/. Biochem. 86, 335-346 (1979)

The Primary Structure of Cytoplasmic Initiator Transfer Ribonucleic Acid from Torulopsis utilis1 Shigeko YAMASHIRO-MATSUMURA and Shosuke TAKEMURA Institute of Molecular Biology, School of Science, Nagoya University, Chikusa-ku, Nagoya, Aichi 464 Received for publication, January 31, 1979

Cytoplasmic initiator transfer ribonucleic acid (tRNAinit) was purified from bulk Torulopsis (Candida) utilis3 tRNA by a series of column chromatography procedures. Sequence analysis of the products of complete and partial digestion of this tRNA with ribonuclease A [EC 3.1.4.22] and ribonuclease Tx [EC 3.1.4.8] enabled us to determine the complete primary structure of the molecule. The chain length of this tRNA was 76, including 11 modified nucleotides. The structure of the tRNA was arranged into a cloverleaf model and compared with those of other initiator tRNA species. As in the cytoplasmic initiator tRNA's of most other eukaryotic cells, the sequence -A-U-C-G- is contained in this tRNA in place of the usual -T-y-C-G(or A)- found in other tRNA's.

In a living cell, there are two functional types of methionine-specific tRNA, one of which (tRNAinu) is utilized in the initiation of protein biosynthesis and the other (non-initiator methionine tRNA) in peptide chain elongation. Recently, it has been suggested that initiator tRNA may interact with 5S ribosomal RNA in protein biosynthesis. In our laboratory, the total nucleotide sequence of T. utilis 5S ribosomal RNA has been determined, and a possible model for the secondary structure of this RNA was proposed (/). This model

suggests that eukaryotic 5S ribosomal RNA may function as one of the sites of tRNAinit binding to the ribosome. As a first approach in our studies on the structure-function relationship of tRNAjnit, the complete sequence of this tRNA was determined and compared with those of eukaryotic tRNA'sinit from bakers' yeast (2), Neurospora crassa (3), starfish (Kuchino, Y., Kato, M., Sugisaki, H., & Nishimura, S., unpublished data) and several vertebrate sources (4-8). EXPERIMENTAL PROCEDURE

1

This work was supported in part by grants-in-aid for scientific research (Nos. 11402 & 147129) from the Ministry of Education, Science and Culture of Japan. Abbreviations and symbols for nucleic acid sequences and constituents follow the 1970 recommendations of the IUPAC-IUB Commission of Biochemical Nomenclature. tRNAinit, initiator methionine tRNA. 1

Torulopsis utilis is also known as Candida utilis.

Vol. 86, No. 2, 1979

Isolation of tRNAinit—Initiator tRNA was purified by a series of column chromatography procedures from bulk T. utilis tRNA.* The first * Crude tRNA mixture was supplied by the Research Laboratory, JujO Paper Co., Ltd., and Sanyo Pulp Co., Ltd., Japan.

335

336

S. YAMASHIRO-MATSUMURA and S. TAKEMURA

step of chromatography was performed on a column of DEAE-Sephadex A-50 (9). The tRNA Met -rich fraction was then fractionated on a column of DEAE-Sephadex A-25 with the phosphate system (70). Fractions containing tRNA Met were pooled and then applied to a benzoylated DEAE-cellulose (BD-cellulose) column (2.0x110 cm). The procedure of Gillam et al. (11) was used for the preparation of BD-cellulose. Methionine acceptor activity was separated into two peaks (I and II) when assayed with crude yeast aminoacyl-tRNA synthetase, one of which, peak I, was assigned as the tRNAinn species for the following reasons, (a) Peak I, methionine tRNA could be charged with methionine by either crude yeast or E. coli aminoacyl-tRNA synthetases. (b) The methionyl-tRNA thus formed

