reduces the precursor to fragments which have a mobility comparable to mono- and dinucleotides. Residual amounts of the specific precursor-cleaving activity remain as- sociated with the ribosomes after washing in 0.5 M NH,Cl, but tliey are uot apparent after further rounds of washing in buffer

contaiuing 1.0 or 2.0 ~1 NH,Cl (Lanes 8 to 12). (The apparent nonspecific enzymatic activity seen in Lane 13 is a variable phe- uomenon and is not important for our discussion here.)

Itlspection of Lanes 3 to 5 of Fig. 1 reveals a slight additional

c*h:ruge in the mobility of the band running in the position of t>-rosine tRNA. This observation is the result of the elimina- tiou frorn t’he 0.2 M ribosomal pellet of activities responsible for trinnniiig the 3’ end of the precursor. Such trimming of the 3’ e11tl of the precursor was observed in crude extracts by Altman :uid Srnit,h (2). Fractionation of tRNA precursor cleavage activity in /?. coli strain 180 (see “AIaterials”) revealed similar

intracellular localization nud ribosomal attachment propert,ies to those shown in Fig. 1 for strain MREBOO.

We chose to continue the purification of the precursor cleaving activity removed from the ribosomes by 0.2 M NH&l. The activity in this supernatant fraction is stable for months wheii stored frozen at -20”. Ammonium sulfate fractionation was carried out as follows: 0.1 gram of solid ammonium sulfate per ml of original solution was added slowly with stirring at 4”. The resulting precipitate was removed by centrifugation for 20 min at 12,000 rpm in the SS34 rotor of a Sorvall RC2-B centrifuge. Additional ammonium sulfate (0.1 g per ml of original solution) was then added to the supernatant, and the mixture was stirred and centrifuged as above. This process was repeated once more. Finally, 0.2 gram of ammonium sulfate per ml of original solution was added. Each of the four ammonium sulfate precipitates so obtained (designated 0 to lo%, 10 to 20%, 20 to 30%, and 30 to 507;) was resuspended in Buffer A (0.5 ml) and dialyzed, along with an aliquot of the final ammonium sulfate supernatant solu- tion, overnight against three l-liter changes of Buffer B (0.02 hi Tris-HCl, pH 7.6; 0.02 M NH&l; 0.015 M MgClz; and 0.006 M 2-mercaptoethanol). Assays of these five ammonium sulfate fractions are shown in Fig. 2. It is evident that the activit,J precipitates between 30 and 50 g of ammonium sulfate per 100 ml of original solution (30 to 5074). At this stage of the purifica- tion we can detect a second specific fragment which has the mobil- ity expected for the undegraded 5’.terminal fragment of the tRNA precursor (Lane 8). This component is absent in the reaction analyzed in Lane 11, which contained the unfractiouated 0.2 hf NH&l ribosomal wash.

The 30 to 507; ammoniurn sulfate fraction was then fraction- ated stepwise on DEAE-Sephades as follows. DEAE-Sephades was poured illto a column (1 x 8 cm) and equilibrated with Buffer I<. The dialyzed 30 to 50(7; ammonium sulfate fraction was added and 4 column volumes were collected. These frac- tions were made 5c/;, in sucrose, and the NHdCl concentration of the washing buffer was raised to 0.05 M. hgain, 4 column vol- urnes were collected. This process was repeated at NH&l con- centrations of 0.10, 0.15, 0.20, and 0.50 M. Each time, the collected fractions were rnade 5c/;, in sucrose. Assays of these fractions from DEAE-Sephadex are shown in Fig. 3. The specific endonucleolytic activity does not elute until 0.5 M NH&l is added (Lanes 16 to 18), and the relative yield of the putative 5’.terminal fragment is markedly improved in com- parison to that observed with the 30 to 5Oyc ammonium sulfate fraction (cf. Lanes 16 aud 19). Subsequent experiments (not shown) have demonstrated that the specific RNase activit,y does not elute from such a column in the presence of Buffer 13 contain- ing 0.4 M NH,Cl, but it is eluted at about 0.42 M NII4Cl.

