THY JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 250, No. 16,Issue of August25, PP. 6248-6255,1975

Printed in U.S.A.

In Vitro Synthesis of Transfer RNA

II. IDENTIFICATION OF REQUIRED ENZYMATIC ACTIVITIES*

(Received for publication, January 27, 1975)

ELIZABETH K. BIKOFF'$

From the Department of Biological Xciences, Columbia University, New York, New York 10027

BERNARD F. LARUE! AND MALCOLM L. GEFTER

From the Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

SUMMARY

We have shown that the synthesis of active s& tRNATyr from a +8Ops&1 DNA template requires the action of four distinct enzymatic activities. The first of these, DNA-de- pendent RNA polymerase, catalyzes the formation of a large molecular weight transcript, initiating synthesis at a specific site 41 nucleotides proximal to the 5’ end of the s&r tRNATyr structural gene and continuing at least 100 nucleotides be- yond the 3’ terminus of the S&I tRNATyr sequence. The second required component, designated Fraction V, allows purified DNA-dependent RNA polymerase to function in tRNA synthesis. We have shown that this fraction contains an endonuclease that together with DNA-dependent RNA polymerase is responsible for the synthesis of s& tRNATyr “precursor.” Thus, S&I tRNATyr precursor is not itself the primary transcription product of the S&I tRNATyr gene, but rather, it arises as a result of post-transcriptional cleavage of a much larger transcript by the action of the nuclease present in Fraction V. The third enzymatic activity required for synthesis of active S&I tRNATy’ is a ribonuclease (RNase P III) that specifically catalyzes the removal of the 3’ extra nucleotides from the s&r tRNATyr precursor. The fourth activity required for synthesis of tRNA is a previously identi- fied endonuclease, RNase P, that specifically catalyzes the removal of the 5’ extra nucleotides from tRNA precursors. The properties of RNase P purified according to the pro- cedure developed in this laboratory have been compared with those of the enzyme purified from ribosomes according to the procedure described by Robertson et al. (ROBERTSON, H. D., ALTMAN, S.,AND SMITH, F. D. (1972) J. Biol. Chem.

247, 5243-5251.)

Post-transcriptional cleavage and modification of larger mo- lecular weight precursors appears to be a general cellular mecha-

* This work was supported in part by Grants NPGB and NPGC from the American Cancer Society.

$ Supported by United States Public Health Service Grant in Developmental Biology 5.TOl-GM0287-04. Present address, On- tario Cancer Institute, 500 Sherbourne Street, Toronto, Ontario M4K1, Canada.

$ Supported by the Medical Research Council of Canada.

nism for the production of RNA. Such processing has been clearly demonstrated for tRNAs in mammalian cells (1, 2) and in Esche- richia coli (3-5), for ribosomal RNA in mammalian cells (6), and in E. coli (7,8), and for messenger RXAs of the bacteriophage T7 (8, 9).

In the case of tRNA biosynthesis in E. coli, such processing is known to involve the removal of extra nucleotides at both the 3’ and 5’ ends of these precursors (3-5). These large RSA species were identified as tRNA precursors on the basis (a) that within the larger molecule was included the nucleotide sequence for a mature tRNA species, and (b) that in vitro nucleolytic cleav- age of the larger molecule with extracts of E. coli resulted in the appearance of the mature 4 8 tRNA. Thus, the conversion of precursor size RNA to 4 S tRNd has been utilized as a means of measuring post-transcriptional cleavage. 011 this basis, an endonuclease (RSase 1’) has been purified from the ribosomes of E. coli which is responsible for cleavage of tRXA precursors at. their 5’ ends (10). The essential role of RXase 1’ in tRNA biosynthesis was conclusively demonstrated by the isolation of conditional lethal mutants of E. coli that contain a temperature- sensitive RNase 1 and that also fail to synthesize mature tRNA at the nonpermissive temperature (11).

Conditional lethal mutants have also been isolated that were defective in the activity responsible for maturation of tRNA pre- cursors at the 3’ ends (11). Extracts prepared from these cells appeared to have low levels of activity of a previously identified exonuclease, RNasc II (12). Therefore, it was suggested that the enzyme that catalyzes the removal of the 3’ extra nucleotides is RNase II (11). However, temperature sensitivity of RNase II purified from these cells has not been demonstrated.

We have described a system for the in vitro synthesis of active S&I tRXATyr from +8Ops& DNA (13). Using this system, we have identified four essential components required for tRNA synthesis: (a) DNA-dependent RXA polymerase, (b) a fraction, designated Fraction V, that allows purified RX’;1 polgmerase to function in tRi\;A synthesis, (c) a ribonucleasc that specifically catalyzes the removal of the 3’ extra nucleotides from tRSA precursors, and (d) a fraction containing RNase I-‘.

We IIOW present evidence that Fraction V is distinct from the termination factor, p (14), and other heretofore described en- zymes, and similarly, RKase P III is distinct from other previ- ously described activities.

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EXPERIMENTAL PROCEDURE Singer and Tolbert (12) utilizing [3H]p~ly(U) as substrate (spe- cific activity 106 cpm/pmol) and measuring the release of soluble

Materials nucleotide. Transfer RNA nucleotidyltransferase activity was determined

Bacterial and Bacteriophage Strains-The bacterial and bacteri- according to the procedure described bv Miller and Philinos (17) ophage strains have been described elsewhere (15).

Chemicals-All chemicals and reagents needed for tRNA syn- thesis have been described previously (13).

&VA-The su&, tRNATYr precursor, uniformly labeled with 32P, was purified from &?Opsu&i-infected Escherichia coli A49 by the procedure described by Altman and Smith (3). Yeast tRNA was purchased from Schwarz/Mann.

Enzymes-Electrophoretically purified pancreatic DNase was obtained from WorthingtonBiochemical Corp. Tyrosyl-tRNAsyn- thetase was purified from E. coli 514 by aslight modification of the procedure described by Calendar and Berg (16). The final specific activity of the enzyme was 820 units/mg.

RNase II was prepared from E. coli MRE600 according to the method described by Singer and Tolbert (12). The final specific activity of the enzyme was 1500 units/mg.

