THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 243, No. 3, Issue of Februsry 10, pp. 575483, 1968

Printed in U.S.A.

Studies on Bolynucleotides

I,XXIX. YEAST PHEK\-LALAKIKE TRAn’SE’ER RIBOKUCLEIC ACID: PRODUCTS OBTAINED BY DEG- RADATIO1\’ WITH PANCREATIC RIBONUCLEASE*

(Received for publication, August 21, 1967)

U. I,. IIAJBHASDARY, R. D. FAULKNER, AKD ALEXAXDER STUART

From the Institute for Enqme Research, University of Wisconsin, Madison, Wlkonsin 53706

SUMMARY

Purified phenylalanine transfer RNA (tRNA) from yeast has been degraded with pancreatic ribonuclease, and the fragments obtained have been separated and identified. The 3’-terminal nucleoside is cytidine, and the 5’4errninal dinucleotide is pGpCp. Fourteen minor nucleosides are present, including 7-methylguanosine, 2’-0-methylcytidine, and a nucleoside the identity of which is not established. The sequences involving some of the minor nucleosides, N2- dimethyl-GpCp, GpTp, and ApGp-dihydro-Up, are common to many other tRNAs. The largest oligonucleotide produced is an octanucleotide with the sequence GpGpGpApGpAp-

GPCP.

The nucleotide sequence analysis of yeast alanine tRNA1 derived by Holley et al. (1) has been followed by the elucidation of the sequences of serine tRNA (2), tyrosine tRNA (X), and phenylalanine tRNA (4) from yeast. The general principles utilized in all these studies were (a) degradation of the tRNA with specific ribonucleases and separation and analysis of the fragments obtained (5-g), and (b) isolation and characterization of larger fragments from partial enzymatic digests (9-11) in order to obtain overlaps of sequences necessary for t’he derivation of a unique sequence. The present paper describes the detailed sepa-

* This work has been supported by the National Science Foun- dation (Grant GB-3342), the National Cancer Institute of the National Institutes of Health, United States Public Health Serv- ice (Grant CA-05178). and the Life Insurance Medical Research Fund (Grant 65-44). ”

1 The abbreviations used are: tRNA, transfer ribonucleic acid; tltNAPhe, phenylalanine tRNA; diHU, 5,6-dihydrouridine; 5MeC, 5.methylcytidine; 2’0MeC. 2’.0-methvlcytidine: 2’0MeG. 2’-0- methycguanosine f 2MeG, N2-met,hyigu&osine’; diMeG, W- dimethvlauanosine: 7MeG. 7.methvlnuanosine: 1MeA. A”-methvl-

” - ” Y I adenosine; BMeA, A-6.methyladenosine; Y, an unidentified minor nucleoside in tRNAPhe which is fluorescent under ultraviolet light; Y’, the presumed conversion product of Y, which is non- fluorescent. One optical density unit is defined as that amount of material per ml of solution which produces an absorbance of 1 in a l-cm-light path cell at 260 rnp.

ration and qualitative and quantitative analyses of oligonucleo- tide fragments produced by the action of pancreatic RNase on tRNAPh”. The following paper (12) reports on the analyses of fragments produced by digestion of tRNAPhe with T1 RNase. Brief reports of these results have been summarized elsewhere (4, 13, 14).

In the preceding paper (15), we reported on the terminal se- quence analyses of two peaks (Peak I and Peak II) of yeast tRNAPhe purified by countercurrent distribution, and concluded that these two peaks represented a single species of tRNAPhe. By the use of radioactive end group-labeling techniques (16,17), it was found that the nucleotide sequences at the 5’ and 3’ termini of tRNAPhe (Peak I) were pGpC- and -CpApCpC, respectively. These results are now confirmed from the data described in this paper and in the following paper (12).

EXPERIMENTAL PROCEDURE

Generab Procedures

Chromatography-Paper chromatography was carried out on Whatman No. 1 or Whatman No. 3MM paper. The following solvent systems were used: Solvent A, 2-propanol-concentrated ammonium hydroxide-water (7 : 1: 2, v/v) ; Solvent B, l-pro- panol-concentrated ammonium hydroxide-water (55 : 10 : 35, v/v) ; Solvent C, isobutyric acid-concentrated ammonium hy- droxide-water (66:1:33, v/v), pH 3.7; Solvent D, l-butanol- concentrated ammonium hydroxide-water (86 : 5 : 14, v/v) ; Solvent E, ethanol-l M ammonium acetate, pH 7.5 (70:30, v/v) ; Solvent F, t-butyl alcohol-methyl ethyl ketone-concentrated ammonium hydroxide-water (40: 30: 10: 20, v/v) ; Solvent G, ethyl acetate-l-propanol-water (4: 1:2, v/v; upper layer); and Solvent H, ethanol-water (70:30, v/v).

EZectrophores&-Paper electrophoresis was performed on What- man No. 3MM paper with a voltage gradient of 40 volts per cm for 1 hour in 0.05 M ammonium formate buffer, pH 2.7. The pa- per was immersed in Varsol (a high boiling petroleum fraction) kept cold with running water.

Column Chromatography-DEAE-cellulose (Whatman DE-23) was washed successively with 0.2 N sodium hydroxide, water, 0.2 N hydrochloric acid, and water. A fine suspension of the resin in 1 M ammonium carbonate was used for packing the column; the

575

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576 Studies on Polynucleotides. LXXIX Vol. 243, No. 3

column was then washed thoroughly with 0.01 M ammonium carbonate before use.

For chromatography in the presence of 7 M urea (18), a fine suspension of the resin in 0.5 M sodium chloride containing 7 M

urea and 0.02 M Tris buffer, pH 7.3, was used for packing the column.