It is also evident in Fig. 3 that Buffer B containing 0.1 M NH&l has removed significant amounts of RNase activit,y with proper- ties different from that eluted in 0.5 M KH,Cl. In light of the results shown in Fig. 1, it is likely that the activity eluted in 0.1 M NH&l corresponds to the latent nonspecific RiYase cornpo- nents, the rnajority of which were not removed from ribosomes until they were washed in 0.5 M NH4C1. Activity present in fract,ions eluted from DEAE-Sephades in 0.5 RI NIIdCl could be conceiitrated by addition of 0.6 g of ammonium sulfate per ml of enzyme solution, followed by centrifugation as described in the legend to Fig. 2, and dialysis of the resuspended pellet against Buffer 1% containing 0.02 h1 KH4Cl. This activity could be stored unfrozen on ice and is stable for several weeks.

by guest on June 18, 2018http://w

ww

.jbc.org/D

ownloaded from

5246

f 12 3 45 6 --_

78 9 10111213l4

FIG. 1 (top left). Subcell&r fractionation of tyrosine tRNA precursor cleavage activity. Ribosomes were prepared from 5 ml of an S 30 supernatant of Escherichia coli MRE600 and washed with the indicated NH&l concentrations as described under “Experimental Procedure.” At each stage small aliquots were retained for assay purposes, and equivalent volumes, normalized to the S 30 starting volume, were added to each reaction. This means, for example, that about 100 pg of protein were added to the reaction in Lane I and correspondingly lesser amounts to the other reactions. Each reaction contained 2 X lo4 cpm of s2P- labeled tyrosine tRNA precursor, and the assays were incubated and analyzed as described under “Experimental Procedure.”

FIG. 3 (bottom). DEAE-SeDhadex fractionation of the 30 to 50% ammonium sulfite fraction. - A DEAE-Sephadex column (1 X’s cm) in Buffer B containing 0.02 M NH&I was set up as described in the text, and 0.2 ml of the dialyzed 30 to 50% ammonium sulfate fraction (Fig. 2, Lanes 7 and 8) was added. Four column volumes of Buffer B with 0.02 M NH&l were collected in 2-ml fractions, followed by equal volumes of Buffer B containing increasing amounts of NH&l as indicated. Each fraction was made 5% in sucrose upon collection, and 25 ~1 of the first three 2-ml fractions to be collected at each NH&l concentration were assayed for activity against tyrosine tRNA precursor (1 X lo4 cpm) as de- scribed under “Experimental Procedure.”

FIG. 2 (top right). Ammonium sul- fate fractionation of t.he 0.2 M NH&l ribosomal wash. Four milliliters of a 0.2 M NH&l ribosomal wash (Fig. 1, Lane 4) were subjected to ammonium sulfate fractionation as described in the text, and the precipitates were resuspended in 0.5 ml of Buffer A. After dialysis of each resuspended fraction and the final supernatant against Buffer B containing 0.02 M NH,Cl, 10-J fractions were assayed for activity against 1 X lo4 cpm of ty- rosine tRNA precursor as described under “Experimental Procedure.” The ammonium sulfate percentages in the figure refer to the number of grams of ammonium sulfate added per 100 ml of original enzyme solution. Each ammonium sulfate fraction was as- sayed twice. Those reactions de- picted in the left of each pair re- ceived 1~1 of enzyme solution, whereas those on the right received 5 ~1.

by guest on June 18, 2018http://w

ww

.jbc.org/D

ownloaded from

\

‘tRNA’- I

mature tRNA

5’ fragment -

precursor by 1 FIG. 4. Products of digestion of 32P-labeled A25

the precursor-cleaving enzyme. a, separation of the proaucts on polyacrylamide gel electrophoresis together with a marker of mature tRNAQ’r. b, ribonuclease T1 products from the ‘tRNA’ band. The 3’ end includes the additional 3’-terminal nucleotides of the precursor; all other products are from the tRNA sequence. e, ribonuclease T1 products from the 5’ fragment. The numbered

The 0.5 M NH&l fraction from the DEAE-Sephadex column was dialyzed against Buffer B containing 0.02 M NH&l and 5% sucrose. A l-ml aliquot was further analyzed on a phosphocellulose column 1 cm in height prepared in a Pasteur pipette. Stepwise elution of this column was carried out exactly as described for the DEAE-Sephadex step, using Buffer B con- taining 5% sucrose and the NH&l concentrations indicated in Fig. 3. We find that all of the activity is recovered in the flow- through of the column (Buffer B containing 0.02 M NH&l). At this point the amount of protein in the active fractions is not detectable. However, an estimate of the minimum extent of purification through the DEAE-Sephadex step is presented below.