RNase P was purified from E. co/i 514 according to the procedure of Robertson et al. (10). The enzyme was concentrated before use by dialysis for 8 hours at 4’ against Buffer III containing 30% (w/v) polyethylene glycol.

Transfer RNA nuileotidyltransferase purified according to the method described bv Miller and PhiliDas (17) was the kind gift of Dr. Tom Fraser of the Massachusetts Institute of Technology. The -. specific activity of the enzyme was 200 units/mg.

DNA-deoendent RNA nolvmerase was uurified from E. coli 514 by a slight’ modification hf “the procedure described by Burgess (18). The specific activity of the enzyme after the second glycerol gradient was 730 units/mg.

p factor purified according to the procedure described by Rob- erts (14) was the kind gift of Dr. Russ Maurer.

Bu$ers-Buffer II contained 10 InM Tris-acetate (pH 8.2), 14 mM magnesium acetate, 60 mM KCl, and 1 mM dithiothreitol. Buffer IV was the same as Buffer II, but containing 570 (v/v) glycerol. Buffer III contained 20 mizl Tris-HCI (pH 7.4), 10 m&l KCI, 1 mM magnesium acetate, 2 mM dithiothreitol and 10% (v/v) glycerol.

Methods

Protein Determinaliolz-In most cases, protein was determined by the method of Bucher, using crystalline bovine serum albumin as standard (19). However, when the protein concentration was less than 250 pg/ml, the protein was measured by the procedure of Lowry et al. (20).

Aciylamide Gel Electrophoresis--Electrophoresis was carried out on 10% polyacrylamide slab gels (20 X 30 X 0.2 cm) in a con- tinuous buffer svstem containing per liter: 10.8 g of Tris base, 0.93 g of disodium EDTA, and 5.5-g of boric acid-(final pH, 8.3)

_. I

utilizing-yeast tRNA as substrate and measuring the incorpora- tion of [3H]ATP into acid-insoluble material.

Post-transcriptional Cleavage Reactions-Reaction mixtures were incubated for 1 hour at 35” with gentle agitation in a rotary shaking water bath. Each reaction contained in a final volume of 0.5 ml: 40 mnl Tris-acetate (pH 8.2), 2 mM dithiothreitol, 50 mM potassium acetate, 25 mM ammonium acetate, 2 rnhl ATP, 0.18 mM GTP, 0.5 mM CTP, 0.5 rnhf UTP, 0.5 mM cyclic 3’:5’-AMP, 21 mM sodium phosphoenolpyruvate, 29 mM magnesium acetate, 0.1 mM isopentenyl pyrophosphate, [32P]RNA (10’ cpm), and the appropriate amount of the particular enzyme fraction to be tested for post-transcriptional cleavage activity. At the end of the incu- bation, an equal volume (0.5 ml) of redistilled, freshly neutralized phenol was added. and isolation of the RNA nroducts was carried but as described under “Preparation of Active Fractions of tRNA Synthesis.”

Preparation of Active Fractions of tRNA Synthesis-The puri- fication of the four essential components required for the in vitro synthesis of active s&r tRNA Tyr has been described in detail elsewhere (13). A brief summary is presented below.

Extracts were prepared from E. coli 514 by a slight modification of the procedure described by Zubay et al. (22). Ribosomes were removed by centrifugation. The clear supernatant constitutes the SlOO. The extract was adjusted to 0.4 M KC1 and passed over a DEAE-cellulose column to yield the nucleic acid-free SIOO.

Ammonium sulfate fractionation of the nucleic acid-free SlOO yielded two active fractions. Fraction I (the 30 to 50yo precipitate) contained 90 to 95% of the DNA-denendent RNA oolvmerase 1 i activity. Fraction II‘(the 50 to 85yo precipitate) contained RNase P and RNase P III. Further purification of Fraction I was carried out by chromatography on DEAE-cellulose to yield Fraction IA. Fraction IA (containing RNA polymerase) was then applied to a second DEAE-cellulose column. The DNA-dependent RNA polymerase recovered is Fraction IB. Alternatively, DNA-de- pendent RNA nolvmerase was nurified from Fraction IA bv rrlvc- I Y” Errol gradient centrifugation. Fractions having a specific activity of greater than 600 units/mg were pooled to yield Fraction IC. Fractions from the upper part of the gradient were pooled to yield Fraction V.

RNase P and RNase P III were purified from Fraction II by chromatography on DEAE-cellulose. The column was washed with Buffer III containing 0.35 M KCl. Fractions containing an Azso of greater than 1.0 were pooled to yield Fraction III (RNase P III). The column was then washed with Buffer III containing 0.8 M KCI. Fractions having an A zaO greater than 1.0 were pooled to yield Fraction IV (RNase P).

removed by centrcfugation at 10,000 rpm for 30 min. To remove traces of acrylamide, the RNA was adsorbed to a DEAE-cellulose column (0.4 X 2 cm) previously equilibrated with a buffer con-

for 12 hours at 400 volts and 25”. The position of the radioactive

taining 10 mM NaCl, 10 mM MgCl%, 10 mM Tris-HCl (pH 7.4), and 0.5 mM 2-mercaptoethanol. The column was washed with 2 ml of

RNA bands in the gels was determined by autoradiography. Indi-

the same buffer. Then the RNA was eluted with this same buffer containing 1 M NaCl. Poly(U) was added as carrier at a final con-

vidual RNA species were recovered from the gel as follows: the

centration of 50 pg/ml. The RNA was then precipitated by the addition of 0.25 volume of sodium acetate (pH 5) and 2.5 volumes

portion of the gel containing the radioactive band was cut out and

of absolute ethanol.

mechanically homogenized in a buffer containing 10 mM NaCl, 10 mM Tris-HCl (pH 7.4), and 0.1 mM EDTA. Acrylamide was

Fraction III was then applied to and eluted from a second DEAE-cellulose column. The RNase P III thus obtained consti- tutes Fraction IIIA.

Fraction IV was purified on a second DEAE-cellulose column to yield Fraction IVA. Fraction IVA was then applied to a Sephadex G-200 column. The column was washed with Buffer III. The peak of activity was found to elute just behind the void volume (Frac- tion IVB j .