Elution from Paper-Elution of nucleotidic material from pa- per chromatograms or electrophoretograms was carried out with water; in all cases a corresponding area was also eluted and used as blank. Recovery of ultraviolet-absorbing material was usually in the range of 90%.

Bnalytical Methods-Estimation of inorganic phosphate was carried out with the method of Chen, Toribara, and Warner (19) as modified by Ames and Dubin (20). Inorganic phosphate was detected on paper chromatograms with the ammonium molybdate- perchloric acid spray reagent (21), and dihydrouridine either with the sodium metaperiodate-p-rosaniline hydrochloride spray (22) (reagent for vicinal diols) or with the sodium hydroxide-p- dimethylaminobenzaldehyde spray reagent (23).

Ultraviolet Absorption Spectroscopy-A Zeiss PM& II spectro- photometer was used for measurement of absorbance, and a Cary 14 spectrophotometer for recording spectra.2

Enzymes-Pancreatic RNase, snake venom phosphodieeter- ase, micrococcal nuclease, and Escherichia coli alkaline phospha- tase were purchased from Worthington; prior to its use, alkaline phosphatase was treated as described previously (24). T1 RNase either was a gift from Dr. F. Egami (25) or was obtained from Sankyo Company, Ltd., Tokyo, and was purified by column chromatography. T2 RNase (partially purified) was kindly provided by Dr. K. Takahashi. Calf spleen phosphodiesterase was a preparation purified as described previously (26).

Methods

Paper Chromatography of Incubation Jlfixtures-The incuba- tion mixture, containing 2 to 10 O.D. units of nucleotidic material, was evaporated to dryness, and the residue was dissolved in 10 to 50 ~1 of water and applied as a 3- to S-cm-wide band for paper chromatography.

Characterization of Oligonucleotides-This was basically done by degradation of the oligonucleotide with the various enzymes listed above. Unless otherwise mentioned, all the products were obtained in quantities close to equimolar ratios except pseudo- uridine, which was usually in the region of 0.6 to 0.8 mole per mole of oligonucleotide. (For a review of the mode of action of these enzymes, see References 25 and 27.)

General Conditions for Enzymatic L)egradations-All incubations were conducted at 37”. For T1 RNase the mixture, in 0.5 ml, contained the oligonucleotide (4 to 8 O.D. units), Tris buffer, pH 7.5 (5 pmoles), and T1 RNase (20 to 30 units). Incubation time was 5 hours. The mixture for alkaline phosphatase con- tained, in 0.5 ml, the oligonucleotide (5 to 10 O.D. units), am- monium bicarbonate (10 pmoles), and alkaline phosphatase (10 pg). Incubation was conducted at pH 8.0 to 8.5 for 4 to 6 hours. For degradation with T1 RNase and alkaline phosphatase com- bined, the oligonucleotide (5 to 10 O.D. units) was incubated with ammonium bicarbonate (10 Mmoles), T1 RNase (20 to 30 units), and alkaline phosphatase (5 to 10 pg) in 0.5 ml for 6 to 8 hours. For T2 RNase, the mixture, in 0.5 ml, contained the oligonucleotide (2 to 5 O.D. units), sodium acetate, pH 5.0 (5

2 Throughout this paper, spectrum is meant to indicate ultra- violet absorption spectrum.

pmoles), and the enzyme (50 to 150 pg). Incubation was con- ducted for 6 to 8 hours. For snake venom phosphodiesterase, the oligonucleotide (2 to 10 O.D. units) was incubated with am- monium bicarbonate (10 pmoles) and enzyme (5 to 10 pg)3 for 6, to 8 hours. The mixture for micrococcal nuclease, in 0.2 to 0.4 ml, contained the oligonucleotide (2 to 4 O.D. units), calcium chloride (1 to 2 pmoles), glycine-sodium hydroxide buffer, pH 8.6 (5 to 10 pmoles), and the enzyme (50 pg). Incubation time was 5 to 6 hours.

Hydrolysis with .4lkaZi-The oligonucleotide (2 to 5 O.D. units) was dissolved in 1 N sodium hydroxide (0.2 ml) and left at room temperature for 24 hours. Sodium ions were removed with Dowex 50 (pyridinium form) resin, the resin was removed by filtration, and the filtrate and washings were evaporated for chromatography.

Identification of Nucleasides and Nucleotides-Nucleosides or mononucleotides were identified by their ultraviolet absorption spectra at pH 1, 7, and 13 and by their mobilities in the various chromatographic systems. For the final identification, authentic markers of the appropriate nucleosides or nucleotides were al- ways present during chromatography.4 Dihydropyrimidine derivatives were identified from their characteristic loss of ab- sorption in alkaline medium (28).

RESULTS

Purification of tRNAPhe

This included three steps of countercurrent distribution and was essentially the same as described previously (29)) except for scaling up in the amount of tRNA used. Thus, 8 g of crude yeast tRNA were subjected to a 200.cycle countercurrent dis- tribution in an EC 580 countercurrent fractionator (total phase volume per tube, 80 ml), and the partially purified tRNAphe (16,000 O.D. units and 10 to 20% wit’h respect to phenylalanine acceptor activity) was subjected to an 800.cycle distribution in the same machine. The major peak of tRNAPhe (15, 29) (2,500 O.D. units, 55 to 65% pure) was finally purified by a 1,200- cycle distribution in the EC 520 fractionator (total phase volume, 20 ml per tube). Over-all yield of tRNAPhe was usually 60 to 80 mg; unless otherwise stated, phenylalanine acceptor activity of the tRNA used in most of these experiments was higher than 90%.