Specijicity of Precursor-cleaving Activity-We have previously shown that crude E. coli extracts split the precursor to give the tRNA sequence (with partial loss of the extra nucleotides at the 3’ end) a fragment comprising the first 22 to 23 nucleotides from the precursor 5’ end, and mono- and dinucleotides (2). These products could have resulted from a specific single cleavage splitting off the 41 nucleotide fragment which was subsequently partly degraded by other enzymes in the crude extracts. This interpretation has now been shown to be correct.

Fig. 4a shows that the more highly purified enzyme gives only two major RNA products on polyacrylamide gel electrophoresis. One (“tRNA”) migrates slightly behind mature tRNA, while the second (5’ fragment) moves more slowly than did the 22 nucleo-

nucleotides from the precursor segment are: 1, Gp; 2, ApGp; 3, CPAPGP; 4, CPCPAPGP; 5, APUPAPAPGP; 6, UPAPAPAP- APGP; 7, CPUPUPCPCPCPGP; 8, CPAPUPUPAPCPCPCPGP. Separation is by electrophoresis on cellulose acetate in pyridine acetate, 7 M urea, pH 3.5, from right to left; and on DEAE paper in 7y0 formic acid (v/v) from top to bottom.

tide fragment described before (2). The products of digestion of these two bands with Tr and pancreatic ribonucleases were examined; Fig. 4, b and c, shows the separation of the Tr ribo- nuclease products. The “tRNA” band contains the entire tRNA sequence from the terminal pGpG. . . , and includes the extra nucleotides at the 3’ end of the precursor. Partial loss of these nucleotides results in an additional 3’Qerminal T1 ribo- nuclease product which is present when the crude extract’s are used for processing but which is absent in Fig. 4b.

The digestion products of the 5’ fragment (Fig. 4c) are those expected from the complete 41 nucleotide 5’ segment. GpUp is absent from the pancreatic ribonuclease products obtained with this fragment; instead an additional nucleotide migrating in the position of GpU is found. An alkaline hydrolysis of this gave Gp, and it is tentatively identified as GpU. This pancreatic ribonuclease product would be expected if the specific RNase splits the 3’ phosphodiester bond of the last nucleotide in the precursor 5’ segment.

These results indicate that the enzymatic activity which we are studying here has a single simple mode of action on the precursor; we designate this activity ribonuclea,se P. We can further con- clude that RNase P purified to this stage is already free of the activity or activities which degrade the 5’-terminal fragment and remove the extra nucleotides from the 3’ end of the precursor. The sequence of tyrosine tRNA precursor and its cleavage point by RNase P are shown in Fig. 5.

by guest on June 18, 2018http://w

ww

.jbc.org/D

ownloaded from

1 ~CPGPUPGPGPUPGpGpGpGpUp~JpCpCpCpGpApGpCpGpGpCpCpApApApGpApGpApGpCpApGpApCpU

pUpUpCpCpCpCpCpApCpCpApCpCpA$JpCpU

FIG. 5. Primary structure of 480 A25 tyrosine tRNA precursor. The CCTTOW indicates the cleavage point of RNase P and the untler- !i/fetl segments indicate those nucleotides not normally found in the mature tRNA. The nucleotides not underlined are frequentl) depicted in the cloverleaf configuration.