In summary, Fractions IA, IB, and IC contain the DNA-de- pendent RNA polymerase activity at increasing stages of purifica- tion. Fraction V contains an enzymatic activity that allows puri- fied RNA polymerase to function in tRNA synthesis. Fraction II contains both RNase P III and RNase P activitv. Fractions III and IIIA contain RNase P III activity at increasing stages of purification. Fractions IV, IVA, and IVB contain RNase P ac- tivity at increasing stages of purification.

Enzyme Assays-DNA-dependent RNA polymerase activity was determined under the conditions described by Burgess (18) utilizing calf thymus DNA as template and measuring the incorpo- ration of [3H]UTP into acid-precipitable material.

Tyrosine acceptance activity of s&i tRNArVr was determined under the conditions described by Gefter and Russell (21) utilizing purified tyrosyl-tRNA synthetase and measuring the incorpora- tion of [3H]tyrosine into acid-precipitable material.

RNase II activity was determined by the method described by

RESULTS

Four Essential Components for tRNA Synthesis-We have previously reported that synthesis of active S&I tRNATyr re- quires four essential components (13). These results are sum- marized in Table I. As shown in Part A, in addition to Fraction IA (partially purified DNA-dependent RNA polymerase), in vitro synthesis of tRNA is completely dependent upon the addi-

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tion of both Fraction III and Fraction IV. Fraction III contains a ribonuclease (RNase I’ III) that specifically catalyzes the re- moval of the 3’ extra nucleotides from tRNA precursors. Frac- tion IV contains RNase P, the endonuclease that is responsible for the cleavage of the 5’ extra nucleotides from tRNA precur- sors (13).

As shown in Part B of Table I, Fraction IC RNA polymerase or RNA polymerase purified according to the procedure de- scribed by Burgess (18) was unable to replace the requirement for Fraction IA in the in vitro system. On this basis, a fourth es- sential component for tRNA synthesis, designated Fraction V, has been identified (13).

Requirement for RNase P-The identification of Fraction IV as an essential component for tRNA synthesis and its subsequent purification were based solely on the observed activity of this fraction in the in vitro system. However, when assayed directly on the 32P-labeled su& tRNATyr precursor prepared from @Opsu&r-infected Escherichia coli according to the procedure described by Altman and Smith (3), Fraction IV was found to contain high levels of RNase P activity. It was similarly deter-

mined that the RNase I’ activity had been quantitatively re- covered in Fraction IVA and Fraction IVB. As shown in Fig. 1, RNase P purified according to the procedure described by Robert- son et al. (10) was able to replace the requirement for Fraction IV in the complete system. It is therefore concluded that the esserl- tial enzyme for tRNA synthesis provided by Fraction IV is solely RNase P.

In these experiments, the amount of S&I tRNATyp synthe- sized was quantitated by measuring the amount of radioactivity that appeared in the 4 S tRNA band when the products of the in vitro synthesis reactions were analyzed by acrylamide gel elec- trophoresis. This was necessary because of the presence of large amounts of potentially chargeable tRNA in the preparation of RNase P that had been purified according to Robertson et al. (10) .I

We have arbitrarily defined 1 unit of RNase I’ activity as that

amount of enzyme that, in the presence of an excess of the other required components, supports the synthesis of 1 pmol of su&r tRNATyr. On this basis, the specific activity of RNase P purified according to the procedure described by Robertson et al. (10) is

the same as that of Fraction IV. However, further purification

1 Measurements of the ratio of the A26,, to the As80 indicated that RNase P aurified according to the nrocedure described bv Robert- son et al.* (10) contained Urge amounts of nucleic acid”; e.g. the ratio A260/A280 was 1.8. As judged by the failure to find acceptor activity for three different amino acids (valine, glutamic acid, and tyrosine), there was no mature tRNA present. However, treat- ment of the RNA with tRNA nucleotidyltransferase resulted in the appearance of large amounts of amino acid acceptor activity (up to 5 pmol/Anso for tyrosine, 6 pmol/A2so for valine, and 5 pmol/ A2G0 for glutamic acid). Moreover, when incubated with tRNA nucleotidyltransferase, this RNA was found to incorporate [SH]- ATP at a level of 1200 pmol/A260. We therefore conclude that at least 75’$& of the RNA that co-purified with RNase P is material with the structure tltNA-X-C-Con. This conclusion was supported by direct sequence analysis: RNase P purified from @Opsu&I- infected cells was found to contain large amounts of SLI&I tRNATy1.. C-COB. However, there was no evidence for the formation of a complex between RNase P and this RNA. As determined bv su- crose gradient centrifugation under a variety of ionic conditions, the enzyme and t,he RNA did not co-sediment. Therefore, it is un- likely that their co-purification results from a physiological associ- ation between RNase P and this RNA.

2 The appearance of small amount,s of mature 4 S tRNA as a product of in vitro synthesis reactions containing Fraction IA polymerase and Fraction IVB RNase P (Columns S and 4) resulted from the presence in Fraction IA of low levels of contaminating RNase P III activitv. When the DNA-dependent RNA polvmerase activity was further purified by chromatography on a second DEAE-cellulose column to vield Fraction IB nolvmerase. this fraction together with Fraction IVB RNase P iynthesized’ only pre-tRNAn. The synthesis of mature 4 S SU~I tRNATyr was com- pletely dependent upon the addition of Fraction III.

Several partially purified fractions required for in vitro synthesis of tRNA were found to be contaminated with tRNA nucleotidvl- I transferase. Therefore, the use of aminoacvl-tRNA svnthetase reactions as a means oi quantitating de nova tRNA synthesis was prohibited when RNase P purified according to the procedure de- scribed by Robertson et al. (10) was substituted for Fraction IV.

T.WLF: I Four essential components for tRiVA synthesis

In vitro synthesis reactions were carried out as described under “Experimental Procedure,” and the amount of SU$I tRNATyr synthesized was determined by its tyrosine acceptance activity. RNA polymerase was purified according to the procedure de- scribed by Burgess (18).