could be further converted to /V-formylmethionyltRNA in the presence of formyl-group donor (calcium leucovorin=7V5-formyltetrahydrofolate) and crude E. coli methionyl-tRNA transformylase according to the method in ref. (12). (c) Peak II could not be aminoacylated with crude E. coli aminoacyl-tRNA synthetase but could with yeast enzyme, and the methionyl-tRNA thus formed could not be formylated. The initiator tRNA (peak I) was further purified by column chromatography on RPC-5 (75). For the chromatographic conditions, see Fig. 1. The preparation accepted 1,600 pmol of methionine per Aleo unit of tRNA. One Also unit is defined as the amount of tRNA which gives an absorbance reading of 1 at 260 nm in 1 ml of solution in a cell of 1 cm pathlength. The purity of the preparation was higher than 85 % judging from the results of the fractionation of oligonucleotides obtained by RNase digestion. Enzymes—Bovine pancreatic RNase (RNase A) [EC 3.1.4.22], snake venom phosphodiesterase [EC 3.1.4.1], and E. coli alkaline phosphatase [EC 3.1.3.1] were purchased from Worthington

40

60 80 TUBE NUMBER

Fi g . 1

Fig. 1. Preparation of tRNAinit by chromatography. A: 10 g of crude tRNA from T. utilis was charged on a DEAE-Sephadex A-50 column (5.0x150 cm) and eluted at room temperature in 0.02 M Tris-HCl buffer (pH 7.6) with a linear gradient from 0.40 M NaCl0.08 M MgCl, to 0.52 M NaCl-0.16M MgCl, (total volume 24 liters). The flow rate was about 120m]/hr. B: 1.5 g of tRNAMet fraction from Fig. 1A was charged on a DEAE-Sephadex A-25 column (2.1 x 100cm) and eluted at 32CC with a linear gradient from 0.20 to 0.35 M KC1 in 1 M potassium phosphate buffer (pH 6.2) containing 5% (v/v) dimethylformamide (total volume 5 liters). The flow rate was 50m]/hr. C: 150mg of tRNAMet fraction of the phosphate system was charged on a BD-cellulose column (2.0x110 cm) and eluted at room temperature with a linear gradient from 0.4 to 0.7 M NaCl in 0.02 M Tris-HCl buffer (pH 7.2) containing 0.01 M MgCl,. The flow rate was 20 ml/hr. D: Peak I in Fig. 1C was charged on an RPC-5 column (0.8 x 100 cm) and eluted at 35°C with a linear gradient from 0.35 to 1.0 M NaCl 0.02 M Tris-HCl buffer (pH 7.2) containing 0.01 M MgCl, (total volume 400 ml). , Absorbance at 260 nm; , methionine acceptor activity with S. cerevisiae aminoacyl-tRNA synthetase; , methionine acceptor activity with E. coli aminoacyl-tRNA synthetase. / . Biochem.

PRIMARY STRUCTURE OF INITIATOR tRNA Biochemical Corp., Freehold, N.J. (U.S.A.). RNase T, [EC 3.1.4.8], T, [EC 3.1.4.23] and U, [ribonucleate 3 '-purino-gligonucleotidohydrolase] were products of Sankyo Ltd., Tokyo. Silkworm endonuclease was kindly supplied by Dr. K. Nakamura of Daiichi Pure Chemicals Co., Tokyo. Crude yeast aminoacyl-tRNA synthetase was prepared from Saccharomyces cerevisiae as described previously (14), and was made up to a 50% glycerol solution, then stored at —20°C until use. Crude E. coli aminoacyl-tRNA synthetase was prepared from E. coli B/l by a modification (IS) of the procedure of Dickerman et al. (16). This preparation contained methionyl-tRNA transformylase as well. Complete Digestion of tRNAimt with RNases— About 10 mg of the purified RNA was digested with RNase A or RNase Tlt and the digest was chromatographed on a column of DEAE-cellulose under the conditions reported previously (17-19). Rechromatography was usually carried out on 0.25 x (30-50) cm DEAE-cellulose columns, eluting with a linear gradient of NaCl in 7 M urea-0.02 M formic acid (pH ca. 3.7), or on 0.2 x (15-20) cm columns of Dowex AG-1, eluting with a linear concentration gradient of NaCl, usually in 0.008 M0.01 M HC1. The procedures were essentially identical with those described previously (17-19). Partial Digestion of tRNAimt with RNase Tj— Two hundred Ate0 units of the purified tRNAinit was incubated with 1,000 units of RNase Tj in 2.0 ml of 0.05 M MgCl,-0.05 M Tris-HCl buffer (pH 7.6) for 20 min at 0°C. Bovine serum albumin (4 mg/0.2 ml) was added and the reaction was stopped by phenol extraction of the RNase. Phenol was removed from the aqueous layers by ether extraction (20, 21). All procedures were carried out at 0-4°C. Another procedure for preparing fragments of partial digestion was as follows. The initiator tRNA (200 AtK units) dissolved in 2 ml of 0.05 M Tris-HCl buffer (pH 7.6) without MgCl, was divided into two portions. One was digested with 500 units of RNase Tt at 0°C for 30 min, and the other was digested with 500 unks of RNase Tj at 0°C for 18 min. The reaction mixtures were separately shaken with phenol (22), and then combined and treated as above. The digests were made up to pH 1 with hydrochloric acid and stored overnight at 0-4°C to Vol. 86, No. 2, 1979