-6 b

;;

-4 <

72 2

Oo I I I ! I I I I s

10 20 30 40 50 60 70 80 90 100

-5 ‘0

/ I I / I / I I I 10 20 30 40 50 60 70 a0 90 100

Tilne Hans 1

Fro. 6. Kinetics of RNase P digestion. A, kinetics of RNase P activity purified t,hrough DEAE-Sephadex. Tyrosine tRNA precursor (3.6 X lo4 cpm) was incubated with 15 ~1 of RNase P, purified through DE:AE-Sephadex and concentrated by am- monium sulfate precipitation, under standard conditions in a 0.1.ml reaction. At the times indicated, 15.~1 aliquots were taken and added to 10 ~1 of 0.4 M EDTA containing bromphenol blue and left on ice until the desiccation step. The samples were run on an acrylamide gel such as that shown in Figs. 1 to 3, and the bands corresponding to each species were cut out and their radio- activity determined as described under “Experimental Proce- dure.” O-O, tRN;A precursor; O-0, tRNA moiety; A-A, 5’ fragment. Less than one-tenth of the indicated radioactivity found at 90 min in the tRNA or 5’ fragment positions was observed in a sample incubated for 90 min at 37” without added enzyme.

Iowic requirements of purified RLVuse P

RNase P purified through the D@;AE-Sephadex step was con- centrated by ammonium sulfate precipit,ation, and aliquots were assayed under standard conditions except that the assay mixture cont,ained not more than 0.001 M MgClz or 0.0013 M NH,Cl after addition of enzyme. The various subst,ances listed under Addi- tions in the table were present in the following final concentra- tions: 0.005 M MgClI; 0.1 M NaCl; 0.1 M NHaCl; 0.02 M EDTA; 0.1 M KCl; 0.01 M MnClp; 0.001 M ATP. Relative activity was estimated by visual inspection of autoradiographs of slab gels which resemble those shown in Fig. 3. A dash indicates complete absence of the tHNA band and the 5’ fragment band.

-

Tube number Additions Relative activity

I ! None 2 3 4 3 6 7 8 9

10 11 12 13 14

MgCl* &lgCl~, NaCl M&l,, NH,Cl NH&l, El)TB MgCly, KC1 NaCl KC1 NH&l MdXZ ATP, MgCIa, NH&l MgCls, 2 x NH&l MgCl,, NaCl, NH,Cl No enzyme

+

++

++

+ ++ +

Note the change of scale OII the right-hmcl ordinate. B, compari- son of the kinetics of RNase P activity in fractions of different purity. An experiment identical with that described in A was performed using 3 ~1 of the S 30 extract from which the RNase P fraction used in A was obtained. Since the 5’ fragment is de- graded by the S 30 fraction, the total amount of radioactivity recovered in each aliquot m--as estimated by adding the radio- activity in the precursor band to 1.5 times the radioactivity in the tRNA moiety. O-O, precursor band in reaction with S 30; O-0, tRNA moiety in react,ion with S 30. Superimposed on these data are the data from A with the scale on the ordinate magnified 4-fold. m-u, precursor band from A; O-O, tRNA moiety from A.

by guest on June 18, 2018http://w

ww

.jbc.org/D

ownloaded from

FIG. 7. Properties of ribonuclease activity present in 0.5 M

NH&l ribosomal washes assayed by polyacrylamide gel elec- trophoresis. Assay conditions were those described under “Ex- perimental Procedure.” All substrates were used at the same specific radioactivity and were prepared from pulse-labeled cells as described previously (I, 2) and under “Experimental Proce- dure.” The assays depicted in Lanes 1 to 7 contained the 0.5 M ribosomal wash activity shown in Fig. 1, Lane 6, and contained 0.025 mg of protein per ml. Reactions shown in Lanes 8 to 10 contained RNase P activity in the 0.2 M ribosomal wash (Fig. 1, Lane Q), and the final protein concentration was 0.08 mg per ml. No enzyme was added to the reaction shown in Lane 11. Potential substrates and other additions to the assays were as follows.