Digestion of tRNAphe with Pancreatic RNase and Chromatog- raphy on DEAE-cellulose (Carbonate Form)

tRNAPhe (355 O.D. units in 0.02 M phosphate buffer, pH 7.0) was incubated at 37” with pancreatic RNase (0.9 mg) and phos- phate buffer, pH 7.0 (100 pmoles), in a volume of 6 ml. After 16 hours (increase in total O.D. units was 27.87,), the incubation mixture was chromatographed on DEAE-cellulose (carbonate form). The pattern of elution is shown in Fig. 1. The recovery of total nucleotidic material was higher than 92%, and further elution of the column with 1 M sodium chloride did not release any nucleotidic material.

Fractions within a certain peak were pooled, and ammonium carbonate was removed by repeated evaporation from water. Removal of the last traces of ammonium carbonate was effected

3 For degradation of oligonucleotides containing minor nucleo- side components, the amount of enzyme used was increased by a factor of 2- to 5-fold.

4 We thank Dr. Ross Hall for supplies of minor nucleosides used as reference samples.

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Issue of February IO, 1968 RajBhandary, Faulkner, and Stuart 577

-.Ol TO .16M I5

.16TO 2M I .2 TO .8M-

6

i

FIG. 1. Chromatography of a pancreatic RNase digest of tRNAPhe (355 O.D. units) on a column (1 X 104 cm) of DEAE- cellulose (carbonate form). Elution was carried out in three stages. Stage I was elution with an increasing gradient produced from 300 ml each of 0.01 M, 0.05 M, 0.08 M, 0.1 M, and 0.16 M am-

by mixing an aqueous solution of the oligonucleotide with Dowex 50 (pyridinium form) resin. The resin was filtered off and washed with dilute ammonium hydroxide. The filtrate and washings were evaporated to dryness twice from dilute ammonium hy- droxide. No significant difference between the absorbance of the pooled peak before and after this treatment was observed.

The identification of the mononucleotides and the oligonucleo- tides produced and quantitative estimation of these products are summarized in Table I.

Digestion of tRNAphe with Pancreatic RNase and Chromatography on DEAE-cellulose (Chloride Form) in 7 M Urea

In a volume of 4.4 ml, tRNAPhe (550 O.D. units, at least 70% pure) was incubated with pancreatic RNase (3.6 mg) and phos- phate buffer, pH 7.0 (80 /Imoles), for 7 hours at 37”. The reac- tion mixture was adjusted to 7 M with respect to urea and then chromatographed on DEAE-cellulose (chloride form) in the presence of 7 M urea (18). A typical elution pattern is shown in Fig. 2. The pooled fractions were separated from urea and so- dium chloride by passage through columns (2 x 90 cm) of poly- acrylamide gel, Biogel P2 (31).

Identification of Pancreatic RNase Digestion Products (Fig. 1)

Peak 1 (Cytidine)-This was identified as cytidine from its mobility in Solvents A and D and from its spectrum. The amount of adenosine present was 5 to 10%.

Peak 2 (C-cyclic-p; diH U-cyclic-p)-The complete spectrum of a small portion indicated the presence of a cytidine derivative and a dihydropyrimidine derivative (28) ; these were found to be C-cyclic-p and diHU-cyclic-p. The cytidine derivative was characterized by chromatography in Solvent B. The bulk of the peak material was treated further with pancreatic RNase and alkaline phosphatase, and the nucleosides produced were chro- matographed in Solvent F against markers of uridine, pseudo- uridine, and diHU. The area of the chromatogram correspond- ing to diHU (marker on either side of the chromatogram detected by the spray reagent) was eluted. diHU was present in the elu- ate as judged by its spectrum.

monium carbonate in five chambers of a Varigrad (30); Stage II was elution with a linear gradient produced from 250 ml each of 0.16 M and 0.2 M ammonium carbonate; and Stage III was elution with a linear gradient produced from 750 ml each of 0.2 M and 0.8 M ammonium carbonate.

TABLE I

Analysis of products obtained by degradation of tRXAPhe with pancrealic RNase (Fig. 1)

Peak

1 2

3

9

10

11

12

13 14 15

16

Composition

Cytidine C-cyclic-ph diHU-cyclic-p*

CP 5MeCp

UP *P diMeGpCp

APCP GPTP GPCP GPUP PGPCP Ap2MeGpCp GplMeApUp GpGMeApUp ApGpdiHUp 2’OMeGpApApYpApqp ApGpAp2’OMeCpUp

GPGPAPUP APGPAPAPUP APGPAPAPUP GpGpApGp7MeGpUp

GPGPGPAPGPAPGPCP

- Molar ratio

7.6 0.8

7.6 6

10 9.6

21.6 21 19 17.8 20 17.8 33.4 32.4 33.4 25 60 50 42.8 58 58 59 75

7.2 1.7 6.3 0.6 1.0 1.9 1.0 1.1 1.1 0.8 0.9

i

0.9

0.9 0.7 1.0 0.9

0.9

0.85 0.P

a Calculated on the basis of contribution of the individual nucleotides (5, 6) after correction for hypochromicity. Approxi- mate hypochromicity of the oligonucleotides at neutral pH and at 260 rnp was measured after hydrolysis of the oligonucleotides either with alkali or with TP RNase.

b No quantitative determination was attempted, as this peak consisted of a mixture of other materials.

c The yield of this oligonucleotide was low when degradation with pancreatic RNase was carried out for prolonged periods. However, with shorter incubation periods (5 to 7 hours), the yield of this oligonucleotide (Peak 8, Fig. 2) increased to 0.8 to 0.9 mole per mole of tRNAPhe.

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578 Studies on Polynuclectides. LXXIX Vol. 243, Ko. 3

0 25 50 FRACTIOt.