337

cleave cyclic phosphodiester linkages. After neutralization, the digest was diluted tenfold with 7 M urea-0.01 M Tris-HCl buffer (pH 7.6) and subjected to column chromatography. Separation of Large Oligonucleotides—The partial digests with RNase Tx were charged on a DEAE-cellulose column (0.3x110 cm) previously equilibrated with 7 M urea-0.01 M Tris-HCl buffer (pH 7.6). Elution was performed with a linear gradient from 0 to 0.5 M NaCl in the above ureaTris buffer, in a total volume of 700 ml. Each peak was rechromatographed with the following systems: a) DEAE-cellulose column (0.3 x(5060) cm), eluted with a linear gradient of NaCl in 7 M urea-0.1 M formic acid (pH ca. 3.5) in a total volume of 100 ml. b) DEAE-cellulose column (0.3 x (50-60) cm), eluted with a linear gradient of NaCl in 7 M urea-0.06N HC1 (pH ca. 2.7) in a total volume of 100 ml. Desalting—Urea and salts in the oligonucleotide fractions were removed by adsorption on a DEAE-cellulose column and elution with 6 column volumes of 1 M ammonium carbonate as described in previous papers (17-19). The alkali-labile oligonucleotides and large oligonucleotides were desalted by dialysis against 0.001 M Tris-HCl buffer (pH ca. 7) (25). The dialyzed fractions were dried in a vacuum desiccator. Procedure for Sequencing Oligonucleotides— For sequence analysis, pure oligonucleotides obtained by complete RNase digestion of the RNA were cleaved with nucleases of various specificities under the conditions described previously (17-19). One of the large oligonucleotides (T 10) was subjected to partial digestion with snake venom phosphodiesterase in the presence of E. coli alkaline phosphatase (18). The reaction mixture was applied to a DEAE-cellulose column (0.25 x 20 cm) previously equilibrated with 7 M urea-Tris-HCl buffer (pH 7.6). Elution was carried out with a linear gradient from 0 to 0.3 M NaCl in the same buffer (pH 7.6). Each fragment, separated in order of increasing chain length, was digested with RNase A after desalting and drying. High performance liquid chromatography was used for sequence determination of the digestion products. Details of the analysis of ribonucleic acid constituents by high performance liquid chromatography will be published elsewhere by Komiya et al. (24).