1 Tyrosine tRNA precursor 2 Tyrosine tRNA 3 4 S bulk tRNA 4 @80 RNA 5 Tyrosine tRNA precursor

6 Tyrosine tRNA precursor 7 Tyrosine tRNA precursor 8 Tyrosine tRNA precursor 9 Tyrosine tRNA precursor

10 11

Tyrosine tRNA precursor Tyrosine tRNA, 4 S tRNA,

and 480 RNA

Potential substrate Additions other than enzyme

None None None None Denatured DNA (0.33 mg/

ml) 0.04 M EDTA 0.1 M NaCl None Denatured DNA (0.33 mg/

ml) 0.04 M EDTA No enzyme or other addi-

tions

5249

Properties of RNase P--We have studied the kinetics of the cleavage reaction by RNase P, both in crude extracts and after purification through the DEAE-Sephadex step, by quantifying the release of specific RNA products with time. In this way, we hope not only to find whether the properties of the reaction are altered by the removal of components during the purification, but also to estimate the minimum extent of purification. Re- sults obtained with the more highly purified enzyme fraction, shown in Fig. 64, indicate that the recovery of products accounts quantitatively for the loss of tRNA precursor. In addition, the ratio of radioactivity recovered in tRNA to that in the 5’ frag- ment at any given time is close to that expected for two RNA’s of identical specific activity which have their size ratio (88 to 41 nucleotides). Fig. 6B illustrates a comparison of the kinetics of RNase P activity in fractions of different purity. After ap- propriate normalization of the data obtained with the S 30 es- tract, as described in the legend to Fig. 6B, to facilitate com- parison, we find that both the kinetics of disappearance of the tRNA precursor and the appearance of the tRNA are identical. This result is one indication that the properties of the RNase P cleavage reaction have not been grossly altered by the purifica- tion steps. Furthermore, 154 pg of S 30 protein were added to one reaction shown in Fig. 6, while less than 1 pg of protein from the RNase P DEAE-fraction was added to the other. Since the initial rate of tRNA precursor cleavage by S 30 is 2.8 times that of the DEAE-fraction as determined from the data on which this comparison is based, we conclude that the enzyme in this fraction has been purified more than 50.fold, but the real figure is un- doubtedly higher.

The effect of ionic conditions on the RNase P reaction has been surveyed using the same assay system already described for the purification. We have tested various salt and pH conditions with the intention of comparing the RNase P cleavage reaction with those carried out by other E. coli RNases. For this reason, and because of the cumbersome nature of the standard assay, we have only screened certain carefully chosen sets of conditions. The results which we have obtained for a purified RNase P prep- aration, summarized in Table I, show that RNase P has the fol- lowing properties. (a) This enzyme requires magnesium or some divalent cation @In++ may partially replace Mg++) ; (b) this activity is stimulated by K+ and NHd+ (0.1 M) when Mg++ is present. No activity is observed in the presence of K+ and I$H4+ when Mg++ is absent. An equal concentration of Naf (0.1 M)

inhibits the extent of cleavage. ATP has no effect on the reac- tion, nor does the addition of excess bulk tRNA (data not shown). A similar pattern of ionic requirements for RNase P was also observed with cruder preparations (e.g. the activity associated with 0.06 M NH&l-washed ribosomes) as well as with RNase P preparations purified through phosphocellulose and desalted by passage through Sephadex G-25. Separate experiments have shown that the pH optimum for RNase P at various stages of the purification is in the neighborhood of 8.0, with lower activity ob- served both at pH 7.0 and pH 8.5.

The size of RNase P was estimated by a velocity sedimentation through a 7 to 257, sucrose density gradient in the absence of NH&!1 as described under “Experimental Procedure.” Purified catalase and hemoglobin were centrifuged in a separate tube as markers. In some experiments low levels of activity were re- covered moving slightly ahead of catalase, which has a reported

Some skewing to the interior of the gel has occurred with material added to the outside lanes, 1 and 11.

by guest on June 18, 2018http://w

ww

.jbc.org/D

ownloaded from

mined according to “Experimental Procedure” (Fig. 8). Much activity is still observed in the presence of 0.04 M EDTA, suggest- ing that certain aspects of this activity must be independent of divalent cations. Furthermore it is not affected by monovalent cations (data not shown), in contrast to RNase P.

Some selectivity in substrates is shown by this enzyme fraction as demonstrated by the following experiments. The active preparation attacks tyrosine tRNA precursor but. mature tyro- sine tRn’A or bulk tRNA isolated from infected cells are much

.&z 20- more resistant to this digestion (Figs. 7 and 8). Fig. 8 shows that A. the activity degrades a small @O-induced RNA molecule and

Oo 20 40 60 80 100 phage f2 RKA to a more limited extent than tRNA precursor.

