The Biosynthesis of inosinic acid in transfer RNA

352

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 96554

T H E B I O S Y N T H E S I S OF INOSINIC ACID IN T R A N S F E R RNA H A R O L D O. K A M M E N AND S Y L V I A J. S P E N G L E R

Space Sciences Laboratory, University o] CaliJornia, Berkeley, Calf/. (U.S.A.) (Received M a r c h ISth, 197 o)

SUMMARY

IMP constitutes approx, o.14 ~o of the nucleotides of Escherichia coli B transfer RNA (tRNA), which corresponds to I residue per 8- 9 t R N A chains. No significant amount of IMP was found in ribosomal RNA. The biosynthesis of these IMP residues was examined in E. coli B-94, a m u t a n t defective for the enzyme, adenylosuccinase, (adenylosuccinate AMP-lyase, EC 4.3.2,2). The properties of this strain make it possible to preferentially dilute inosine nucleotide pools without a parallel dilution of adenosine nucleotide pools. When strain B-94 was grown in [~Cladenine, the addition of hypoxanthine did not lead to any reduction in the labeling of t R N A IMP. This observation indicates that these residues did not arise b y insertion of IMP monomers during assembly of t R N A but were formed b y modification of the polynucleotide. The data are consistent with a mechanism involving deamination of specific AMP residues of a t R N A precursor. E. coli cells are essentially devoid of a pool of ITP. The absence of this nucleotide can be attributed to the inability of E. coli to phosphorylate 5'-IMP to IDP.

INTRODUCTION

IMP occurs as a normal constituent of Escherichia col# ,2, yeast 3-6 and m a m m a 1fan 7 transfer RNA (tRNA). The IMP residues are localized solely within tile "anticodon loop" of the tRNA, predominantly at the 5'-terminus of the anticodon triplet 8. CRICI~9 has proposed that IMP residues at this position engage in a unique "wobble" pairing with the alternative bases, C, U and A, at the 3'-terminus of the eodon triplet. We consider two major possibilities for the biogenesis of the IMP of tRNA: (A) by enzymatic alteration of a precursor polynucleotide, in a manner analogous to the methylation 1°,~1,~°, thiolation 12,1~ and other modifications 14 of tRNA; (B) by the incorporation of IMP monomers during the assembly of the t R N A chain. These two possibilities should be distinguishable from the labeling of t R N A purines after exposure to an isotopic purine precursor. Mechanism A predicts that the labeling of the t R N A IMP residues should be independent of the specific activity of acid-soluble inosine nucleotides, whereas Mechanism B demands a direct relationship between them. The latter mechanism also implies that I T P should occur as a normal cellular metabolite; that enzymatic reactions should exist for the generation of I T P ; and that a recognition signal should operate for the specific insertion of IMP into the t R N A chain by RNA polymerase. Biochim. Biophys. Acta, 213 (197 o) 352-364

BIOSYNTHESIS OF

t R N A INOSINIC ACID

353

Pur B mutants of E. coli 15 are unable to convert inosine nucleotides to adenosine nucleotides (Fig. 1). This metabolic feature makes it possible to perturb differentially the pools of these nucleotides and thereby to test the above alternatives. The results of such a test indicate that the IMP of E. coli t R N A is formed by a modification of the polynucleotide chain, presumably by a deamination enzyme. A preliminary account has appeared 17. /AD~ A~AMP

+

ATP ~

tRNA AMP

,,

Adenylosuccinete Hx ~ I M P , .

tRNA ITP . . . . IMP

X~ P~IDPt G~GMP

tRNA

GTP~GMP ~, GDP, /

Fig. I. Metabolic r e l a t i o n s h i p s a m o n g p u r i n e n u c l e o t i d e s in E. coli. T h e k n o w n p a t h w a y s are s h o w n w h i c h lead to t h e s y n t h e s i s of t R N A purines. T h e s e h a v e been reviewed in detail b y MAGASANIK 16. D a s h e d lines d e n o t e r e a c t i o n s of q u e s t i o n a b l e occurrence. T h e m u t a t i o n a l block in aden y l o s u c c i n a s e , c h a r a c t e r i s t i c of P u r B m u t a n t s , is i n d i c a t e d b y t h e b a r line.

METHODS AND MATERIALS

Growth and labeling o] bacteria: E. coli strains were grown at 37 ° in the Tris-casamino acids-glucose medium described b y THOMAS AND ABELSONTM, except that the final concentrations of casamino.acids and glucose were o.I and 0.2 ~o, respectively. In the case of strain B-94, the medium was supplemented with 0,25 mM adenine and 5 °/~g]ml of L-arginine.HC1 to satisfy the additional nutritional requirements of the organism jg. Isotopic additions were made early in logarithmic growth (2o-40 Klett units/ml; red filter) and incubations were continued at 37 ° as noted. Extraction and hydrolysis o! nucleic acids The labeled cultures were rapidly chilled and the cells were harvested and washed once with cold o.oi M Tris-HCl-o.ooI M magnesium acetate buffer (pH 7.4)The cells were resuspended in the same buffer and lysed with 2 % sodium lauryl sulfate-o.25 % bentonite. The bulk RNA was extracted and freed of DNA b y the method of MONIER et aI.% In order to assure the complete removal of phenol and all traces of labeled nucleotide, a gel-filtration step with Sephadex G-25 was added after the initial ethanol precipitation of the nucleic acids. The RNA preparations were dissolved in 1.5 ml of o.I M NaCl-o.o2 M TrisHCl-o.ooI M magnesium acetate buffer (pH 7.4), and were fractionated on a IOO cm ×2 cm column of Sephadex G-2oo (ref. 21), containing 0.2 mg/ml of bentonite. The column was preequilibrated and eluted with the above buffer. The ribosomal RNA (rRNA) and t R N A peaks were each pooled and the nucleic acids precipitated with 2 vol. of ethanol at --20 °. The RNA was collected b y centrifugation and dissolved in 1.5 ml of 0.02 M Tris-HC1 buffer (pH 7.4)Biochim. Biophys. Acta, 213 (197 o) 3 5 2 - 3 6