338

S. YAMASHIRO-MATSUMURA and S. TAKEMURA

Sequence Analyses of Large Oligonucleotid.es— nucleotide. This smaller fragment was further The large oligonucleotides obtained by partial digested with snake venom PDase to give adenosine, digestion of the RNA were further hydrolyzed with pGp and a modified nucleotide which had UV RNase Tx or RNase A, and the digests were absorption spectra at pHs 2 and 12 similar to chromatographed on a DEAE-cellulose column those of Gp. Thus, the modified nucleotide was (0.15x20 cm) in 7 M urea at pH7.6. The condi- designated as G*. RNase T t digestion of this tions of enzymatic digestion and of chromatog- trinucleotide fragment yielded Ap and G*-Gp. raphy were as described previously (20). The The position of the spot G*-Gp on a cellulose thinseparated end-products were identified by their layer two-dimensional chromatogram (Avicel SF) elution positions from columns and also from their was close to that of Gm-Gp using isobutyric acid: UV absorption spectra. If necessary, further 0.5 N NH4OH (5 : 3 in v/v) in the first dimension chromatography was performed on a column of and 2-propanol: cone. HC1 : H2O (70 : 15 : 15 in Dowex AG-1 (0.15x 10cm) with an HCl-NaCl v/v) in the second dimension. Although G* is not yet completely identified, it might differ from system. Gm, since the nucleoside (G*) produced by snake venom PDase digestion in the presence of alkaline RESULTS phosphatase gave a spot different from that of Gm Complete RNase A Digestion Products—The in a two-dimensional thin-layer chromatography pancreatic RNase digest of tRNAinit was first system as described by Rogg et al. (25). The fractionated by column chromatography on DEAE- sequence of peak A 14 was established as A-G*cellulose in 7 M urea at pH 7.6 (Fig. 2). Rechro- G-A-G-A-Cp by considering the sequence of the matography showed that peaks A 2, A 5, A 6, A 9 fragment n^A-A-A-C-C-A-G'-Gp found in the and A l l consisted of two or more fragments and RNase T1 digest of the tRNA (Table II), since the other peaks were single components (chro- A-Gp cannot be placed at the 5' end of A-G*-Gp. Complete RNase T/ Digestion Products— matograms not shown). The results of sequence analyses of the purified fragments are summarized Primary separation of the RNase Tx digestion in Table I and our comments are given below. products of tRNAinit was carried out by column Peak A 9-2 consisted of an equimolar mixture chromatography on DEAE-cellulose in 7 M urea of A-G-Dp and G-A-Up, which were not separated at pH 7.6 (Fig. 3). Rechromatography gave two by chromatography on a Dowex AG-1 column. pure fragments from peak T 2 and four from The relative molar yields of the two trinucleotides peak T 5. The other peaks were single comwere estimated from the molar ratio of A-Up to ponents (chromatograms not shown). The results A-Gp obtained by further RNase Tx digestion. of sequence analyses of the purified fragments are summarized in Table n and our comments are RNase Tx digestion of the largest fragment, given below. peak A 14, yielded three peaks, A-Cp, A-Gp, and a smaller fragment which contained an unidentified

»0 TUBE NUMBER

Fig. 2. Chromatography of an RNase A digest of tRNAinit on a DEAE-cellulose column in 7 M urea at pH 7.6.

TU3E NUMBER

Fig. 3. Chromatography of an RNase Tx digest of tRNAinit on a DEAE-cellulose column in 7 M urea at pH 7.6. /. Biochem.

PRIMARY STRUCTURE OF INITIATOR tRNA

339

TABLE I. Fragments obtained by complete RNase A digestion of tRNAinit. The peak numbers correspond to those in Figs. 2 and 3. Peak No. A 1 A 2-1

Identity

Mol per mol of A»