Time (minutes) The synthetic alternating ribonucleotide copolymer poly(AI1) is solubilized by this activity, but the double-stranded homo- polymer pair poly(G) .poly(C) is resistant to this degradation. Some of the implications of these results are discussed below.

DISCUSSION

A nuclease which specifically cleaves the tyrosine tRNA pre- cursor has been isolated from E. coli. This nuclease, which we have ralled RKase P, is loosely associated with ribosomal par& cles, from which it can be readily removed and further purified. It appears to be highly specific for the endonucleolytic cleavage of the tRNA precursor at a single phosphodiester bond. Opti-

‘0 I I

20 40 60 80 100 ma1 activity requires the presence of both monovalent and di- Time (minutes)

FIG. 8. Kinet,ics of degradation of various RNA’s by RNase activity in 0.5 M iYH&l ribosomal wash assayed by acid solubiliza- tion. Kuclease activity was followed by determining the amount of radioactive substrate rendered soluble in 57, trichloroacetic acid (11). Various RNA’s were prepared as described under “Experimental Procedure” and used at the same specific radio- a.ctivity with the exception of poly(AU) and poly(G).poly(C), which had the specific activities noted under “Experimental Procedure.” Since the radioactive 4 S bulk tRNA used was ex- tracted from pulse-labeled cells, this preparation may contain some labile RNA which co-electrophoresed with the tRNA. A, o--o, tyrosine tRNA precursor; A-L,, 4 S bulk tRNA; O-D, f2 viral RNA. B, O-O, tyrosine tRNA precursor; O-O, &X0 RNA (see the text); wm, poly(G).poly(C); A-A, poly(AU).

sedimentation constant of 11.3 S (20). This peak can be well separated from the other contaminating RNase activities present

in cruder RNase P fractions. However, the recovery of active RNase P in the fast moving peak ahead of catalase is variable and seems to depend on the ionic conditions and state of purity of the enzyme.

Experiments on Other Subcellular Fractions Containing Addi- tional RNase Activities-We undertook a partial characterization of the RNase activity found in the supernatant obtained from

ribosomes washed and centrifuged in Buffer A containing 0.5 M

T\;H,Cl (Fig. 1, Lane 6). During our attempts to distinguish the activity or activities in this fraction from RNase P, we have

observed that the RNase activity in this fraction exhibits variable behavior upon ammonium sulfate precipitation or chromato- graphic fractionation, both with respect to recovery and condi- tiorls of elution. Since the chromatographic patterns, unlike t,hose of RNase P, are not simple, this activity may be a mixture of several different enzymes or a complex capable of assuming different active states in different ionic environments. Never- theless, we have succeeded in demonstrating the following. The activity reduces tyrosine tRr\‘h precursor to small fragments, as assayed by polyacrylamide gel electrophoresis (Fig. 7, Lane I),

valent cations. RNase P can be distinguished from previously reported E. coli

ribonucleases as follows. Its requirement for monovalent and divalent cations enables us to distinguish it from RNase I (3) or Rn’ase IV (6). Purified p factor (12) also had no RNase P- like activit,y. The specific endonucleolytic mode of action of RNase I’ rules out its identity with the major exonucleolytic ac- tivity associated with F. coli RNase II (4, 19, 21) or with poly- nucleotide phosphorylase (8). In order to test whether RNase III of E. coli is also responsible for the RNase P activity, we in- cubated an excess of highly purified RNase III (11) with the tyrosine tRNA precursor mlder optimal conditions for both enzymes (which are almost exactly the same). We found that RNase III has no effect on the electrophoretic mobility of the tRNA precursor.

An RNase activity associated with ribosomes which has been called RNase V (7) has been identified with an activity which degrades messenger RKA’s in an exonucleolytic fashion from their 5’ ends. This process apparently depends upon the state of activity of the ribosomes, and our RNase P preparations do not’ appear to have such an acbivity.

Several investigators have reported t,hat subcellular fractions of E. coli can cleave the 17 S precursor to 16 S ribosomal RNA (22, 23). In particular, Corte et al. (23) have described an activ- ity present in RKase II preparations which carries out such a re-

action. However, the endonucleolytic step of this reaction dis- plays different ionic requirements from those we have described for RNase P.2 It would be of interest t,o learn whether RIYase P can also carry out cleavage of 17 S ribosomal RXA precursors.