Additions

A. Complete system” -IA -111 -IV

sufiI tRNATY’ synthesized

- pm01

2.5 <O.l

0.2 <O.l

B. Complete system* -IA + IC -IA + RNA polymerase +v -IA + IC + V -IA + RNA polymerase + V

2.2 0.3

<O.l 1.0 0.9 0.7

Q The complete system contained 0.8 mg of Fraction IA, 2.8 mg of Fraction III, and 0.1 mg of Fraction IV.

b The complete system contained 0.8 mg of Fraction IA, 2.8 mg of Fraction III, and 0.1 mg of Fraction IV. All reactions con- tained 120 units of l>NA-dependent RNA polymerase activity. Where indicated, 160 pg of Fraction IC or 100 /Ig of purified RNA polymerase were added in place of Fraction IA. In addition, where indicated, reactions also contained 0.1 mg of Fraction V.

of Fraction IV by DEAE-cellulose chromatography to yield Fraction IVA, followed by gel filtration on Sephadex G-200 to yield Fraction IVB, has increased the specific activity of the RNase 1’ in these fractions 50.fold and loo-fold, respectively (relative to Fraction IV). Th e yield of RNase I’ activity purified from the 100,000 X g supernatant fraction according to the pro- cedure described above is significantly greater than that obtained from ribosomes according to the procedure described by Robert- son et al. (10). Furthermore, Fractions IVA and IVB are free of detectable nonspecific nuclease activity. ils shown in Fig. 4, prolonged incubation of pre-tRNA, with Fraction IV13 resulted in the specific cleavage of this RNA, and equivalent amounts of the pre-tRNAz and the 5’ fragment are the only hydrolysis products.

Identification of RNase P III Activity-As shown in Fig. 2, in the absence of Fraction III, the in vitro synthesized RNA was found to contain significant amounts of a species designated pre- tRNA2, which migrated slightly slower than the mature 4 S suhr tRNATy’. It has previously been demonstrated by sequence analysis that this RNA species is S&I tRNATyr containing extra nucleotides beyond the -CCAon3’ terminus of the mature tRNA (13). Addition of Fraction III to the in vitro synthesis reactions resulted in the disappearance of pre-tRNA2 and the appearance of mature 4 S su;‘Ir tRNATyr (Fig. 2). Therefore, the essential function provided by Fraction III is the removal of the 3’ extra

nucleotides from the tRKA precursor.*

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FIG. 1 (left). Identification and purification of RNase P ac- tivity. In vitro synthesis reactions and acrylamide gel electro- phoresis of the RNA products were carried out as described under “Experimental Procedure.” All reactions contained [Q-~~P]GTP at a final specific activity of 100 mCi/mmol, 0.2 mg of Fraction IA polymerase, and 0.7 mg of Fraction III (RNase P III). In addi- tion, where indicated, either 0.4 mg of Fraction II, 25 pg of Frac- tion II, 25 pg of Fraction IV, 1 pg of Fraction IVA, or RNase P purified according to the procedure described by Robertson et al. (10) was added as the source of RNase P activity.

RNase P III and RNase II Are Distinct Activities-It has been suggested that the enzyme that catalyzes the removal of the 3’ extra nucleotides from tRNA precursors is RNase II (11). We have also found that RNase II is capable of catalyzing the re- moval of the 3’ extra nucleotides from su&r tRNA precursor previously treated with RNase P. However, at enzyme concen- trations as high as 18 units/ml, onlv 30 ‘% of the RNase P-treated s&r tRNATy’ precursor was found to contain the -CCAon3’ terminus of the mature 4 S species, while 70% of the material retained the 3’ extra nucleotides. In contrast, quantitative re- moval of the 3’ extra nucleotides was achieved upon the addition of crude preparations of RNase P III (Fraction III) contaminated with RNase II activity at a level of 6 units/ml. These results suggest that the activity in Fraction III that is responsible for the hydrolysis of the 3’ extra nucleotides is distinct from RNase II.

As shown in Fig. 3, separation of these two activities was sub- sequently achieved by chromatography on DEAE-cellulose. The

FIG. 2 (right). Requirement for RNase P III for synthesis of 4 S S&I tRNArYr. In vitro synthesis reactions and acrylamide gel electrophoresis of the RNA products were carried out as described under “Experimental Procedure.” All reactions contained [CZ- 32P]GTP at a final specific activity of 100 mCi/mmol and 0.2 mg of Fraction IA polymerase. In addition, where indicated, reactions also included 0.4 mg of Fraction II (2), 1 or 2 fig of Fraction IVB (RNase P); reactions in Columns 5 and 6 also included 0.7 mg of Fraction III (RNase P III).

RNase P III was found to elute at a lower salt concentration than RNase II. Moreover, RNase II was unable to replace the requirement for RNase P III for synthesis of mature s&r tRNATyr in the in vitro system (Table II). We therefore conclude that RNase P III and RNase II are distinct enzymatic activities.

RNase P III Is Distinct from tRNA Nucleotidyltransjerase- Crude preparations of RNase P III (Fraction III) were found to contain high levels of tRNA nucleotidyltransferase activity. In addition, as shown in Fig. 3, the peak of RNase P III activity was found to be coincident with the peak of tRNA nucleotidyl- transferase activity when elution profiles of these enzymes on DEAE-cellulose were compared. For this reason, the possibility that these two enzyme activities were associated with the same protein molecule was considered.

As shown in Table III, tRNA nucleotidyltransferase purified according to the procedure described by Miller and Philipps (17) was unable to replace the requirement for RNase P III in the in vitro system. This result was confirmed by acrylamide gel elec-

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8 -160

2 22 -140

is

0 IO 20 30 40 50 60

Fraction No.

FIG. 3. DEAE-chromatography of RNase P III. DEAE-chro- matography of Fraction III (RNase P III) was carried out as previously described (13). RNase III (O-O), tRNA nucleo- tidyltransferase (O-O), and RNase P III ( t - 1 ) activities of each fraction were determined as described under “Experi- mental Procedure.”

