Amino acids in yeast acceptor ribonucleic acid

BIOCHIMICA ET BIOPHYSICA ACTA

583

BBA 8155

AMINO ACIDS IN YEAST ACCEPTOR RIBONUCLEIC ACID v. M. INGRAM AND EMELIE SULLIVAN* Division of Biochemistry, Department o/ Biology, Massachusetts Institute o~ Technology, Cambridge, Mass. (U.S.A.)

(Received May 7th, 1962)

SUMMARY Yeast acceptor RNA was found to contain considerable quantities of amino acids bound to the RNA in a manner unknown, but different from the enzymically attached amino acids. INTRODUCTION In the course of other investigations on the chemical properties of yeast (amino acid) acceptor RNA, we have detected the presence of amino acids in our preparations, which were extraordinarily hard to remove. The present experiments were undertaken to see whether the amino acids were merely adsorbed to the acceptor RNA and whether they were distinguishable from the enzymically attached amino acids which are intermediates in protein synthesis. METHODS AND RESULTS The yeast acceptor RNA was prepared according to a modification 1 of MONIER'S method 2. To determine the total quantity of amino acids present, aliquots of solutions of the nucleic acid in water, previously incubated with a trace of pancreatic RNAase, were reacted with the ninhydrin reagent described by MOORE AND STEIN3. It was usual to find between 0.6 and I/zmole total amino acids per /,mole RNA (mol. wt. 30 ooo). These amounts could not be reduced by further purification on DEAEcellulose columns 1, which separated added amino acids very easily. A sample of acceptor RNA (40 mg = 1.3/~moles) was dissolved in 4.0 ml of water; the pH was raised to IO at 38 ° with 0. 5 N NaOH and held for I h. This is the conventional method for liberating socalled "bound" amino acids from acceptor RNA. The amino acids were allowed to dialyze without previous adjustment of pH at 4 ° into 2 x 25 ml of water. The dried combined dialysates were dissolved in 3.0 ml of 0.20 M citrate buffer (pH 2.2) and were analyzed for amino acids on the Spinco Model-MS automatic analyzer (Table I). In addition to the amino acids listed, there were three small unidentified peaks emerging before aspartic acid on the chromatogram. The presence of the amino acids in our acceptor RNA preparations could also be demonstrated by paper ionophoresis as illustrated in Fig. I. The astonishing difference between dry and wet loading at pH 3.5 is worth noting; it would seem that the especially high local loading produced by the dry-loading process, and/or some other factor, * Present address: Arthur D. Little Company, Cambridge, Mass. (U.S.A.). Biochim. Biophys. Acta, 61 (1962) 583-587

584

V. M. INGRAM, E. SULLIVAN

TABLE I ANALYSIS

OF AMINO ACIDS OBTAINED FROM ACCEPTOR B Y D I A L Y S I S O F AN A L K A L I N E S O L U T I O N

Amino acids

ml~moles

Lysine Histidine Arginine A s p a r t i c acid Threonine Serine G l u t a m i c acid Proline total

I{.2~TA

Amino acids

mr*moles

2o6 Glycine 39 Alanine trace Valine 84 Isoleucine 76 Leucine 380 Tyrosine 36 Phenylalanine trace I. 3 4 / , m o l e s I.O/*mole//*mole R N A (mol. wt. 3 ° ooo)

216 i 12 5i 42 48 3° 23

Point of Applicotion Lys r

CI

A.0

d,

~

/

7-

--

Cyfidine

0

;',~:\T-,,

..cleo~ide~

Adenosinel Guonosine

b

~ %~ (~ ys

Ar9

LyS

Gy {~ ff

Cytldin~

RNA - S(t.nple

~

NeLJtroI Glu Amino Acids

+

~

A.ninoAcids

Asp

+

Io Set

Amino

$0~ ~

~0 O --

Amino Acids

~-3

A~denosine Guo/nosine\Uridine

Acids

R,,,~-So,n,,,, +

Fig. I. P a p e r i o n o p h o r e s i s of acceptor R N A p r e p a r a t i o n s p e r f o r m e d on W h a t m a n 3 MM p a p e r on 24-in water-cooled b r a s s p l a t e 5, u s i n g a double t h i c k n e s s of p a p e r a t each end d i p p i n g into t h e b u f f e r vessels. Solid line: n i n h y d r i n - p o s i t i v e areas; i n t e r r u p t e d line: u l t r a v i o l e t - a b s o r b i n g areas. D e v e l o p m e n t a t r o o m t e m p e r a t u r e w i t h 0. 5 % n i n h y d r i n in a c e t o n e c o n t a i n i n g 5 % (v/v) of o.05 M p h o s p h a t e b u f f e r (pH 7.2). a, Buffer: a m m o n i u m f o r m a t e (pl-{ 3.5) (RusHIZKY A~D KNIGHT G); d r y loading: R N A , 0. 5 m g in 5 °/*1 of w a t e r on I in; 5/*1 of a solution of 17 a m i n o acids, 2. 5 / * m o l e s / m l ( B e c k m a n Spinco Co., c a l i b r a t i n g m i x t u r e , cat. No. 12o-22o); 5/~1 of a n a q u e o u s s u s p e n s i o n of adenosine, cytidine, g u a n o s i n e a n d uridine, 1 % in each. b, B u f f e r a n d s a m p l e s a s (a). W e t l o a d i n g (lysine a p p a r e n t l y too low to be visible in t h i s s a m p l e ) , c. Buffer: a m m o n i u m f o r m a t e (pH 2.7) (RusHIZKY AND KNIGHTe). S a m p l e s as in (a).