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H.O. KAMMV.N,S. J. SPENGLER

The rRNA and t R N A preparations were hydrolyzed with T 2 ribonuclease (ribonuclease CB, Calbiochem., lot No. 76II3), under conditions similar to those described b y UCHIDA~. A typical hydrolysis contained 5° mM sodium acetate buffer (pH 4.5), 2 mM EDTA, 65 A~,0 m~ units of RNA and 33o units of enzyme in a total volume of 3.o ml. The mixture was incubated for 18 h at 37 °.

Chromatographic procedures The RNA hydrolysates were diluted with 3 vol. of water and 2 #moles of carrier 2'(3')-IMP were added. The mixture was applied to a 23 cm × I cm column of Dowex I (formate) and was washed into the resin with a few ml of water. Separation of the major ribonucleotide components was achieved with a linear gradient of ammonium formate buffer (pH 4.75), ranging from o.2 to o. 9 M, with an initial volume of 4oo-6oo ml (see Figs. 2 and 4)- This gradient was chosen to elute the IMP fraction well ahead of most other purine nucleotides. The IMP peak emerged immediately after UMP. When all of the IMP was eluted (about 3/4 of the total volmne of ammonium formate buffer was required), an exponential gradient was instituted, with IOO ml of I M formic acid in the mixing vessel and 5 M formic acid in the reservoir. The AMP and GMP emerged as single sharp peaks, although the latter nucleotide was sometimes partially resolved into the 2'- and 3'-isomers. The Dowex-I column fractions were pooled and assayed for radioactivity. As a preliminary to further purification, the pooled IMP fractions were desalted by adsorption to Norit A and elution with ethanolic ammonia 2a. The eluates were taken to dryness under reduced pressure at 4 o°, redissolved in water and ultimately concentrated to a final volume of o.2 ml. Recovery of radioactivity after desalting was generally 8o %*. Traces of phosphomonoesterase in the T, ribonuclease led to the generation of significant amounts of atP1 during the prolonged hydrolysis of 32P-labeled nucleic acids. The PI eluted as a shoulder on the leading edge of the CMP peak and was quantitated by its failure to adsorb to Norit A. Additional purification of the desalted IMP fraction was carried out by chromatography on W h a t m a n 3 MM paper, or on silica gel and cellulose thin-layer sheets (Polygram-Cel and Polygram-Sil sheets, 25o/*, Brinkmann Instruments). The solvent systems employed were: (A) isobutyric a c i d - a m m o n i a - w a t e r (67:3:3o; by vol.); (B) ethanol-i M ammonium acetate buffer (pH 7.o) (7o:3o; by vol.); (C) butanol-water (86:14; by vol.); and (D) isopropanol-ammonia-water (7o:1:29; by vol.) 24. Enzymatic dephosphorylation of nucleotides was carried out in a total volume of o.i ml, which contained 5/,moles of Tris-HC1 buffer, p H 8.5, up to o.25/,mole of nucleotide and 2o/,g of E. coli alkaline phosphatase. The mixture was incubated for 6o min at 37 °, after which 25-/.I portions were removed and applied to Polygram sheets by streaking. The nucleosides were separated b y one-dimensional chromatography in Solvents C or D.

* Occasionally, w i t h 3~P-labeled t R N A digests, significant a m o u n t s of 32p in t h e I M P peak failed to adsorb to Norit A. The n a t u r e of this material has n o t been determined.

Biochim. Biophys. Acta, 213 (197 o) 352-364

BIOSYNTHESIS OF t R N A INOSINIC ACID

355

Examination o/nucleoside triphosphates The relative size of ribonucleoside triphosphate pools was estimated b y a modification of the method of NEUHARD et al. ~5. Acid-soluble extracts were prepared from IO-i5-ml cultures, labeled with 3~p at 37 °. The cells were collected on 47-mm Millipore membranes and were washed with 30 ml of 0.05 M Tris-HC1 buffer (pH 7-4). The membranes were then suspended in 5 ml of cold 5 ~o trichloroacetic acid and were agitated with a Vortex-type mixer. After 15 rain at 2 °, the acid-soluble fraction was recovered b y filtration through 0.45-# membrane filters. A portion of the filtrate (4.5 ml) was extracted 8 times with diethyl ether and was then freeze-dried. The final residues were dissolved in 0.2 ml of water. The nucleoside triphosphates were separated by two-dimensional chromatography on 20 cm × 20 cm PEI-cellulose sheets (Polygram MN-Cel 300 P E I , Brinkm a n n Instruments). Samples (IO or 2o/~1) were applied to one corner of the sheet and after drying, 3 #1 of a carrier mixture, containing 25 nmoles each of ATP, UTP, CTP, GTP and ITP, were spotted at the starting point. The chromatograms were developed in the first dimension with 1. 5 M LiCl-I.O M formic acid and dried in air. Desalting of the sheets with methanol, as recommended elsewhere 26, caused gross distortions in the migration of nucleotides during subsequent chromatography. However, the sheets were successfully desalted by gentle agitation in distilled water for 15 rain. The upper 1/3 of the sheets was cut off and discarded and the chromatograms were developed in the second dimension with 4 M sodium formate buffer (pH 5.0). The sheets were dried and subjected to radioautography for 2-3 days (Kodak No-Screen X-ray film), in order to define the location of labeled compounds. The identity of the labeled spots was readily correlated with the positions of the carrier triphosphates, as viewed with a 2537-~. Mineralite lamp (see Fig. 5). The areas corresponding to individual triphosphates were carefully cut from the sheets, immersed in scintillation fluor and counted.