TABLE II

RNase P III is distinct jrom RNase II

Each in vitro synthesis reaction contained 0.8 mg of Fraction IA polymerase and 0.1 mg of Fraction IV RNase P. In addition, where indicated, reactions also included either 2.8 mg of Fraction III, 0.6 mg of Fraction IIIA, or purified RNase II. RNase II was puri- fied according to the procedure described by Singer and Tolbert (12)) and RNase II activity was determined by the release of solu- ble nucleotide utilizing [3H]poly(U) as substrate (see “Experi- mental Procedure”). In vitro synthesis reactions were carried out as described under “Experimental Procedure,” and the amount of su& tRNATyr synthesized was determined by its tyrosine ac- ceptance activity.

Addition

I

None Fraction III. Fraction IIIA. . RNase II.. .

RNase II activity s+ tRNATY’ (units added) synthesized

13 <.05 15

pm01

<0.2 2.8 1.6

<0.2

trophoresis; purified tRNA nucleotidyltransferase did not sup- port the conversion of pre-tRNAz to mature 4 S s&r t.RNATrr. Moreover, tRNA nucleotidyltransferase further purified from Fraction IIIA by chromatography on hydroxylapatite did not contain measurable RNase P III activity. In addition, the two activities had different rates of heat inactivation (data not shown). Therefore, we conclude that RNase P III and tRNA nucleotidyltransferase are distinct enzymatic activities.

Post-transcriptional Cleavage of S&I tRNATg’ Precursor-As shown in Column 1 of Fig. 2, transcription of @Opsu&r DNA by Fraction IA polymerase resulted in the appearance of an RNA species designated pre-tRNAi. This RNA has previously been shown to be a mixture of two RNA species: s&r tRNATyr pre- cursor and a @O-specified RNA of unknown function (13). As shown in Columns 5 and 6 of Fig. 4, separate incubation of the total RNA transcription product of Fraction IA polymerase with Fraction IVB (RNase P) resulted in the disappearance of

TABLE III RNase P III is distinct from tRNA nucleotidyltransjerase

Each in vitro synthesis reaction contained 0.8 mg of Fraction IA polymerase and 0.1 mg of Fraction IV RNase P. In addition, where indicated, reactions also included either 2.8 mg of Fraction III, 0.6 mg of Fraction IIIA, or purified tRNA nucleotidyltransferase.

tRNA nucleotidyltransferase was purified according to the procedure described by Miller and Philipps (17) and tRNA nucleo- tidyltransferase activity was determined by incorporation of [3H]ATP into acid-insoluble material utilizing yeast tRNA as substrate (see “Experimental Procedure”). In vitro synthesis reactions were carried out as described under “Experimental Procedure,” and the amount of s&r tRNATyr synthesized was determined by its tyrosine acceptance activity.

Addition tRNA nucleotidyl-

transferase activity (units added)

None. Fraction III Fraction IIIA tRNA nucleotidyltransferase

4.8 4.6 5.0

SU&~ tRNATYr

synthesized

pm01

co.2 2.8 1.6

<0.2

- m

FIG. 4. Post-transcriptional cleavage of in vitro synthesized SU&I tRNATYr precursor. In vitro synthesis reactions containing 0.2 mg of Fraction IA polymerase and 100 mCi/mmol of [oI-~~P]- GTP were carried out as described under “Experimental Pro- cedure.” A portion of the total RNA product remained untreated, and the remainder was analyzed by acrylamide gel electrophoresis. The position of the pre-tRNAl band was determined by autoradi- ography and this RNA species was recovered from the gel as de- scribed under “Experimental Procedure.” Next, post-transcrip- tional cleavage reactions were carried out as described under “Experimental Procedure.” Reactions shown in Columns 1 to 4 cont,ained 104 cpm of the pre-tRNA, species. Reactions shown in Columns 5 and 6 contained 105 cpm of the original total RNA prod- uct. In addition, where indicated, post-transcriptional cleavage reactions also contained Fraction IVB RNase P (150 pg/ml). The reaction shown in Column 7 is a control in which 0.4 mg of Frac- tion II was included with Fraction IA polymerase in the original in vitro synthesis reaction. The intense 4 S band in this panel shows the position of mature s&i tRNAryr.

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the pre-tRNAl band and in the appearance of pre-tRNAz (s&r tRNATyr with extra nucleotides at its 3’ end). Moreover, as shown in Columns 1 to 4, incubation of the isolated pre-tRNAl species with Fract,ions IVIS RNase 1’ yielded only two hydrolysis products: pre-tRNAz and the 41.residue 5’ fragment. The residue of material not degraded by Fraction IVB RNase P is the #30- specified RNA. When post-transcriptional cleavage reactions were similarly carried out with individual RNA transcription products of Fraction IL4 polymerase, no change in the mobility of these other RNA bands was observed. We therefore conclude that the pre-tRNAi species is the only @Ops& transcription product of Fraction IA polymerase that is specifically hydrolyzed by Fraction IVB (RNase 1’) to yield pre-tRKAz.

Enzymatic Activity of Fraction V-It has previously been re- ported that when the DNA-dependent RNA polymerase activity was further purified from Fraction IA by glycerol gradient cen- trifugation to yield Fraction IC polymerase, this latter fraction, together with Fraction III RNase P III and Fraction IV RNase P, was unable to support detectable levels of tRNA synthesis (13). However, tRXA synthet.ic activity was restored by t,he addition of a fourth essential component, designated Fract’ion V, which was separable from RNA polymerase by glycerol gradient centrifugation (13). This fraction was also able to reconstitute tRNA synthetic activity of in vitro synthesis reactions contain- ing DNA-dependent RNA polymerase purified according to the procedure described by Burgess (13, 18). These results are sum- marized in Table I.

The data presented in Fig. 5 demonstrate that the synthesis

of s&r tRNATy’ precursor by Fraction IC polymerase is de- pendent upon the addition of Fraction V. As shown in Columns 2 and 3, the s&r tRNATyr precursor (pre-tRNAl) band synthe- sized by Fraction IA polymerase was not observed as a trans- scription product of either Fraction IC polymerase or DNA- dependent RNA polymerase purified according to the procedure described by Burgess (18). Indeed, the RNA synthesized by Fraction IC polymerase or purified RNA polymerase was suffi- ciently large that greater than 90% of this RNA did not enter the 10% acrylamide gel. However, as shown in Columns 4 and 5, addition of Fraction V resulted in the appearance of pre-tRNAi. We therefore conclude that Fraction V together with DNA-de- pendent RNA polymerase is responsible for the synthesis of s& tRNATyr precursor.