Analyses6

A

1.0

a

m'Cp

b

2-2

Cp

1.5C 8.8=

A 3 A 4 A 5-1

Up

5.6

b

m'G-Dp A-Cp m.Kj-Cp G-Cp A-Up G-Up rnKJ-rnKJ-Cp t»A-A-Cp G-A-Up A-G-Dp

0.6i

c, d

1.2

e

5-2

A 6-1 6-2

A 7 A 8 A 9-1 9-2

A 10

G-m'-A-A-A-Cp

b

1.0

e

3.0

e

0.9

e

0.8 0.8

e c

0.8

c

0.9

It

0.9

\d

0.7

f c

A 11-1

pA-G-Cp

0.9

f

e 11-2

G-G-A-Up

0.9

f

e A 12 A 13

A-G-G-G-Cp G-G-A-A-G-Cp

0.7 0.8

c f e g

A 14

A-G*-G-A-G-A-Cp

0.7

f c e e

m'G 0.8, pGp 1.0 Ap 1.1, Cp 1.0 m.'Gp 0.9, Cp 1.0 Gp 0.8, Cp 1.0 Ap 0.8, Up 1.0 Gp 1.3, Up 1.0 m'G 0.9, pm«G1.0, pGp 1.0 t'A 1.2, pA 1.3, pGp 1.0 Gpl.2, A-Upl.0, A-Gpl.O Dp 1.0 Gp 1.0, (m'A-A-A-Cp 0.8) m'A-A-A-Cp — m'A 1.3, pA 2.3, pCp 1.0 Cp 1.0, (pA-Gp 0.9) pA-Gp-»pAp 1.1, Gp 1.0 Gp 2.1, (A-Up 1.0) A-Up-»Ap 1.2, Up 1.0 A 1.2, pG 3.0, pCp 1.0 Gp 1.8, Cp 1.0, (A-A-Gp 1.0) A-A-Gp — Ap 2.2, Gp 1.0 G-G, pA-A-G, pCp (A-G*-Gp 1.0) (A-Gp 1.0) (A-Cp 1.0) A-G*-Gp —Ap 1.1, pG* 1.0, pGp 1.0 A-Gp-»Ap 1.1, Gp 1.0 A-Cp— Ap 1.0, Cpl.O

* For most millimolar extinction coefficients, see Ref. (IT). In addition, the following millimolar extinction coefficients were used: f Ap, 16; m'G, 10.3 (at pH 2 and 260 ran); m'G*p in Ref. (IS). b The following methods were used for analysis of the products: a, identification by chromatography on a Dowex-50 column and from the ultraviolet absorption spectra at pHs 2 and 12; b, identification by chromatography on a Dowex AG-1 column with an NaCl-HCl system and from the ultraviolet absorption spectra at pHs 2 and 12; c, complete digestion with snake venom phosphodiesterase, followed by b; d, phosphorus determination; e, RNase T, digestion, followed by b; f, RNase T, digestion, followed by b; g, silkworm endonuclease digestion, followed by b. Parentheses indicate that the sequence of this fragment was determined in a subsequent step. c Molar ratios of m'Cp and Cp were estimated after rechromatography of peak A 2 on a Dowex AG-1 column, eluting with 0.001 N HC1 or 0.002 N HC1. m5C-m»C-C, (A-U-m'G-D>m'C-miC-C-U, Cp 1.0, (m'A-A-A-Cp 1.0) (A-G*-Gp 0.8) n^A-A-A-Cp -• m J A 1.3, pA 1.9, pCp 1.0 A-G'-Gp-. A p 1.0, G*-Gp 0.9 Cp 3.7, U p 2.0, G p 1.0, A-Up 0.8 (t'A-A-Cp 0.9) t'A-A-Cp-• t'A 1.1, pA 1.1, pCp 1.0 (C-U-C-Ap 1.0) (C-C-C-U-Gp 1.0) (U-t'A-Ap 0.9) C-U-C-Ap->Cp 1.7, U p 1.1, A p 1.0 C-U-C-Ap — C 1.1, pC 1.2, p U 0.9, pAp 1.0 C-U, C-U-C, pAp, etc. C-C-C-U-Gp-• Cp 3.1, U p 0.9, G p 1.0 C-C, C-C-C, pU-Gp, etc. U - f A - A p — U 0.9, pt'A 0.9, pAp 1.0

b

» For methods of analyses a through g, see footnote to Table I, h, RNase A digestion, followed by b; i, RNase U, digestion, followed by b; j , Partial digestion with snake venom phosphodiesterase in the presence of E. coli alkaline phosphatase, and fractionation on a DEAE-cellulose column followed by analysis by high performance liquid chromatography after digestion with RNase A. b See footnote