Enzymatic phosphorylation assays Crude extracts of E. coli strains were prepared from cells grown to late logarithmic phase with Tris-casamino acids-glucose medium. The cells were harvested and washed once with 0.05 M Tris-HC1 buffer (pH 7-4). After resuspension in the same buffer, the cells were broken by sonic disruption with a Branson Sonifier, Model G-75 (45-sec treatment at power level No. 6). Debris was removed by centrifugation at 30 ooe ×g for 30 min and the supernatant fraction employed for enzyme assays. Nucleoside monophosphokinase and nucleoside diphosphokinase assays were performed in a total volume of o.I ml, which contained 25 mM Tris-HC1 buffer (pH 8.o), io mM magnesium acetate, 4 mM ATP, 3 mM labeled nucleotide (3oo-8oo counts/min per nmole) and enzyme. Aliquots (5-/A) were removed after o, IO, 2o and 4o min at 37 ° and were applied to Polygram PEI-cellulose sheets. Carrier mixtures, containing 2o nmoles of each of the appropriate nucleoside di- and triphosphates were then added to the origin. Resolution of the mono-, di- and triphosphates was achieved b y development in I.O M LiCl-I.O M acetic acid 2~. The nucleotides were visualized with a Mineralite lamp and were cut from the sheets and counted as above.

Radioactivity measurements Samples were counted with a Nuclear-Chicago low-background gas-flow counter Biochim. Biophys. Acta, 213 (197o) 352-364

356

H . O . KAMMEN, S. J. SPENGLER

or with a Nuclear-Chicago scintillation counter. The scintillation fluor consisted of a 25-fold dilution of Liquiflor (New England Nuclear Corp.) in toluene. Liquid samples were prepared for scintillation counting by mixing 0.25 ml of sample with 3 ml of ethylene glycol monomethyl ether and 5 ml of scintillation fluor.

Materials Carrier-free 32P1 was purchased from the New England Nuclear Corp. and all isotopically labeled nucleotides or polynucleotides from Schwarz Bioresearch. Unlabeled nucleotides were obtained from Calbiochem. and from P-L Laboratories; enzymes were from Worthington Biochemical Corp., except as noted. Polyadenylic acid was purchased from Miles Laboratories and tRNA (E. coli B) from General Biochemicals. 2'(3')-IMP and 5'-IMP were prepared from the respective adenosine nucleotides by the procedure of KAPLAN27.

RESULTS

Chemical deamination o/adenosine derivatives In order to develop reliable quantitative procedures, it was necessary to estimate the deamination of adenosine compounds arising from chemical methods of RNA degradation. Accordingly, a number of radioactive adenosine derivatives was subjected to treatments comparable to those employed for the hydrolysis of RNA. The results (Table I) indicated that the conditions of alkaline hydrolysis led TABLE i CHEMICAL AND ENZYMATIC DEAMINATION

OF ADENOSINE

DERIVATIVES

The t r e a t m e n t s were carried o u t w i t h 2-2. 5 Ftmoles of each c o m p o u n d in a final volume of I.O ml. E x p o s u r e to T~ ribonuclease (io 5 units) was in 5 ° mM acetate buffer (pH 4.5), containing 2 mM E D T A , and was t e r m i n a t e d b y the addition of 5 vol. of water. After t r e a t m e n t s w i t h N a O H , the N a + was r e m o v e d b y passing t h e samples t h r o u g h I - m l columns of Dowex-5o-NH,+. Two #moles of the a p p r o p r i a t e inosine derivative were t h e n added to the m i x t u r e (eitherinosine, 5'-IMP, or 2'(3')-IMP), and the respective adenosine and inosine derivatives resolved b y ionexchange c h r o m a t o g r a p h y on D o w e x - i - f o r m a t e columns. The p e r c e n t d e a m i n a t i o n is derived from the recovery of isotope in the respective nucleoside or nucleotide fractions.