The termination factor, p, purified according to the procedure described by Roberts (14)) was unable to replace the requirement for Fraction V; s&r tRNATyr precursor was not observed as a transcription product of in vitro synthesis reactions containing purified DNA-dependent RNA polymerase and purified p factor. Moreover, as shown in Columns 6 and 7, incubation of the total RNA transcription product of Fraction IC polymerase with Fraction V in post-transcriptional cleavage reactions resulted in the appearauce of su& tRNATyr precursor (pre-tRNAJ. Identi- cal results were obtained when Fraction V was incubated in post-transcriptional cleavage reactions with the total RNA transcription product of DNA-dependent RNA polymerase puri- fied according to the procedure described by Burgess (18) (data not shown). We therefore conclude that Fraction V contains a nucleolytic activity that is essential for the synthesis of s&r tRNATyr precursor. On the basis of these results, we also suggest that the s&r tRNATyr precursor molecule isolated in viva by Altman and Smith (3) and observed in this laboratory as the pre- tRNAl product of Fraction IA polymerase is not itself the pri- mary transcription product of the s&r tRNATyr gene. Rather, we suggest that it arises as a result of post-transcriptional cleav- age of a much larger molecular weight precursor molecule by the action of the nuclease present in Fraction V.

In conclusion, the data presented in Table I demonstrate that Fraction V contains an enzymatic activity that is essential for the synthesis of active s&r tRNATyr. The data presented in Fig. 5 demonstrate that Fraction V contains a nucleolytic ac- tivity that is required for the synthesis of s&r tRNATyr pre- cursor. Thus, the essential activity of Fraction V is the generation of the intermediate, s&r tRNATyr precursor, which is converted to mature suhr tRNATyr by the action of RNase P and RNase P III.

DISCUSSION

We have shown that the synthesis of active s&r tRNATyr from a 480psu&-DNA template requires the action of four distinct enzymatic activities. The first of these, DNA-dependent

FIG. 5. Fraction V together with RNA polymerase synthesizes RNA polymerase, catalyzes the formation of a large molecular

su,fr tRNATyr precursor. In vitro synthesis reactions were carried weight transcript, initiating synthesis at a specific site 41 nucleo-

out in the presence of 100 mCi/mmol Iol-32PlGTP as described under tides proximal to the 5’ end of the s&r tRNATyr structural gene “Experimental Procedure.” All reactions contained 30 units of and continuing at least 100 nucleotides beyond the 3’ terminus DNA-dependent RNA polymerase activity (either 200 pg of Frac- tion IA, 40pg of Fraction IC, or 25 rg of RNA polymerase purified

of the s&r tRNATyr sequence. The second required component

according to the procedure described by Burgess (18)). In adc’i- is an endonuclease that catalyzes the hydrolysis of this transcript

tion, reactions shown in Columns 4 and 5 also included 25 fig of to yield a smaller RNA molecule (s&r tRNATyr precursor), Fraction V. The RNA products were isolated by phenol extraction which contains, in addition to the s&r tRNATyr sequence, addi- and et,hanol precipitation. These RNA products were then incu- bated in post-transcriptional cleavage reactions as described under

tional nucleotides at both its 5’ and 3’ ends. The third enzymatic

“Experimental Procedure.” Reactions shown in Columns 1 to 5 activity required for synthesis of active s&r tRNATyr is a previ-

did not contain any other additions. Reactions shown in Columns ously identified endonuclease, RNase P, which catalyzes the re- 6 and 7 contained 25pg of Fraction V. moval of the 5’ extra nucleotides from tRNA precursors (10).

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6254

The fourth activity is RNase P III, a ribonuclease that specifi- cally catalyzes the removal of the 3’ extra nucleotides from the s&r tRNATyr precursor (13). These four enzymes have been purified on the basis of their required activities in the in vitro system for tRNA synthesis (13).

It has been reported that RNase P is associated with the ribo- somes (10). Indeed, we have also found that when extracts were prepared according to the procedure described by Robertson et al. (lo), and cell components were separated by differential centrifugation, up to 70% of the RNase P activity remained with the ribosomes. However, when extracts were prepared according to the procedure described by Zubay et al. (22) RNase P activity was quantitatively retained in the supernatant fraction. The in- tegrity of the ribosomes in these extracts is indicated by their ability to support protein synthesis in a DNA-directed cell-free system. We therefore conclude that the distribution of RNase P activity between the ribosomes and the supernatant fract.ion de- pends on the method used to prepare the extracts, and that the previously observed association of RNase P activity with ribo- somes does not necessarily reflect in vivo compartmentalization of enzyme activity.

When purified from ribosomes, RNase P was found to contain large amounts of nucleic acid. We have shown that at least 75% of this material is RNA with the structure tRNA-X-C-Con. However, as determined by sucrose gradient centrifugation, there was no evidence for the formation of a complex between RNase P and this RNA. Therefore, it is unlikely that their co-purification reflects any physiological association.

It was suggested by Robertson et al. (10) that the unusual affinity of RNase P for DEAE-Sephadex (the enzyme was eluted at a salt concentration of 0.42 M NH,Cl) was a reflection of its association with nucleic acid. However, quantitative removal of nucleic acid was achieved at a very early stage of the purification procedure developed in this laboratory. We have also found that RNase P interacts strongly with DEAE; the enzyme was eluted from DEAE-cellulose at 0.5 M KCI. Thus, it is likely that this unusual property of the enzyme reflects the presence of acidic amino acids on the exterior of the molecule.

It has been suggested that the enzyme that catalyzes the re- moval of the 3’ extra nucleotides from tRNA precursors is RN- ase II (11). We have also observed that RNase II is capable of catalyzing the removal of the 3’ extra nucleotides from s&r tRNATyr precursor, which has been treated with RNase P but only at very high enzyme concentrations. We have shown that RNase P III activity was clearly separated from RNase II ac- tivity during further purification. Finally, we have demonstrated that purified RNase II was unable to replace the requirement for RNase P III in the in vitro system for tRNA synthesis. We there- fore conclude that RNase P III is distinct from RNase II.