FIQ. 2. Chromatography of a pancreatic IZNase digest of t,ltNAP’Le (550 0.1). units) on a column (0.7 X 90 cm) of DEAE- cell\llose (chloride form) in the presence of 7 M urea and 0.02 M

Tris buffer, pH 7.5. Elution was performed with a linear gradient, of sodium chloride produced from 300 ml each of 7 M urea in 0.02 M

Tris buffer, pH 7.5, and 0.3 M sodium chloride containing 7 M urea and 0.02 M Tris buffer, pH 7.5. Fractions were collected every 10 min; fract,ion volumes were around 3.5 ml.

Peak S (Cp; SLkleCp)-This peak consisted of a mixture of Cp and 5MeCp, which were identified as follows. The spectrum in- dicated that the predominant constituent, was a cytidine nucleo- tide. Chromatography in Solvent A gave a single band of ultra- violet-absorbing material with the mobility of a mononucleotide, and no fast traveling material in the nucleoside 2’,3’-cyclic phosphate region could be detected. Dephosphorylation fol- lowed by chromat,ography in Solvent D yielded cytidine and 5MeC.

The presence of any diHUp was ruled out on the basis of the following experiment. An aliquot (15.2 O.D. units) of material from Peak 3 was subjected to paper electrophoresis at pH 2.7, and a single, ultraviolet-absorbing component with the same mobility as Cp was observed. The area corresponding to the re- gion of a marker of Cp was eluted, treated with alkaline phospha- tase, and assayed for release of any inorganic phosphate. No in- organic phosphate was produced; under identical conditions the band of Cp eluted from the same electrophoretogram produced 1 mole of inorganic phosphate per mole of cytidine.

Peak 4 (Up)-This peak was identified as Up from its spectrum and mobility in Solvent A. Also, a portion (13 O.D. units) of material from this peak, on treatment with alkaline phosphatase and subsequent chromatography in Solvent F, showed a single band of uridine. No other nucleosidic material was detected by the use of either spray reagent; markers of uridine, diHU, and thymine ribonucleoside showed a positive response to one or both of these reagents.

Peak 5 (qp)-This was characterized by its spectrum and by chromatography subsequent to dephosphorylation in Solvent A.

Peak 6 (diiMeG$$)-The material from this peak was found to be diMeGpCp. Thus, a single dinucleoside phosphate (Sol- vent B) was obtained after dephosphorylation. This, on elution and treatment with snake venom phosphodiesterase, yielded diMeG and pC (separation in Solvent A).

Peak ‘?’ (ApCp)-Treatment with alkaline phosphatase pro- duced a compound identical with ApC (Solvent 13). Subsequent digestion wit,h venom diesterase and chromatography in Solvent

A gave adenosine and PC, indicating that the dinucleotide in this peak was ApCp.

Peak 8 (GpTp; GpCp)-The components of this peak were sep- arated by paper electrophoresis at pH 2.7 and characterized as GpTp and GpCp by procedures identical with those used for DiMeGpCp and ApCp.

Peak 9 (GpUp; pGpCp)-This peak consisted of a mixture of GpUp and pGpCp, which were easily separated by chromatog- raphy in Solvent B. The faster traveling component was char- acterized as GpUp, in much the same way as the dinucleotides described above. The structure of the slower moving component as pGpCp was deduced from the following considerations: (a) it had a lower mobility in Solvent B compared to GpCp; (b) on de- phosphorylation it produced GpC; (c) on alkaline hydrolysis the products were pGp and Cp (separation in Solvent B).

Peak 10 (ApdMeGpCp; GplMerlpUp)-The components of this peak were found to be Ap2MeGpCp and GplMeApUp. Characterization of the latter compound was complicated by the fact that the amount of this trinucleotide was small (part of this trinucleotide was isolated as GpGMeApUp; see data on Peak 11) ; furthermore, during incubations at 37” for enzymatic digestions or chromatography in ammoniacal solvent systems, conversion of 1MeA to 6MeA (32) produced two bands from a single oligonu- cleotide. The combined yield of GplMeApUp and GpSMeApUp was, however, close to 1 mole per mole of tRNAPhe, and, since the ready conversion of 1MeA to 6MeA is well established (33), the trinucleotide GplMeApUp must be a genuine constitutent of tRNAPhe. The evidence for the structure of the trinucleotides is presented below.

Dephosphorylation and chromatography in Solvent C produced a major component (Band I) and two minor components travel- ing closely together but separated (Bands II and III). The ratio of nucleotidic material in Band I to that in Bands II and III combined was 2.4. The fastest traveling compound (Band I) was characterized as Ap2MeGpC by degradation with snake venom phosphodiesterase and chromatography in Solvent A. The products obtained were adenosine, p2MeG, and PC; the mononucleotides could be resolved in Solvent G after dephos- phorylation into the corresponding nucleosides and identified by their spectra and mobilities.

The structure of material in Band II was established as Gp6- MeApU from the products of snake venom phosphodiesterase, which were guanosine, pGMeA, and pU (1: 1: 1, separation in Sol- vent a). The component of Band III was similarly identified as GplMeApU; the products in this case were guanosine, p6MeA (small amount), and a mixture of plMeA and pU, subsequently separated as their nucleosides by chromatography for 24 hours in Solvent A.