We conclude that RNase P is a novel RNase activity of E. coli. This enzyme may cleave all l?. coli tRNA precursors or only the one for tyrosine tRSA. The choice of one of these alternat’ives will await the isolation of additional E. coli tRNA precursors.

The subcellular fractionation of RNase P shown in Fig. 1 dem

and also renders it soluble in .5c/; trichloroacetic acid as deter- 2 D. Schlessinger, personal communication.

by guest on June 18, 2018http://w

ww

.jbc.org/D

ownloaded from

onstrates that processing of the two ends of the tRNA pre- cursor is carried out by different enzymatic activities. This find- ing is confirmed by the fingerprinting data shown in Fig. 4. The results shown in Fig. 1 also show that RNase P shares with sev- eral other important E. coli proteins the property of loose associa- tion with ribosomes within crude extracts capable of biologically important reactions. For example, RNase III (11) and the various factors required for the initiation of protein synthesis (24) show behavior very similar to that of RNase P with regard to their removal from ribosomes by NH&l. While association with ribosomes in estracts does not necessarily mean that these enzymes and factors are located upon these particles in viuo, analysis of the behavior of the other E. coli RNases during such gentle subcellular fractionation as that described here might re- veal other imeresting functional associations within the cell.

The behavior of RNase P upon ammonium sulfate fractiona- tion (Fig. 2) and chromatography upon DEAE-Sephadex (Fig. 3) and phosphocellulose is different from that of the bulk of E. coli proteins and has greatly facilitated our purification of this enzyme. In light of these properties, it is possible that the active form of RNase P, which must have a strong negative charge, could be associated with some nucleic acid.

In light of the specific cleavage of a single phosphodiester bond within the 129 nucleotide tRNA precursor, in contrast to the behavior of other RNases, we conclude that RXase P is the first, ribonuclease to be described which has such a high degree of specificity. Several examples of specific DNases have been reported (25-27). The fact that RNase P creates a 5’ phos- phate end group also makes it unique among E. coli endonucle- olytic RNases so far characterized. Altman and Smith (2) sug- gested on the basis of studying various mutated tyrosine tRKA precursors that baxe changes both near and far away from the point of cleavage may affect the rate of cleavage. Those studies as well as the ones reported here suggest that RNA secondary and tertiary structure may be as important as sequence in the action of RNase P. -4n assessment of the relative importance of these substrate properties in determining RNase P specificity could also be important in a more general study of RNA to protein inter- actions.

Although we have only performed a few experiments on the additional RXase activities present in 0.5 M NH&l ribosomal washes (Figs. 7 and 8), their results are worthy of some comment. From their studies of mutant tRNA’s, Altman and Smith (2) proposed the existence of a degradative pathway for tRNA pre- cursor other than the one leading to mature tRNA. RNase activity present in 0.5 M NH&l ribosomal wash fractions which can degrade tyrosine tRNA precursor but not mature tRNA (Figs. 7 and 8) may correspond to this proposed scavenger en- zyme. In addition, the activity or activities responsible for degrading the 5’.terminal fragment of the tyrosine tRNA pre- cursor in less pure RNase P fractions, as well as that which proc

5251

esses the 3’ end of the precursor molecule (see Figs. 1 and 2), may also be present in this 0.5 M NH&l wash. An interesting feature of this RNase activity is its latent nature. The existence of such latent RNases associated with ribosomes might help to account for reports of R1Jase activities which are dependent upon ribo- somal configuration or protein synthetic activity (7).

Finally, we conclude that the use of natural substrates such as tRNA precursors as one aspect of the rigorous characterization of E. coli ribonucleases should be instrumental in revealing new aspects of the regulation of RNA metabolism.

Acknowledgments-We thank Mr. ‘I‘. V. Smith and Miss E. Higgins for expert technical assistance.

1. 2. 3.

ALTM.\N, S. (1971) Nature New Biol. 229, 19 ALTMAN, S., AND SMITH, J. D. (1971) Nature New Biol. 233, 35 SP~HR, P. F., IND HOLLINGWORTH, B. 12. (1961) J. Biol. Chem.