Treatment

Compound

Deamination (o/~)

0. 3 M N a O H , 18 h, 37 °

[l~ClAdenosine 5'- [14C1AMP 2'(3')- [aH] AMP all-labeled poly A

0.37 0.33 0.29 o.35

o. 3 M N a O H , 2o rain, 8o °

aH-labeled poly A

o.3o

T 2 ribonuclease, 18 h, 37 °

5'-[14C]AMP 2' (3")- [3H] AMP 3H-labeled poly A

0.03 o.oi o.o 5

to o.3o-o.35 ~o deamination of the adenosine moiety, regardless of whether it was present as a nucleoside, nucleotide, or polynucleotide. The ratio of inosine derivative/ adenosine derivative produced under these conditions was about 45 % of the ratio Biochgm. Biophys. Acla, 213 (197 o) 352-364

BIOSYNTHESIS OF t R N A

357

INOSINIC ACID

which was found in E. coli tRNA (Table II) and represented an unacceptable blank. In contrast, a comparable treatment with T 2 ribonuclease led to much lower levels of deamination. The enzymatic method was therefore selected as a more suitable hydrolysis procedure for the following experiments. TABLE II I M P CONTI~I','TOF r R N A AND t R N A FROM E. coli B A logarithmic culture of E. coli B (8o ml) was g r o w n for four generations in T r i s - c a s a m i n o acidsglucose m e d i u m containing I o p C / m l of 66P1. The p r e p a r a t i o n and hydrolysis of the R N A fractions are described in the text. C h r o m a t o g r a p h y on D o w e x - i - f o r m a t e was carried o u t as s h o w n in Fig. 2. The I M P fraction was desalted and concentrated to o.2 ml volume. Aliqucts (6o-/~1) were spotted on 46 cm × 57 cm sheets of W h a t m a n 3 MM p a p e r and subjected to two-dimensional c h r o m a t o g r a p h y with Solvents A (first dimension) and 13 (second dimension). The radiochemical p u r i t y of the I M P spots was verified b y s u r v e y i n g an a p p r o p r i a t e section of the c h r o m a t o g r a m with a V a n g u a r d c h r o m a t o g r a p h i c scanner. The I M P spots were cut from the sheets, i m m e r s e d in scintillation fluor and counted,

RNA

Counts/rnin

rRNA tRNA

Ribonuclease digest

I M P fraction (Dowex-z)

Recovered in I M P after paper chromatography

3.39" lO3 1.71' lO 6

9 800 19 o4o

361 2397

Percent o/total phosphate recovered as I M P *

O.Oli o.155

Corrected for d e p h o s p h o r y l a t i o n of nucleotides during enzymatic hydrolysis.

I M P content of E. coli t R N A Although IMP has been found in E. coli tRNA ~,2, only limited quantitative information has appeared 2. An estimate of the IMP content of E. coli B tRNA was made by the 3~P-labeling technique, rRNA and tRNA were prepared from cells grown in supplemented Tris-casamino acids-glucose medium containing 3zp~; the analysis of 5-S rRNA was not attempted, since this RNA does not contain IMP ~. The RNA preparations were hydrolyzed to completion with T 2 ribonuelease and the

20

40

60

80

100

FRACTION NO.

Fig. 2. C h r o m a t o g r a p h i c resolution of T 6 ribonuclease digests of 36P-labeled t R N A f r o m E.

coli B. The labeled t R N A h y d r o l y s a t e was prepared, hydrolyzed and Iractionated on D o w e x - i formate, as described in the text. The initial v o l u m e of t h e linear gradient w a s 400 nil and t h e exponential gradient of formic acid w a s b e g u n a t the position of the arrow. The a b s o r b a n c e at 253. 7 mff was m o n i t o r e d w i t h an L K B Uvicord scanner. 5-ml fractions were collected and assayed for s6p. This same procedure was used for the resolution of the 82P-labeled r R N A h y d r o l y s a t e . 96-98 % of the 3,p applied to the c o l u m n s was recovered in t h e combined PI and mononucleotide peaks. The ultraviolet a b s o r p t i o n of t h e I M P p e a k is essentially t h a t of the carrier I M P added to the hydrolysate.

Biochim. Biophys. Acta, 213 (197 o) 352-364

358

a . O. KAMMEN, S. J. SPENGLER

proportion of the total 82p appearing in the IMP was determined by a combination of ion-exchange and paper chromatography (Fig. 2 and Table II). IMP was found to account for o.155 ~o of the total phosphate of the tRNA. Parallel digests of the rRNA contained i residue of IMP per 95oo nucleotides (O.Oli °/o). This is equivalent to o.5 residue per 7o-S equivalent, which would not appear to be structurally significant. If the IMP content of the rRNA digest is regarded as the chemical and enzymatic blank for both rRNA and tRNA, then the IMP content of the t R N A is corrected to o.144 % of the total phosphate. This value corresponds to I residue per 697 nucleotides and, assuming an average length of 8o nucleotides per tRNA chain, amounts to I IMP residue per 8. 7 chains.

Properties o/E. coli B-94 The absence of adenylosuccinase in this strain has been well documented l°. As a consequence of this enzymatic defect, strain B-94 shows an absolute growth requirement for adenine or adenine derivatives; this requirement cannot be filled by any other purines. In addition, the growth of strain B-94 also varies with tile concentration of adenine in the medium and is maximal at approx, o.4 mM adenine. The generation time for this strain, in Tris-casalnino acids--glucose medium, supplemented with L-arginine and with o.o5, o.I, o.25 and o. 4 mM adenine was IOO, 68, 53 and 43 rain, respectively, at 37 °. It should be emphasized that the ability of this strain to fill all of its purine requirements from exogenous adenine indicated that the metabolic pathways for the conversion of adenine derivatives to inosine derivatives and thence, to guanine derivatives, were intact (see Fig. I). Fig. 3 illustrates the utilization of several labeled purine precursors by strain B-94. As might be expected from the growth response, no incorporation of [14C]hypoxanthine into nucleic acids took place, unless adenine was present in the medium. When the cells were incubated with L14C]adenine (o.25 mM), the presence of an equal concentration of hypoxanthine reduced the rate of incorporation by about 4 ° ~/o throughout the course of the experiment. Under these conditions of suboptimal