Peak 11 (GpGMeApUp; ApGpdiHUp)-Part of the trinucleo- tide GplMeApUp from Peak 10 was present in this peak as Gp6MeApUp (the products of snake venom phosphodiesterase degradation were guanosine, pGMeA, and pU) ; the other com- ponent of this peak was ApGpdiHUp. The two trinucleotides were resolved by chromatography either in Solvent B or in Sol- vent C. Initially the presence of a dihydropyrimidine in the slower moving trinucleotide band was indicated by the fact that on dephosphorylation and subsequent treatment with snake venom phosphodiesterase the only ultraviolet-absorbing prod- ucts were adenosine and pG. The sequence of this compound was established as ApGpdiHUp from work on the dephosphoryl- ated compound as follows. (a) On digestion with T1 RNase and

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Issue of February 10, 1968 RajBhandary, Faulkner, and Stuart

(b) &I dOMeGpAp+2Ap+Yp+Y

2’OMaGpApApYpApYm

I

L SLOW DEGRADATION

Cd) Wp-EXHAUSTIVE 2~OMeG c2Ds Y,ELD,

1 , T2 -RN; c 2’OMcGpAp+Ap+Y 2’OMaGpA~ApY+pA+pY

M. NUCLEASE 2’OMaGpAp+.4pY

(a) T,-RNasa d NO DEGRADATION

579

/(fl T2-RNasa 1 WRN-a -.9p+y

c 2’0MaGpAp+Ap+ApY-CYCLIC-p+Y

FIG. 3. Scheme of degradative experiments used during analysis of the hexanucleotide 2’OMeGpApApYpApqp. SPD, spleen phos- phodiesterase; SVP, snake venom phosphodiesterase; M. n&ease, micrococcal nuclease.

alkaline phosphatase, the products were ApG and diHU (ApG dientified by degradation to adenosine and pG with snake venom phosphodiesterase, and diHU from its spectrum and mobility in Solvents A and F) ; (b) treatment with snake venom phospho- diesterase and chromatography in Solvent A yielded adenosine, pdiHU, and pG. pdiHU had the same mobility as pU and was converted to inorganic phosphate and diHU by the action of alkaline phosphatase. Inorganic phosphate was detected by spraying the lower half of the chromatogram (close to the origin) run in Solvent A with the molybdate spray reagent (21), and diHU (present in the upper half) by the periodate spray reagent (22) for glycols. A paper blank eluted from the previous chro- matogram and treated similarly gave neither inorganic phosphate nor any diHU.

Peak I2 (ApGpApb’OMeCpUp; 2’OMeGpApApYpAp*p)- The major components of this peak were a pentanucleotide, ApGpAp2’OMeCpUp (the rest of this oligonucleotide was also found in Peak 13), and a hexanucleotide, 2’OMeGpApApYp- Ap\kp. In addition, a minor component was the tetranucleotide GpGpApUp, the bulk of which was present in Peak 13. Sepa- ration of the dephosphorylated oligonucleotides required two steps of paper chromatography; Solvent C completely resolved GpGpApU (slowest mobility) from ApGpAp2’0MeCpU and 2’OMeGpApApYpApq, which were partially separated, the oligo- nucleotide containing the minor nucleoside Y being strongly fluorescent under ultraviolet light. A clear separation of ApGp- Ap2’OMeCpU from 2’0MeGpApApYpAp@ was obtained only after prolonged chromatography (68 hours) in Solvent E.

ApGpAp2’OMeCpU was characterized by degradation with T1 RNase to give ApGp and Ap2’OMeCpU (separation in Sol- vent B), or by combined degradation with T1 RNase and alkaline phosphatase, in which case the products were ApG and Ap2’- OMeCpU (separation in Solvent A). Ap2’0MeCpU was further identified by digestion with snake venom phosphodiesterase to adenosine, p2’OMeC, and pU, or by alkaline hydrolysis to Ap and 2’0MeCpU (separations in Solvent A).

The sequence analysis of the hexanucleotide 2’OMeGpApAp- YpApqp was a more difficult problem because of (a) the presence of minor nucleosides at either end, which resulted in a slow rate of degradation with exonucleases; (b) the presence of the minor

CA) 2’OMoGpApApYpApYp M. NUCLEASE

-2!OMoGpAp+ApYp+ApYp

+SMALL AMOUNTS OF Ap AND Yp

BAP APYP -ApY

T2-RNaso -Ap+Y

(8) 2’OMoGpApApY’pApP PAN. RNarc

-2’OMoGpApApY’-CYCLIC-p+ApY

2bM(oGpApApYkYCLIC-p Gl

I E’OMoGpAp+Ap+Yb ?

M. NUCLEASE - 2’0MoGpAp+ApY’-CYCLIC-p

FIG. 4. Scheme of degradative experiments used during analy- sis of the hexanucleotide 2’OMeGpApApYpAptip and its presumed conversion product, 2’OMeGpApApY’pAptip. M. nuclease, mi- crococcal nuclease; BAP, bacterial alkaline phosphatase; PAN, pancreatic.

nucleoside Y, the identity of which is not known; and (c) the ab- sence of any phosphodiester linkages hydrolyzable by T1 RNase. The total evidence for the sequence of this hexanucleotide came from a combination of degradative studies, which are summarized in Figs. 3 and 4 and are described in detail below. (In some of the enzymatic digests, small amounts (15 to 20%) of fluorescent material distinct from the major band of fluorescence were ob- served upon paper chromatography in ammoniacal systems. The possibility, therefore, that Y was a labile nucleoside that could undergo rearrangement or partial degradation to another fluorescent material either during incubation at 37” or during chromatography could not be ruled out,.)