236, 823-831 4. SPAHIZ, P. F., AND SCHLESSINGER, D. (1963) J. Biol. Chem. 238,

PC2251-PC2253 5. RORERTSON, H. D., WEHSTER, R. YE., AND ZINDER, N. D. (1967)

Virology 32, 718 6.

7.

SP.ZHIZ, P. F., AND GESTEL.YND, R. F. (1968) Proc. *Vat. Acad. Sci. U. S. A. 69, 876

Ku~~No, M., KWAN, C. N., APIRION, I>., .\ND SCHLESSINGIX,

8. D. (1969) &x. Nat. Acad. Sci. U.‘S. A. 64, 693

LITTXUER. U. Z.. .IND KORNBERG. A. (1957) J. Biol. Chem. 226.

9. 1077

PRIM.\I<OFF, P., ANI) BJ~~RG, P. (1970) Cold Spring Harbor Swm. Quant. Biol. 36. 391

10.

Il.

SMX’I’H~ J.-D., BIIRNETT,’ L., BILICNNER, S., AND RUSSELI,, 11. I,. (1970) J. itfol. Biol. 64, l-14

ROEERTSON, H. D., W~~SI~JXI, R. E., a~~I) ZINDJ<:R, N. D. (1968) J. Biol. Chem. 243, 82-91

12. ROBI~TS, J. W. (1969) Nature 224, 1168 13. DAHI,BERG, J. E. (1968) Zature 220, 548 14. PIECZENIK, G., B~I~RELL, B. G., .~ND GEIVJCR, M. L. (1972) Arch.

15. Biochem. Bibphys., in press

SANGNR. F.. BROWNLEJS. (+. cf.. AND B.z~t~u:r,r,. B. G. (1965)

16. J. Mo’l. B;ol. 13, 373-398 '

~

BRO\VNLI,:P, G. G., S.YNGJ’X, F., .YND B.\ILI~I~LL, B. G. (l!K%) J. Mol. Biol. 34, 379-412

17.

18.

19. 20. 21. 22. 23.

24.

25. 26.

GOOUM.~N, H. M., AEELSON, J., L.ZNDY, A., %.~I)J~.\zIL, S., LSD SMITH, J. D. (1970) Eur. J. B&hem. 13, 461

Lo\\-RY, 0. H., ROSEIXIoUGr-l, N. J., FARI~, A. L., .‘.NII I:.\NI).\LI,, 1:. J. (1951) J. Biol. Chem. 193, 265-275

SP.IHIL, I?. F. (1964) J. Biol. Chem. 239, 3716 SUMNI~X, J. B., BND GRALEN, N. J. (1938) J. Biol. Chem. 126,33 SINGJZIZ, M., .\SD TOLLIICILT, G. (1965) Biochemistry 4, 1319 YUICI, A. (1971) J. Mol. Biol. 62, 321 COI~TE, G., SCHLFXSINGER, D., LONGO, I>., .\ND VENE(U\-, P.

(1971) J. lV!ol. Biol. 60, 325-338 STBNLEY, W. M., JR., S.i~as, M., WAHIU, A. J., .IND OCHO~, H.

(1966) Proc. Nut. Acad. Sci. U. S. A. 66, 290 ~LIICSIZLSON, M. S., .%ND YUAN, R. (1968) Nature 217, 1110 KELLY, T. J., JR., .\ND SMIT~I, H. 0. (1970) J. Mol. Rio!. 61,

393-409 27. LINK, S., .\XD ARBER, W. (1968) hoc. Xat. Acad. Sci. U. S. A.

69, 1300

REFERENCES

by guest on June 18, 2018http://w

ww

.jbc.org/D

ownloaded from

Hugh D. Robertson, Sidney Altman and John D. SmithCleaves a Tyrosine Transfer Ribonucleic Acid Precursor

Ribonuclease whichEscherichia coliPurification and Properties of a Specific

1972, 247:5243-5251.J. Biol. Chem. 

  http://www.jbc.org/content/247/16/5243Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/247/16/5243.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on June 18, 2018http://w

ww

.jbc.org/D

ownloaded from