o*200

/

Ld_

~E ox a:m 100 O_~ OO u_ZE c ~r

0

0

10 20 30 4 0 5 0 INCUBATION TIME,rain

Fig. 3- I n c o r p o r a t i o n of [14C]adenine and [14C]hypoxanthine into the nucleic acids of E. coli B-94. The cells were g r o w n in T r i s - c a s a m i n o acids-glucose-arginine m e d i u m containing o.25 mM adenine and were h a r v e s t e d in mid-log phase. T h e y were washed twice with T r i s - c a s a m i n o acidsglucose-arginine m e d i u m (minus adenine) and resuspended in the same m e d i u m plus the following p u r i n e supplements, each at a final concentration of o.25 mM: O - O , [l*C]adenine; @ - 0 , [14C]adenineplus unlabeled h y p o x a n t h i n e ; A - & , [14C]hypoxanthine; A - A , [14C]hypoxanthin¢ plus unlabeled adenine. Aliquots were w i t h d r a w n as shown, mixed with cold 5 % trichloroacetic acid and assayed for acid-precipitable 14C b y the m e m b r a n e filtration technique.

Biochim. Biophys. ,4eta, 2i 3 (197 o) 352-364

BIOSYNTHESIS OF t R N A

INOSINIC ACID

359

adenine concentration, the addition of hypoxanthine also promoted a slight increase in the growth rate, probably by sparing the adenine requirement for the synthesis of guanine derivatives. The e//ect o/ pool dilutions on the/ormation o~ t R N A I M P The metabolic properties of strain B-94 make it possible to attain a preferential dilution of inosine nucleotide pools without a parallel effect on the adenosine nucleotide pools. If strain B-94 were grown with a continuous source of [14C]adenine, all purine nucleotide pools would become labeled and would approach maximum specific activities after an extended period. If nonradioactive hypoxanthine were simultaneously supplied to the cells, only the inosine nucleotide and guanosine nucleotide pools would be diluted, since these are the only purine nucleotides which could be formed from hypoxanthine. Dilution of adenosine nucleotides could not occur, since strain B-94 cannot convert hypoxanthine to adenosine nucleotides (see Fig. I). This type of perturbation should indicate whether the labeling of tRNA IMP is responsive to changes in the inosine nucleotide pools. Strain B-94 was subjected to such a labeling experiment. The tRNA fractions were isolated, hydrolyzed with T 2 ribonuclease and the hydrolysates resolved on Dowex-I (Fig. 4). The labeling of the significant tRNA purine nucleotides is sum-

08

0.4

0 c

4

~ 1 2

3

4

5

, I

0.0 46

7

8

08 ~

04

20

40

60 80 FRACTION NO.

100

00

Fig. 4. F r a c t i o n a t i o n of digests of 14C-labeled t R N A f r o m E. coli B-94. An overnight culture of E. coli B-94 w a s diluted 4o-fold into fresh T r i s - e a s a m i n o acids-glucose-arginine medium, s u p p l e m e n t e d w i t h o.25 mM adenine, and was g r o w n at 37 °. W h e n the cell d e n s i t y reached 20 K l e t t units/ml, [14C~adenine was added to a final specific a c t i v i t y of o. 5 #C/ml. The i n c u b a t i o n was continued for an additional t w o generations, either in the absence or presence of o.25 mM h y p o x a n t h i n e . The t R N A was p r e p a r e d a n d hydrolyzed w i t h T 2 ribonuclease as described in METHODS AND MATERIALS. The digests were fractionated on D o w e x i (formate), w i t h an initial v o l u m e of a m m o n i u m f o r m a t e buffer of 600 ml. The exponential formic acid gradient was s t a r t e d at the point s h o w n b y t h e arrow. The fractions (5 ml) were assayed for 14C and those corresponding to labeled peaks were pooled. The s u m m a r y of the labeling of each pooled fraction is given in Table I I I .

marized in Table III. In view of the small amounts of radioactivity involved, efforts were made to assure the isotopic purity of the IMP fractions. The IMP peak recovered from Dowex-I chromatography was desalted, concentrated and subjected to further chromatographic and degradative procedures. Approx. 5 ° % of the 1~C recovered from the column was found to be associated with IMP after additional two-dimensionBiochim. Biophys. Acta, 213 (197 o) 352-364

360

H . O . KAMMEN, S. J. SPENGLER

TABLE III THE EFFECT OF HYPOXANTHINE

ON T H E L A B E L I N G O F t R N A

PURINE NUCLEOTIDES

OF

E . eoli B-94

T h e f r a c t i o n s c o r r e s p o n d to t h o s e s h o w n in Fig. 4.