The oligonucleotide was not degraded by T1 RNase and hence does not contain guanosine, inosine, 2MeG, or diMeG. The nucleotide composition of the oligonucleotide and the 3’-terminal end group was determined by alkaline hydrolysis and chroma- tography in Solvent B. The products were pseudouridine (in- dicating that the 3’-terminal end was pseudouridine), a fluores- cent material with mobility similar to that of a mononucleotide, Ap (2 moles), and 2’OMeGpAp (1 mole). 2’OMeGpAp was char- acterized by digestion with snake venom phosphodiesterase sub- sequent to dephosphorylation. Digestion of the oligonucleotide with spleen phosphodiesteraseproceeded very slowly, with the re- lease of some 2’OMeGp and Ap. The slow rate of degradation

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0.7

y 0.6

$0.5

5: $ 04

0.3

0.2

01

I 1 I I I I \d

I / / 220 240 260

‘1- 280 300

?.. 329 240

,L j 260 200 300 320

WAVELENGTH ;ny

FIG. 5. Ultraviolet absorpt,ion spect,ra at pH 7.0 of the hexa- nucleotide 2’0MeGpApApYpAptip (A) and that of its presumed conversion product, 2’0MeC;pApApY’pApfip (R).

of this oligonucleotide indicated that’ 2’0MeG might be the 5’-terminal end group of this oligonucleotide. Further evidence that 2’0MeG represented the 5’-terminal end group was ob- tained by exhaustive treatment of the oligonucleotide with snake venom ghosphodiesterase. The only nucleoside product detected was 2’0MeG (20% yield), and, since it was already known that alkaline hydrolysis gives 2’0JIeGpAp, the partial sequence of the oligonucleotide is 2’0MeGp9p(Ap, Yp, Ap)\kp, in which the order of the nucleotides shown within parentheses is not known. The information for the nucleotide sequence came from the iso- lation of a fluorescent tetranucleoside triphosphate, pA, and p@ (chromatographic separation in Solvent 13) as the major products from the action of snake venom phosphodiesterase on the oligonucleotide. The fluorescent product was characterized as 2’OMeGpApApY by degradation either with micrococcal nu- clease to 2’0MeGpAp and ,4pY (separation in Solvent A, where ApY travels faster and is fluorescent) or with Tz RNase to 2’0MeGpAp, Ap, and Y (separation in Solvent A). On diges- tion with Tz RNase, the products from ApY were Ap and Y (separation in Solvent A).

Further support for the structure of the oligonucleotide was ob- tained by treatment with Tz RNase. The products which were completely resolved by paper chromatography (Solvent E) were 2’OMeGpAp, Ap, ApY-cyclic-p, pseudouridine, and also a small amount of Y-cyclic-p. ApY-cyclic-p was identified by degrada- tion with spleen phosphodiesterase to -4p and Y-cyclic-p (separa- tion in Solvent E or A). It should be noted that, whereas ApY was cleaved completely by Tz RNase to -4p and Y, ApY-cyclic-p was largely resistant to the action of this enzyme: furthermore, it appears that ApY-cyclic-p is not further hydrolyzed by Tz RNase to ApYp. Products obtained by the action of micrococcal nuclease on the hexanucleotide (Fig. 4) were in agreement with the structure deduced and also provide direct proof of the pres- ence of the dinucleotide sequence Ap\Ep. After chromatography of such a digest in Solvent B, the oligonucleotides identified were mostly 2’0MeGpAp, BpYp, and Ap\kp, with some Ap and \kp.

The final supporting evidence for the structure of the hexa- nucleotide came from a study on large oligonucleotide fragments

obtained by partial T1 RNase treatment of tRNAPhe (34). It was found that, during resolution of these large oligonucleotides by column chromatography on DEAE-cellulose in 7 M urea and at acidic pH, the characteristic bright greenish blue fluorescence of Y (under ultraviolet light) in some of the oligonucleotides was no longer present, and also that the corresponding hexanu- cleotide isolated from further degradation of these oligonucleo- tides had a distinctly altered spectrum from that of the hexanucleotide containing Y (Fig. 5). Furthermore, the dephos- phorylated hexanucleotide could now be partially degraded5 with pancreatic RNase to yield 2’OMeGpApApY’-cyclic-p and Ap@ (separation in Solvents A and B). The characterization of 2’OMeGpApApY’-cyclic-p (Z&o in Solvent B = 0.92) is shown in Fig. 4. Thus (a) the presence of a 2’,3’-cyclic phosphate end group is inferred from experiments with alkaline phosphatase; (b) on alkaline hydrolysis the ultraviolet-absorbing products were equimolar amounts of 2’OMeGpAp and Ap (it should be noted that Yp itself has low absorbance at 260 rnp, approximate ~260 = 4000 to 5000 per mmole, and was detected on paper chromatograms because of the exhibition of fluorescence under ultraviolet light; it is not surprising, therefore, that the presumed conversion product, Y’p, could not be detected); and (c) the ac- tion of micrococcal nuclease yielded 2’0MeGpAp and ApY’- cyclic-p; the structure of the latter compound was established from its spectral properties, similar to those of adenosine at neu- tral and acidic pH; from its mobility in Solvent L4 (RAP = l.l), which showed that it was not adenosine or A-cyclic-p but could be either Ap or =IpY’-cyclic-p; and, finally, from its lack of sus- ceptibility to alkaline phosphatase, which ruled out the possi- bility that it was Ap.

Peak IS (GpGp.lpUp; -~pG~Apb’OA~eCpUp)-The constitu- ents of this peak comprised the bulk of total GpGpApUp pro- duced and part of ApGpAp2’OMeCpUp, also found in Peak 12. These were dephosphorylated and resolved by chromatography in Solvent C. The faster traveling component was identified as ApGpAp2’OMeCpU as described above. The slower traveling band, on degradation with T1 RNase alone, gave 2 moles of Gp and 1 mole of ApU (separation in Solvent B) ; with T1 RNase and alkaline phosphatase, the products were 2 moles of guanosine and 1 mole of ApU (separation in Solvent A).