causes the amino acids to run as a group close to the same position as the adenosine marker. The position of adenosine itself, but not those of the other nucleosides, is greatly altered by dry loading. In other experiments it was found that digests of acceptor RNA by pancreatic RNAase behave similarly under conditions of dry loading; the amino acids, even lysine, occur close together and almost, but not quite, coincide with the adenosine present in these digests. However, at pH 2.7 the nucleosides and the amino acids in these digests are well spread out and separated. Perhaps this finding may explain in part the report by LIPMANN et al. 4, that amino Biochim. Biophys. Acta, 61 (1962) 5 8 3 - 5 8 7

585

YEAST ACCEPTOR R N A

acids are found covalently linked to liver acceptor RNA without previous labeling and that they remain combined with the adenosine fragment produced by the action of pancreatic RNAase on this RNA. The conditions of their ionophoretic separation may well approximate our conditions of dry loading; the fact that adenosine and the amino acids, including even lysine and histidine, were found together on their ionogram 4 may be a reflection of their experimental conditions rather than evidence for covalent linkage. It is interesting to note that the relative frequencies of various amino acids found in liver acceptor RNA by these authors is rather similar to the spectrum observed by us in yeast acceptor RNA (Table I). As shown below, however, we have reason to believe that these amino acids are not those which can be attached enzymically to the RN-A. Attempts were made to see whether the amino acids present in our RNA could be exchanged with 14C-labeled amino acids. In one such experiment 7.0 mg of acceptor RNA in 5.0 ml of water, pH 7.1, were heated with 2o/zl containing 1.5 #C (O.Ol9 rag) of mixed ~4C-labeled amino acids (Isotopes Specialties Co., "Algal Protein Hydrolysate") to 9°0 for 15 min, followed by slow cooling to 720 in 15 rain and rapid cooling to room temperature. The acceptor RNA was reisolated on the usual DEAEcellulose column (Fig. 2a) and finally dialyzed and lyophilized. No radioactivity became associated with the RNA. On the other hand, paper ionophoresis as in Fig. 3a of the recovered RNA showed the usual distribution of free amino acids by ninhydrin staining while the RNA itself remained adsorbed at the origin. A number of 344000

~,~

2oo

(oi

'

2 HO00

b~: .905 00%

(bl

1"

,25ooo....../~,

!

~24

i Fr oction Number

Froc]i

Number

Fig. 2. E l u t i o n diagram of acceptor R N A on DEAE-cellulose columns ~. Interrupted line: absorbancy at 26o m#; solid line: t o t a l counts per rain per fraction, a, Acceptor R N A (5 mg in 2.0 ml water) and mixed x4C-labeled amino acids (1. 5/~1 as in b), heated to 9 °0 at pH 7.1 for 15 rain b, Enzymic labeling of acceptor RNA with 14C-labeled amino acids at 380 for 15 min. The conditions were i j : 5 mg RNA in 2.0 ml of water, 6.2 mg Na2ATP, 5 °/~1 of Na2CTP solution (2. 5 rag/ ml), 5o/~1 of i M MgC12; the p t t adjusted at 38° to 7.9 with I N NaOH; IOO/zl of mixed 14C-labeled amino acids (Isotopes Specialties Co., Algal protein hydrolysate, 15/*C, o.19 mg); 5° #1 of crude yeast enzymeL The first ultraviolet-absorbing peak presumably contains the residual ATP and CTP and their degradation products.

other conditions were investigated in a similar manner to try and obtain exchange of the amino acids already present with l~C-labeled amino acids, but without any success whatsoever; these conditions were: pH IO.I, 38°, 15 rain; pH 1. 9, 38°, 15 min in the presence of cellulose fibers; the usual activating system 1, which includes ATP, CTP, MgC12, but with the enzyme solution previously boiled. In none of these experiments were radioactive amino acids found in the recovered acceptor RNA. In most cases, the RNA recovered by chromatography on DEAE-cellulose was tested Biochim. Biophys. Acta, 61 (1962) 583-587

586

V.M.