Fraction

I 2 3 4 5 6 7 8

Major constituents

Not identified Adenosine Guanosine+CMP UMP IMP AMP Not identified GMP

IMP (after r e c h r o m a t o g r a p h y ) " IMP (after c o n v e r s i o n t o nucleoside)~" AMP counts/min/IMP counts/min ratio G.'V[P c o u n t s / m i n / I M P c o u n t s / r a i n r a t i o

:~C (counts~rain) Minus hypoxanthine

Plus hypoxanthine

2 4oo 9 730 5 080 35 ° 82I 67 8o0 3 57 ° :13 57 °

64o i i 246 i 54 ° 53 ° I oIo 77 22o i 5o4 23 5o7

442

536

42o

487

153 266

144 43.9

" T h e D o w e x - I p e a k w a s d e s a l t e d w i t h N o r i t A, c o n c e n t r a t e d , a n d s u b j e c t e d to t w o - d i m e n s i o n a l t h i n - l a y e r c h r o m a t o g r a p h y w i t h S o l v e n t A a n d B. The v a l u e s are t h e a v e r a g e of t h r e e separate determinations. "* T h e d e s a l t e d I M P p e a k w a s t r e a t e d w i t h a l k a l i n e p h o s p h a t a s e , a n d t h e d i g e s t c h r o m a t o g r a p h e d on t h i n - l a y e r s h e e t s in S o l v e n t s C or D.

al chromatography. A comparable proportion was recovered as inosine after treatment of the desalted column fraction with E. coli alkaline phosphatase (Table I I I ) . Since direct measurements of the specific activities of the IMP fractions were not practical, we have also presented the labeling data in Table I I I as the ratios of :4C in the AMP and GMP fractions to that in the IMP fraction. This procedure also served to normalize the results for any differences in the recovery of tRNA. Three major features of this experiment are notable: (a) no change in the labeling of the t R N A IMP occurred under conditions which would selectively dilute the inosine nucleotide and guanosine nucleotide pools; (b) the labeling of the t R N A GMP was markedly reduced. This change served as an internal control in the experiment and attested to the success of the dilution attempt; (c) the labeling of the IMP fraction paralleled that of the AMP. These changes might possibly occur as a consequeloce of wide fluctuations in the size of nucleoside triphosphate pools subsequent to the addition of hypoxanthine. The relative size of these pools was examined in separate experiments and was found to be independent of the addition of hypoxanthine (Table IV). Since the formation of the t R N A IMP did not respond to changes in the inosine nucleotide pool, it must have occurred by a mechanism independent of this pool, namely by a polynucleotide modification reaction. The simplest mechanism of this type would be the deamination of specific AMP residues of a t R N A precursor. Such a mechanism would require a constant A M P : I M P ratio in the dilution experiment. This constancy was observed, in fact (Table I I I ) , Bioehim. Biophys. Aeta, 213 (197 o) 352-364

BIOSYNTHESIS OF

tRNA

361

INOSINIC ACID

TABLE IV R E L A T I V E SIZES OF N U C L E O S I D E T R I P H O S P H A T E

POOLS

2o ml of cells were g r o w n in T r i s - c a s a m i n o acids-glucose m e d i u m at 37 °. At a cell density of 4 ° K l e t t units/ml, 32Pt was added (final activity 6. 7 ffC/ml) and the culture was divided and inc u b a t e d for an additional generation in the absence or presence of o.25 mlV[ h y p o x a n t h i n e . T h e extraction and separation of nueleoside t r i p h o s p h a t e s are described in METHODS AND MATERIALS, and are s h o w n in Fig. 4.

Strain

Nucleoside triphosphate

z2p (counts~rain per ml o/culture) 3/linus hypoxanthine

Plus hypoxanlhine

E. coli 13

ATP UTP CTP GTP ITP

544 ° 1896 81 o 486 lO

4800 1454 708 388 12

E. coli ]3-94

ATP UTP CTP GTP ITP

6450 1874 984 260 15

615o 248o lO3O 208 28

Examination o~ nucleotide pools in E. coli There is no evidence that RNA polymerase can distinguish between GTP and I T P on the basis of coding specificity. In the absence of such a discrimination, it follows that the presence of I T P in a cell could lead to the incorporation of IMP residues into all species of RNA, with possible lethal consequences. Since IMP is not a normal constituent of all species of RNA and is introduced into tRNA only after assembly of the polynucleotide, there would appear to be metabolic restrictions for the availability of I T P in cells. Although I T P is known to function in a variety of enzymatic reactions (see ref. 29), there is little unambiguous evidence that it occurs naturally 29, except, perhaps, in erythrocytes a°. An a t t e m p t was therefore made to ascertain whether I T P could be detected in the acid-soluble fraction of E. coli strains. The cells were grown in a2P1 and the

=TPc) @O UTP

"" L,j/"'~

g 2

2 4 M Na F o r m a t e

Or. 1314 5 0

Fig. 5. Thin-layer c h r o m a t o g r a p h i c separation of ribonucleoside t r i p h o s p h a t e s . The ~2P-labeled e x t r a c t s were s e p a r a t e d on PEr-cellulose sheets as described in METHODS AND MATERIALs.The i d e n t i t y of the m a j o r labeled ribonucleoside t r i p h o s p h a t e s is shown. The position of I T P , as determined with a NIineralite lamp, is s h o w n b y the dashed line. The i d e n t i t y of the o t h e r labeled spots was not determined.

Biochim. Biophys. Acta, 213 (197 o) 352-364

362

H . O . KAMMEN, S. J. SPENGLER

acid-soluble pools of nucleoside triphosphates separate by two-dimensional chromatography on PEI-cellulose (Fig. 5)- No significant pool of I T P was detectable in E. coli B or E. coli B-94. The m a x i m u m level of I T P was < o.3 % of the amount of ATP (Table IV). This experiment also confirmed that there was little change in the size of individual ribonucleoside triphosphate pools after extended exposure of the cells to o.25 mM hypoxanthine. The relative content of each ribonucleoside triphosphate was similar in both strains, with the exception of a somewhat smaller GTP pool in strain B-94.