Peak 14 (ApGpapSpUp)-Except for a small amount (5 to 10%) of contaminant, this peak was mostly ApGpApApUp (part of this pentanucleotide was also present in Peak 15). The oligo- nucleotide was purified by chromatography in Solvent C and then subjected to combined degradation with T1 RNase and alkaline phosphatase, and the products, ApG and ApApU, were separated in Solvent A. ApApU was characterized either by treatment with snake venom phosphodiesterase (adenosine, pA, and pU were formed in equimolar amounts) or by hydrolysis with alkali (2 moles of Ap and 1 mole of uridine were formed).

Alternatively, the pentanucleotide was first dephosphorylated,

5 The degradation of this hexanucleotide requires the presence of pancreatic RNase; under identical conditions of incubation, no cleavage of internucleotidic bonds was observed when alkaline phosphatase instead of pancreatic RNase was present. The possibility that the fluorescence of the hexanucleotide had disap- peared because of the cleavage of the glycosidic bond in Y, result- ing in the release of free base, could t,hus be ruled out. Moreover, if this had happened and the hydrolysis of an internucleotidic bond at this point had occurred by a mechanism of p elimination, one of the products would be pAp# and not Ap+, as was observed.

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Issue of February 10, 1968 RajBhandary, Faulkner, and Stuart 581

and the isolated pentanucleoside tetraphosphate then was treated with Ti RNase alone. The products ApGp and ApapU were then resolved by chromatography in Solvent B.

Peak 15 (ApGpApApUp; GpGpApGp?‘MeGpUp)-This peak consisted of some ApGpApApUp (also found in the preceding peak) and the hexanucleotide GpGpApGp7MeGpUp. These oligonucleotides were easily separated in Solvent C prior to or after removal of phosphomonoester groups. Structural studies on the hexanucleotide are described below.

Earlier analyses (13, 35) by combined degradation with Ti RNase and alkaline phosphatase indicated that this compound could be a heptanucleotide; on the other hand, its elution from the column close to a pentanucleotide suggested that it was at the most a hexanucleotide. A further difficulty in the analysis of this compound was due to the ring opening of the imidazole moiety of the 7MeG present in this oligonucleotide during chro- matography in ammoniacal systems or during prolonged incuba- tion at 37” (36-38), which gave rise to a nucleoside previously designated as X (13). This degradation of one of the constituent nucleosides was kept at a minimum by avoiding the use of am- moniacal solvent systems. Thus (a) combined action of T1 RNase and alkaline phosphatase produced 2 moles of guanosine, 1 mole of ApG (these were not separated in Solvent C) and 1 mole of 7MeGpU (seen as a dark blue fluorescent spot different from the bright greenish blue fluorescence of Y on the chromato- gram). The faster traveling band consisted of guanosine and ApG; these could be separated in Solvent A, and the amounts of guanosine and ApG produced were estimated (moles of guanosine to moles of ApG produced was 2.04:1). 7MeGpU was charac- terized by degradation with snake venom phosphodiesterase (excess enzyme and prolonged incubation were found necessary for complete digestion) to 7MeG and pU. 7MeG was identified by its mobility in Solvents C and H, its characteristic dark blue fluorescence under ultraviolet light, and its spectrum. (b) The 5’ end group of the hexanucleotide was shown to be guanosine, by dephosphorylation and subsequent treatment with snake venom phosphodiesterase. The only nucleoside produced was guano- sine. The above experiments permitted a deduction of the se- quence Gp(Gp , ApGp)‘iLMeGpUp for this oligonucleotide. The ordering of Gp and ApGp shown within parentheses was made possible from studies on large oligonucleotide fragments isolated by a limited digestion of tRNAPhe with T1 RNase and identifi- cation of ApGp7MeGpUp as the 5’-terminal sequence of a large oligonucleotide (34).

Peak 16 (GpGpGpApGpdpGpCp)-The neutral spectrum of this oligonucleotide showed a maximum at 253 rnp and indicated a high content of guanosine residues. From its position of elu- tion from the column, this oligonucleotide was expected to be free of contaminants. For this reason and in view of the expected difficulty of recovery from paper chromatograms, small aliquots (2 O.D. units) were chromatographed in Solvents B and C over prolonged periods to check for the purity of this oligonucleotide. Only a single sharp band at the origin was present. Combined degradation of the oligonucleotide (5 O.D. units) with T1 RNase and alkaline phosphatase yielded cytidine, guanosine, and ApG in the molar ratio 1:3.2:2.1 (separation in Solvent A), and indi- cated that this compound was an octanucleotide with the nucleo- tide composition 3Gp, 2ApGp, Cp. The characterization of this as GpGpGpApGpApGpCp by a method involving dephos- phorylation, partial degradation with snake venom phosphodi-

esterase, and subsequent labeling of the 2’,3’-diol end groups in the oligonucleotides produced is described in an accompanying paper (17).

DISCUSSION

As a first major step toward the elucidation of the sequence of yeast tRNA Phe this paper has reported on the qualitative and quantitative analyses of mono- and oligonucleotides produced by the action of pancreatic RNase on tRNAPhe.

Methods commonly used for the separation of oligonucleotides have been either two-dimensional paper chromatography or electrophoresis (or both) (39, 40), or column chromatography on DEAE-cellulose (18, 41, 42). The former technique was used mostly for the small scale analysis of fragments from valine tRNA (43, 44), alanine tRNA (43, 45), and tyrosine tRNA (45) and is normally limited to oligonucleotides up to the pentanucleo- tide level; recently, modified techniques have made possible the separation of small amounts of a large number of oligonucleotides obtained by degradation of tRNAs (46). The technique of column chromatography was applied (5-7) to the sequence analysis of alanine, serine, and tyrosine tRNAs and, in the pres- ent work, proved adequate for resolution of fragments (Fig. 1) obtained from tRNAPhe. In cases when more than one compo- nent was present within a peak, a single step of paper chromatog- raphy usually separated the constituent oligonucleotides.