INGRAM, E. SULLIVAN

Fig. 3. P a p e r i o n o p h o r e s i s a t pI-I 1.9 of a c c e p t o r R N A e n z y m i c a l l y labeled w i t h m i x e d 1*C-labeled a m i n o acids. T h e R N A (4.2 m g ; specific a c t i v i t y = 25 70o c o u n t s / r a i n / r a g ; Fig. 2b), in 4 °/~1 of w a t e r w a s loaded o n t o I in of t h e s t a r t i n g line on W h a t m a n 3 MM p a p e r m o i s t w i t h t h e p H 1.9 buffer 8. Controls were io-/~1 s a m p l e s of I : io a n d I : 25 d i l u t i o n s of t h e s t a n d a r d a m i n o acid m i x t u r e (Fig. Ia). T h e i o n o p h o r e s i s (Fig. I) w a s a t 20o0 V for 4 h w h i c h allowed t h e basic a m i n o acids to leave t h e paper. T h e s t r o n g a m i n o acid s p o t s in (a) are, f r o m t h e c a t h o d e side, glycine, a l a n i n e a n d serine (see also T a b l e I). a, T h e dried i o n o g r a m w a s developed w i t h n i n h y d r i n as in Fig. I; dipped t h r e e t i m e s ; b, (on t h e n e x t page) r a d i o a u t o g l a p h

for free "cold" amino acids by paper ionophoresis at pH 1. 9 and was found to contain them in the usual proportions. For comparison, Fig. 2b shows the DEAE-cellulose chromatogram of acceptor RNA incubated with the mixed 14C-labeled amino acids, native activating enzymes, etc., in which case the acceptor RNA became labeled. The properties of the original amino acids in the RNA and those attached enzymically, were compared by paper ionophoresis (Fig. 3)- In Fig. 3a it can be seen that the basic amino acids lysine, histidine, and arginine have moved off the paper, but that the remainder are well separated; the yellow color of proline was definitely present in its correct place. Examination under ultraviolet light before ninhydrin development showed that all the RNA had remained adsorbed at the origin. A radioautograph of the same ionogram (Fig. 3b) was made by exposure of Ansco non-screen X-ray film against the ionogram for 5 days. As all the radioactivity remained at the origin with the acceptor RNA, it would appear that the enzymically attached amino acids occupy a different linkage on the RNA than do the amino acids originally present in the preparation. As before, glycine, alanine, serine (or isoleucine) are the commonest amino acids. This method of ionophoresis does not distinguish between serine and isoleucine, but it is presumably mainly the Biochim. Biophys. Acta, 6I (1962) 5 8 3 - 5 8 7

YEAST ACCEPTOR R N A

587

former amino acid in view of the results in Table I. Fig. 3b The only effective way to rid our R N A of amino acids was by passing a sample of the nucleic acid previously incubated at pH Io, 38°, for I h through a Sephadex G-25 column in water without first adjusting the pH of the solution. The experiments show that our preparations of yeast acceptor R N A contain a characteristic spectrum of residual amino acids, which, because we have been unable to exchange them for added 14C-labeled amino acids, appear to be bound to the R N A in a manner unknown, but different to that by which the enzymically attached amino acids are held. They are, however, released by incubation at pH Io, as are the enzymically attached ones. The significance of the presence of these amino acids is obscure. ACKNOWLEDGEMENT

This investigation was supported by grant No. A-34o9(CI} from the U. S. Public Health Service. 1 2 3 4 5 6 7 s

REFERENCES M. INGRAM AND J. G. PIERCE, Biochemistry, I ( I 9 6 2 ) 580. ~'ONIER, M. L. STEPHENSON AND P. C. ZAMECNIK, Biochim. Biophys. ,4cta, 43 (I96o) I. MOORE AND ~V. H . STEIN, J. Biol. Chem., 211 (1954) 907. LIPMANN, VV. C. HULSMANN, G. ]-~ARTMANN, H. G. BOMAN AND Cr. A c s . Syrup. Enz. React. 34eehanism, O a k R i d g e , 1959, p. 75A. O. \~7-. STRETTON AND V. M. [NGRAM, Biochim. Biophys. Acta, 62 ( 1 9 6 2 ) 456. j . W. RUSHIZKY AND C. A. KNIGHT, Virology, II (196o) 236. p. C. ZAMECNIK, M, L. STEPHENSON AND J. F. SCOTT, Proc. Natl. ,4cad. Sci. U.S., 46 (196o) 8 i t . G. N. ATFIELD AND C. J. o . R. MORRIS, Biochem. J., 74 (196°) 37. V. R. S. F.

Biochim. Biophys. Acta, 6 i (1962) 5 8 3 - 5 8 7