Phosphorylation o/ ribonucleotides by extracts/rom E. coli B The absence of a demonstrable I T P pool in E. coli led us to examine the potential enzymatic restrictions. Several possibilities were entertained: (I) failure to phosphorylate 5'-IMP to tile di- or triphosphate; (2) failure to phosphorylate I D P to ITP; (3) the presence of a powerful, specific ITPase, which would cleave I T P more rapidly than it was formed. An examination of crude extracts of E. coli was sufficient to disclose the limitation in the formation of I T P (Tables V, VI). These extracts readily phosphoTABLE V PHOSPHORYLATION

OF RIBONUCLEOTIDES

BY EXTRACTS

FROM

Substrate

nmoles phosphorylated per rain per mg protein

[14CLUMP [aH] CMP [aH]GMP [14C] AMP E14CjIMP

21.o 5.54 2o.1 377.5 <0.02

[aH]CDP [all] G D P [14C]ADP

846.0 945-o 1389.o

[t4C]IDP

119o.o

E. coli 13

T A B L E VI RATE

OF DEPIIOSPHORYLATION OF NUCLEOSIDE TRIPHOSPHATES B Y

EXTRACTS FROM

E . coli ]3

I n c u b a t i o n Inixtures (o.i-ml) contained io InM MgC1v 25 ulM Tris-HC1 buffer (pH 8.0); 3 nlM uucleotide and E. coli e x t r a c t (53/zg protein). W h e r e indicated, A T P was added at a concentration of 4 raM. D e p h o s p h o r y l a t i o n was determined from the a m o u n t of t r i p h o s p h a t e remaining after 2o-min incubation at 37 °.

Nucleotide

ATP UTP CTP GTP 1TP

nmoles o/substrate degraded per rain per mg protein --ATP

+ATP

25. 3 32.3 47.7 34-7 40.2

---

.Biochim. Biophys. Acta, 213 (197 o) 352-364

15.1 12.6

BIOSYNTHESIS OF t R N A INOSINIC ACID

363

rylated I D P to I T P (Table V) and showed no preference for the breakdown of I T P (Table VI). However, the extracts were totally incapable of phosphorylating IMP to I D P (Table V). The absence of this activity would appear to be the chief restriction in the formation of I T P in E. coli. DISCUSSION The amount of IMP found in the tRNA of E. coli is small but m a y represent about 60 % of the content reported for yeast t R N A 3. Our estimate that IMP accounts for 0.144 O/,oof the phosphate of E. cull B t R N A agrees closely with the findings of OFENGAND 2, who determined the IMP content to be o . I I residue per chain, from their reactivity with acrylonitrile. The reactivity with acrylonitrile also indicated that the IMP residues were situated solely within unpaired regions of the t R N A 2. In this type of work, it should be remembered that conventional alkaline hydrolysis of RNA leads to significant deamination of adenosine compounds. This problem can be minimized b y the use of enzymatic hydrolysis with T 2 ribonuclease, or with mixtures of T 2, pancreatic and T 1 ribonucleases. It must be emphasized that these estimates represent the IMP content in the total population of t R N A molecules present under certain experimental conditions and do not directly reflect the number of t R N A species which contain this nucleotide. E.g., it is conceivable that only one or a few species of E. coli t R N A m a y contain IMP, but that these make up a relatively large percentage of the population of t R N A molecules. Furthermore, this distribution might change during the growth cycle al or as a function of other physiological variables 3-°. The fact that the formation of tRNA IMP is insensitive to fluctuations in the inosine nucleotide pool provides strong evidence that these residues are generated by the enzymatic modification of a polynucleotide precursor. The constant ratio of AMP/IMP labeling in the E. cull t R N A (Table I I I ) points to the origin of the IMP residues from specific adenylate residues of a latent tRNA, via a polynucleotide deaminase reaction (an "anticodon deaminase"). The reaction might also be more complex, involving an addition reaction, followed by an elimination step. WOLFENDEN et al. aa have observed that the adenosine deaminase from Aspergilhts oryzae is weakly active toward short oligonucleotides of AMP. However, polyadenylic acid and the terminal adenosine of t R N A were not deaminated and it seems unlikely that this type of enzyme participates in the deamination of tRNA. A primary difficulty in attempting to characterize the polynucleotide deaminase would be the availability of a suitable substrate. One of the major corollaries of the "wobble" hypothesis is that anticodons should not normally occur with AMP at the 5'-terminus TM. Thus far, no exceptions to this corollary have been found among any of the tRNA species whose sequence has been elucidated 8 (see ref. 35)- A presumptive tRNA precursor has been detected in mammalian cells ~n,37, but no evidence has been presented as to whether it is fully aminated. A further difficulty is that it is not possible, at present, to chemically generate deaminated forms of t R N A by reactions which retain the integrity of the polynncleotide backbone. The most hopeful approaches to the characterization of the enzyme would probably involve the use of t R N A precursor species, produced enzymatically by the transcription of t R N A cistrons as or of synthetic t R N A genes 39. Biochim. Biophys. Acta, 213 (197o) 352-.364

364

H. O. KAMMEN, S. J. S P E N G L E R

ACKNOWLEDGMENTS

The authors are indebted to Dr. J. S. GOTS for supplying E. coli Dr. T. H. JOKES for his supporting interest and to Drs. J. S. KRAI
B-94, to J. OFENNational Berkeley