Sequential analysis of oligonucleotides was carried out by the use of standard techniques (for a review, see Reference 35), and except in the case of oligonucleotides containing some minor nucleosides, was straightforward. The major problems en- countered in these oligonucleotides were mainly the interconver- sion of 1MeA to 6MeA t,hrough ring opening and ring closure (32, 33), the labile nature of 7MeG (36-38), and a much slower rate of attack by exonucleases (47).

The isolation of 7MeGpU from ‘l’i RiSase digestion of GpGp- ApGp7MeGpU and the isolation of 7MeGpUpCpSMeCpUpGp from Ti RNase digestion of tRNBPhe (12) indicate that 7AMe- GpX (X = nucleoside) bonds are not susceptible to the action of this enzyme.

Fourteen minor nucleosides were found in tRNAphe, including 2’0MeC and 7MeG, which have been characterized for the first time from a purified RNA. A new minor nucleoside, which has not yet been identified (here designated Y), was also present. This nucleoside exhibited a bright fluorescence in ultraviolet light, and the fluorescence persisted very strongly in large oligo- nucleotides isolated from tRNA Phe ( 34, 48) and even to a certain extent in the tRNAPhe itself. Y displayed a highly characteris- tic spectrum (Fig. 6), with a pronounced maximum at 240 rnp in neutral solutions. Conversion of Y to another unidentified and nonfluorescent nucleoside (Y’, see above) caused a distinct change in the spectral characteristics of the hexanucleotide 2’0MeGpApApYpAp’kp (Fig. 5). Furthermore, the oligonu- cleotide could now be degraded to 2’OMeGpApApY’cyclicp and Ap\k, indicating that Y’ (and hence possibly Y) could be a pyrimidine derivative. Some evidence either that Y is cationic at neutral pH or that conversion to Y’ generates an anion can be deduced from the observation that oligonucleotides containing Y were eluted earlier from a DEAE-cellulose column than the cor- responding oligonucleotides in which Y was changed to Y’ (34, 48). The exact chemical nature of Y remains unknown at this time.

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582 Studies on I’olynucleotides. LXXIX Vol. 243, No. 3

0.2

0.1 1

I III

230 250 270 290 310 3YJz: 210 230 250 270 290 310 330 3.

-ENGTH mp 10

Frg. 6. Ultraviolet absorption spectra at pH 7.0 of the dinucleoside phosphate ApY(A) and of the nucleoside Y (B)

The identification of cytidine at the 3’ end and pGpCp at the 5’ end of tRNAPhe supports the conclusion arrived at (15) by means of end group-labeling techniques. The sequences GpTp, di- MeGpCp, and ApGpdiHUp present in tRNAPhe have been found in other tRNAs (5-7). The largest oligonucleotide obtained from pancreatic RNase digests of tRNAPhe was the octanucleo- tide GpGpGpApGpApGpCp, the characterization of which was described before (17). While closely related oligonucleotides such as GpGpGpApGpApGpUp (5) and GpGpGpApGpApCp (6) are present in alanine and tyrosine tRNAs, respectively, the posi- tions of these oligonucleotides in the three tRNAs are different (1, 3, 4).

(Table I). This is also indicated by the data reported in an ac- companying paper (12), in which very good correlations between fragments produced by pancreatic RNase and those produced by Tr RNase on tRNAPhe were observed.

Acknowledgments-We are grateful to Dr. H. G. Khorana for his generous support and helpful suggestions. We would also

The yield of the oligonucleotides obtained from tRNAPhe shows that these compounds are genuine constituents of tRN,4Phe

like t,o thank I>. Davies and J. Sneider for technical assistance.

7. MELCHERS, F., AND ZACHAU, H. G., Biochim. Biophys. Acta, 91, 559 (1964).

8. D~~TTING, D., AND ZACHAU, H. G., Biochim. Biophys. Acta, 91. 573 (1964).

9. APGAR, J., EVERETT, G. A., AND HOLLEY, R. W., J. Biol. Chem., 241, 1206 (1966).

10. DUTTINO, D., FELDMAN, H., AND ZACHAU, H., Hoppe-Seyler’s 2. Physiol. Chem., 347, 249 (1966).

11. MADISON, J. T., AND KUNG, H., J. Biol. Chem., 242, 1324 (1967).

12. RAJBHANDARY, U. L., STUART, A., AND CHANG, S. H., J. Biol. Chem., 243, 584 (1968).

13. RAJBHANDARY, U. L., AND STUART, A., Fed. Proc., 26, 520 (1966).

15. RA.IBHANDARY’, U. L., STUART, A., HOSKINSON, R. M., AND KHORANA, H. G., J. Viol. Chem., 243, 565 (1968).

16. RAJBHBNDARY, U. L., YOUNG, R. J., AND KHORANA, H. G., J. Hiol. Chem., 239, 3875 (1964).

17. RAJBHANDARY. U. L.. J. Riol. Chem.. 243. 556 (1968’1. 18. TOMLINSON, 1~: V., AND TENER, G. M., j. Am&. Chem. Sot.,

84, 2644 (1962).

14. RAJBHANDARY, U. L., STUART, A., FAULKNER, R. D., CHANG,

19. CHEN, P. S., TORIBARA, T. Y., AND WARNER, H., Anal. Chem.,

S. H., AND KHOR,4NA, H. G., Cold Spring Harbor Symp. Quant. Biol., 31, 425 (1966).

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U. L. RajBhandary, R. D. Faulkner and Alexander StuartWITH PANCREATIC RIBONUCLEASE

RIBONUCLEIC ACID: PRODUCTS OBTAINED BY DEGRADATION Studies on Polynucleotides: LXXIX. YEAST PHENYLALANINE TRANSFER

1968, 243:575-583.J. Biol. Chem. 

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