All tRNAs have the common sequence -CCAon at their amino acid acceptor terminus. The enzyme, tRNA nucleotidyl- transferase, specifically catalyzes the addition of CMP and AMP to the 3’ ends of tRNAs lacking the -CCAon sequence (17, 23, 24). The enzyme has been purified from extracts of E. coli to near homogeneity, and it has been shown to consist of a single poly- peptide chain of molecular weight 50,000 (17). However, the physiological role of this enzyme has remained unclear,

It has been shown that the dimeric tRNA precursor for the T4-specific tRNAs, tRNASer and tRNAPro, does not contain the sequence for the 3’ CCAon termini of these tRNAs (5). When phage T4 was grown in E. coli mutants that possess low levels of tRNA nucleotidyltransferase (29, very little activity of the ma-

ture tRNAs was detected.3 Thus, for these tRNAs at least, it is clear that the 3’ CCAoH must be added post-transcriptionally, and it is strongly suggested that tRNA nucleotidyltransferase is involved in this enzymatic process. However, the mutants de- fective in tRNA nucleotidyltransferase are viable (25), implying either that this enzyme is not essential for tRNA biosynthesis or that the low level of enzymatic activity present in these strains is sufficient for the production of normal levels of tRNA. In the case of s&r tRNATyr, it is known that the -CCAon3’-terminal sequence is included in the precursor and therefore appears to be coded on the tRNA gene (3). The possible involvement of tRNA nucleotidyltransferase in the biosynthesis of s&r tRNATyr is considered below.

That the peak of activity of RNase P III was found to be coin- cident with the peak of activity of tRNA nucleotidyltransferase when the elution profiles of these enzymes on DEAE-cellulose were compared suggested the possibility that these two ac- tivities were associated with the same protein molecule. However, we have also shown that purified tRNA nucleotidyltransferase was unable to replace the requirement for RNase P III in the in vitro system for synthesis of tRNA. Moreover, when purified tRKA nucleotidyltransferase was incubated with pre-tRNAz (s&r tRNATyr precursor previously treated with RNase P), as determined by acrylamide gel electrophoresis, there was no con- version of this material into mature 4 S tRNA. On the basis of these experiments, we conclude that RNase P III is distinct from tRNA nucleotidyltransferase. We cannot, however, exclude the possibility that RNase P III is, in fact, a complex protein, con- sisting of the 50,000-dalton polypeptide chain which has been characterized to be tRKA nucleotidyltransferase and an addi- tional associated protein component. If such a complex protein exists, it must be relatively unstable because as we have shown, further purification of the tRNA nucleotidyltransferase activity associated with RNase 1’ III by chromatography on hydroxyl- apatite resulted in the loss of RNase 1’ III activity. Further purification of RNase 1 III will be necessary in order to resolve this question.

The details of the mechanism by which the 3’ extra nucleotides are removed from tRNA precursors have not yet been determined. Analysis of the hydrolysis products after RNase P III treatment of pre-tRNA9 should yield information as to whether RNase P III is an endonuclease or an exonuclease. Further studies will also be necessary in order to determine whether or not the -CCAon3’ terminal sequence of the mature tRNA is the direct product of RNase P III nucleolytic cleavage. Another possibility is that the direct product of RNase P III hydrolysis is tRNA-X-C-Con or tRNA-Xon, and that the activity of tRNA nucleotidyltransfer- ase is required to yield the mature tRNA-X-C-C-Aon species. Such questions cannot be approached in a meaningful way until further purification of RNase P III has been achieved.

In addition to RNase P and RNase P III, synthesis of tRNA has been shown to require DNA-dependent RNA polymerase and a fourth essential component, designated Fraction V. We have demonstrated that Fraction V contains a nuclease that together with RNA polymerase is responsible for the synthesis of s&r tRNATyr precursor. We have also presented evidence sug- gesting that s&r tRNATyr precursor is not itself the primary transcription product of the s&r tRNATyr gene, but rather that it arises as a result of post-transcriptional cleavage of a much larger molecular weight precursor molecule by the action of the nuclease present in Fraction V. In these experiments, incubation

3M. P. Deutscher and W. H. McClain, personal communication.

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of the total RNA transcription product synthesized by purified DNA-dependent RNA polymerase from @Ops& DNA in post-transcriptional cleavage reactions with Fraction V resulted not only in the appearance of s&r tRNATyr precursor but also in the appearance of several 480.specified RNAs as well. These results imply that the nuclease activity present in Fraction V is not only specific for cleavage of the s&r tRNATy’ precursor but that it may also be involved in post-transcriptional processing of other RNA molecules as well.

As originally described by Robertson et al., RNase III is an E. coli endonuclease with high specificity for double-stranded RNA molecules (26). This enzyme has been shown to be involved in the processing of a precursor for ribosomal RNA in E. coli

(7,8) and in the post-transcriptional cleavage of a high molecular weight precursor for the T7 early messenger RNAs (8, 9). >Iore recently, it has been demonstrated that T7 early messenger RNAs are the direct products of RNase III cleavage (27). In these ex- periments, transcription of T7 DNA with purified RNA polymer- ase resulted in the synthesis of the high molecular weight pre- cursor molecule. Subsequent treatment of this RNA with puri- fied RNase III yielded T7 early messenger RNAs identical with those produced in vivo.

The 3’ termini of the RNase III-cleaved T7 early messenger RNAs all contain the sequence CC(C)UUUAUon (28). Prelimi- nary sequence analysis of the RNase III cleavage site on the ribo- somal RNA precursor from E. coli indicate that it also contains a pyrimidine-rich sequence that is similar to but not identical with that reported for the T7 early messenger RNAs4 Although the precise sequence of the 3’ terminus of the in vitro synthesized S&I tRNATy’ precursor has not yet been determined, pre- liminary results indicate that it also contains a pyrimidine-rich sequence.” The consistency of these observations suggests the possibility that the nuclease act.ivity present in Fraction V that is required for synthesis of s&r tRNATyr precursor may be RNase III.