REFERENCES i F. GANGER AND 0. O. BROVCNLEE,in L. GROSSMAN AND I{. MOLDAVE, Methods in Jinzymology,

Vol. 12, Academic Press, New York, 1967, p. 3792 J. OEENGAND, Cold Spring Harbor Syrup. Quant. Biol.: 31 (1966) 465 , 3 1¢.. H. HALL, Biochemistry, 4 (I965) 661. 4 ]{' xvV" HOLLEY, J. APGAR, G. A. EVERETT, J. T. MADISON, M. MARQUISEE, S. H. MERILL, J. R. PENSWlCK AND A. ZA~IIR, Science, 147 (1965) 1462. 5 H. G. ZACHAI:, D. DUTTING AND H. FELDMANN, Z. Physiol. Chem., 347 (1966) 212. 6 A. A. ]]AEV, T. V. VENKSTERN, A. D. MIRZABEKOV, A. ]. t{RUTIL1NA, L. LI AND V. D. AXELROD, ~a¢ol. Biol., I (1967) 754. 7 M. STAEHLIN, H. ROGG, B. C. BAGULEY, T. GINS~ERG AND "vV. ~VEHRLI, Nature, 219 (1968) 1363. 8 J. T. MADISON, Ann. Rev. Biochem., 37 (1968) 131. 9 F. H. C. CRICK, J. ~,Vlol. Biol., 19 (1966) 546. IO E. FLEISSNER AND ]~. BOREK, Biochemistry, 2 (1963) lO93. i i J. HURWlTZ, M. GOLD AND M, ANDERS, J . Biol. Chem., 239 (1964) 3462. 12 M. N. LIPSETT AND i . PETERKOFSKY, Proc. Natl. Acad. Sci. U.S., 55 (1966) 1169. 13 R. S. I-{AYWARDAND S. B. ~VEISS, Proc. Natl. Acad. Sci. U.S., 55 (1966) 1161. 14 L. K. KLINE, F. FITTLER AND R. I-1. HALL, Biochemistry, 8 (1969) 4361. 15 A. L. TAYLOR AND C. D. TROTTER, Bacteriol. Rev., 31 (1967) 33716 B. MAGASANIK, in I. C. GUNSALUS AND R. Y. GTANIER, The Bacteria, Vol. 3 ,Academic Press, New York, 1962, p. 295. 17 H. O. KAMMI~N, Federation Proc., 28 (1969) 865. i8 C. A. THOMAS AND J. ABELSON, in G. L. CANTONI AND D. it. DAVIES, Procedures in Nucleie Acid Research, H a r p e r and Row, N e w York, 1966, p. 55319 E. G. GOLLUB AND J. S. GOTS, J. Bacteriol., 78 (I959) 320. 20 It. MONIER, G. NAONO, D. HAYES, F. HAYES AND F. GROS, J. Mol. Biol., 5 (1962) 311. 21 T. SCHLEICVl AND J. GOLDSTEIN, J. ~/Iol. Biol., 15 (1966) 136. 22 T. UCHIDA, J. Biochem. Tokyo, 60 (1966) 115. 23 S. MANDELES AND H. O. KAMMEN, Anal. Biochem., 17 (1966) 54 o. 24 J. OFENGAKD, J. Biol. Chem., 242 (1967) 5034 . 25 J. NEUIIARD, E. ]tANDERATH AND K. RANDERATH, Anal. Biochem., 13 (1965) 2~I. 26 K. I{ANDERATH AND E . ]~_ANDERATH, J. Chromatog., 16 (1964) I I I . 27 N. O. KAPLAY, in S. P. COLOWICK AND N. O. I{APLAN, 3/Iethods in Enzymology, Vol. 3, Acade mic Press, N e w York, 1957, p. 873. 28 (~. G. BROV.rNLEE, F. GANGER AND B. G. BARRELL, Nature, 215 (1967) 735. 29 M. F. UTTER, in P. D. ]3DYER, t{. LARDY AND K. MYRBXCK, The Enzymes, Vol. 2 Academi( Press, N e w York, 196o, p. 75. 3 ° B. S. VANDERHEIDEN, Biochem. Biophys. Res. Commun., 21 (1965) 265. 31 1{. H. DoI, I. KANEKO AND R. T. IGARASHI, J. Biol. Chem., 243 (1968) 94532 M. GEFTER AND }t. L. I{USSELL, J. 1~/10l. Biol., 39 (1969) 14533 I{. \¥OLFENDEN, T. K. SHARPLESS AND R. ALLAN, J. Biol. Chem., 242 (1967) 977. 34 R. 1,2. RALPH, Biochem. Biophys. Res. Commun., 3° (1968) 192. 35 G. l{. PHILIPPS, Nature, 223 (I969) 374. 36 I{. IA. BURDON, B. J. MARTIN AND B. M. LEE, J. 3!rol. Biol.,28 (1967) 357' 37 J. E. K:tv AND H. L. COOPER, Biochim. Biophys. Acta, 186 (1969) 62. 38 A. MARKS AND J. H. SPENCER, Federation Proc, 28 (1969) 531. 39 V. SGARAMELLA, N. GUPTA AND H. G. KHORANA, Federation. Proc., 28 (1969) 532. 4 ° J. HURWlTZ, M. GOLD AND M, ANDERS, J. Biol. Chem., 239 (1964) 3474-

Biochim. Biophys. Acta, 213 (197 o) 352-364