However, according to the purification procedure described by Robertson et al. (26)) RNase III was found to have no affinity for DEAE-cellulose. In contrast, according to the procedure that we have described, the endonuclease activity present in Fraction V was purified from Fraction IA polymerase, which was eluted from DEAE-cellulose at 0.25 M KCl. These observations imply that the endonuclease activity present in Fraction V may be dis- tinct from RNase III. Preliminary experiments have shown that RNase III cannot replace Fraction V for the synthesis of pre- tRNA1, thus strengthening the suggestion that the Fraction V activity is distinct from a heretofore described activity.6

4 J. A. Steitz, personal communication. 5 The in vitro synthesized SLI&, tliNATyr precursor contains at

least 4 and no more than 12 addition residues beyond the -CCAc.n 3’ terminus of the mature tRNA. H. G. Khorana and his co-work- ers have determined the nucleotide sequence of that region on the &ODSLI&~ DNA that extends 23 residues hevond the 3’ end of the su:I; t RNAr=

I sequence (29). According to this information, the

sequence of the RNA must be -UCACUUUC4AAGon. 0 In accordance with precedent, we suggest the name ItNase P

IV for this activity.

6255

The activity of the various fractions described above appears to be sufficient to explain the requirements in tRNA biosynthesis. The influence of any additional components (e.g. modification enzymes, phosphorylated nucleotides) on the specific act,ivity of the tRNA or on the regulation of its synthesis is a subject for further study.

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REFERENCES

BURDON, It. H. (1971) Prog. Nucl. Acid RFS. Mol. Biol. 11, 33-79

BERNH.~RDT, I>., AND DIRNELL, J. (1969) J. Mol. Biol. 42, 43-56

AI,TM.\N, S., .~ND SMITH, J. L). (1971) iVature A’cw Viol. 233, 35-39

G~~HRIE, C., SISIDX\N, J. G., ALTM.\N, R., B.\RKI.L, B. G., SMITH, J. D., .\KD MCCUIN, W. II. (1973) LVature Xew niol. 246, G-11

B.\Rw:LL, B. G., SIXDX\N, J. G., GUTHRII:, C., .\ED McCI,.~IN, W. H. (1974) Proc. I\‘&. Acad. Sci. b’. S. A. 71, 413-410

I).\RiXI*:I,L, J. E., JR. (1908) L(actcriol. Rev. 32, 262-290 NII<oL:~I~:v, N., SILI:XGO, L., AND SCHI,I,:SSINGI,:R, D. (1973)

Proc. i\‘atl. Acad. Sci. U. S. A. 70, 3361-33ti5 DUNN, J. J., .~ND STUDIER, W. F. (1973) Proc. Xatl. Acad. Sci.

U. S. A. 70, 3296-3300 DUNN, J. J., .\ND STUDI~IE, W. F. (1973) Proc. Natl. Acad. Sci.

U. S. A. 70, 1559-1563 ROBKRTSON, I-I. Il., AI,TM.\N, S., AND SMITH, J. I>. (1972) J.

Biol. Chem. 247, 524335251 SCHEDL, P., .\ND PRIM.\KOFF, P. (1973) Proc. Aatl. Acad. Sci.

U. S. A. 70, 2091-2095 SINGI:~, M. F., .\ND TOLBER~~, G. (1965) Biochemistry 4, 1319-

1330 BIICOFF, E. K., .~ND G~:FTI:R, M. L. (1975) J. Hiol. Chcm. 260,

G240-G247 ROBERTS, J. W. (1969) Nature 224, 1168-1174 ZUB.\Y, G., CIIEONG, L., .\ND GI:FTI~:R, M. L. (1971) Proc. i\‘atl.

Acad. Sci. U. S. A. 68, 2195-2197 C.~I,I:ND.\R, IL., .\ND BERG, P. (19G6) Bzochemistry 6, lG81-

lG90 MILLER, J. R., l~~~ PHIUPPS, G. It. (1971) J. Riol. Chem. 246,

1274-1279 Bu~c,nss, 11. It. (1969) J. Eel. Chcm. 244, GlGO-6167 BUCHI,:R, T. (1947) Biochim. Biophlys. Acta 1, 292-296 LOWRY. 0. H.. ROSI,:BROUCH. N. J.. F.wR. A. I,.. .\xI) RON-

D.\LL; R. J. (1951) J. Biol.‘Chem. i93, 2(;5-275 ’ GKFTKR, M. L., .\KD Itusser,~, It. L. (19G9) J. 11101. Biol. 39,

145-157 ZUB.~Y, G., CH.\MBI:RS, I>. A., .\ND CHI,:OXG, L. C. (1970) in

The Lactose Oprrojr (BI,:CIC\VITN, J. I<., .IND ZII~I.:R, I>., eds) pp. 375-391, Cold Spring Harbor Press, Cold Spring Harbor, New York

DI,:UTSCHI,:R, M. P. (1973) Prog. ~Yucl. Acid Rcs. Mol. Biol. 13, 51-92

C.\~tm:, 11. S., LIYV~K, S., .\NI) CHIIV~XILIX, F. (1970) Biochim. Biophyw. Acta 224, 371-381

HII~DI~:RM.\N, I<. H., I\ND DIXTTSCHXR, M. P. (1973) Biochrm. Biophys. Rcs. Commzltl. 64, 205-215

HOB~<TSOX, H. L)., WI:BSTI,X, Ii. E., .ZND ZINDKR, N. I>. (1968) J. Biol. Chcm. 243, 82-91

ROSENBERG, M., KR.\MI,:R, R. A., .~ND STKITZ, J. A. (1974) J. Mol. Biol. 89, 777-782

KR.\MEI~, R. A., ROSV:NBI,:RG, M., .WD STISITZ, J. A. (1974) J. Mol. Biol. 89, 767-776

Lo~;.~I<N, P. C., >~ND KHOIVX~, H. G. (1973) J. Biol. Chem. 248.3489-3499

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E K Bikoff, B F LaRue and M L Gefteractivities.

In vitro synthesis of transfer RNA. II. Identification of required enzymatic

1975, 250:6248-6255.J. Biol. Chem. 

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