V O L . 5, N O .
11,
NOVEMBER
1966
Resolution of Cytidine- and Adenosine-Terminal Transfer Ribonucleic Acids” Paul Lebowitz, Pier L. Ipata,? Maynard H. Makman,: Henry H. Richards, and Giulio L. Cantoni
ABSTRACT :
Transfer ribonucleic acid (t-RNA) serine from baker’s yeast, purified by repeated partition chromatography on Sephadex, was fractionated further in three different countercurrent distribution systems. In each of these, t-RNA serine was resolved into two species which were detected and distinguished in studies utilizing purified seryl-t-RNA synthetase and cytidine and adenosine monophosphate incorporating enzyme. One of the t-RNA serine species terminated
A
number of observations indicate that a transfer ribonucleic acid (t-RNA) preparation, specific in its acceptor function for a single amino acid, may be resolved into two or more fractions by various chromatographic or biochemical procedures. The nature of this multiplicity of species has been explored by a number of investigators. On the basis of the important contribution of Weisblum et al. (1962), who found that two distinct leucyl-t-RNA’s (Escherichia coli) would respond to different code words in a ribosomal amino acid incorporation system, it has generally been assumed that multiplicity of peaks for a given amino acid in chromatography or countercurrent distribution of t-RNA is equivalent to coding degeneracy. The extent of the difference in nucleotide composition or sequence between degenerate t-RNA species is not known; it may be recalled, however, that Berg et ai. (1962) found that t-RNA leucine 1 and t-RNA leucine 2 (E. coli) differed in the sequence of nucleotides adjacent to the amino acid acceptor terminal CpCpA (cytidine-cytidine-adenosine),’ and that Berg et ul.
* From the Laboratory of General and Comparative Biochemistry, National Institute of Mental Health, U. S. Department of Health, Education, and Welfare, U. S . Public Health Service, National Institutes of Health, Bethesda, Maryland 20014. Receioed June 21, 1966. Some aspects of this work were presented previously at the 6th International Congress of Biochemistry, New York, N. Y., July 1964 (see also Makman and Cantoni, 1966). t Present Address: Institute of Biochemistry, University of Pisa, Italy. $ Present Address: Departments of Biochemistry and Pharmacology, Albert Einstein College of Medicine, New York, N. Y . 1 Abbreviations used are: C M P and AMP, cytidine and adenosine monophosphates; CTP and ATP, cytidine and adenosine triphosphates; CpCpA, 3’-OH-terminal nucleotide sequence common to all t-RNA’s; TCA, trichloroacetic acid; PPO, 2,s-diphenyloxazole; POPOP, 1,4-bis-2-(5-phenyloxazolyl)benzene; PCA, perchloric acid.
RESOLUTION
OF CYTIDINE-
AND
at the 3’-OH end in adenosine, the other in cytidine. Analogous results were obtained for t-RNA leucine (yeast). Conversion of the cytidine-terminal to the adenosine-terminal species of t-RNA serine was accompanied by change in the chromatographic behavior. It is concluded that differential migration of these two species is related to the nature of the 3’-OH-terminal nucleoside.
(1961) and Bennett et al. (1963, 1965) demonstrated differential responses of multiple methionine t-RNA’s, leucine t-RNA’s, and serine t-RNA’s (E. coli) to homologous and heterologous aminoacyl-t-RNA synthetases. Furthermore, Marcker (1965) has found that only one of the two degenerate methioninyl-t-RNA’s ( E . coli) was capable of being formylated. These observations indicate that differences in degenerate species of t-RNA may extend beyond the “coding site.” Moreover, Zachau et al. (1966) have recently published the complete base sequence of t-RNA serine I and I1 from brewer’s yeast, presumably identical at the “coding site,” but differing in three nucleotides over a span of 19 nucleotides. Berquist and Robertson (1965) and Rushisky et al. (1965) have also reported multiple species of t-RNA serine (baker’s yeast) that exhibit slightly different nucleotide compositions by partial analysis of digestion fragments. This paper will describe a different and previously unreported type of t-RNA heterogeneity that results in the resolution of t-RNA serine and t-RNA leucine from baker’s yeast into two species depending on the presence or absence of adenosine in the CpCpAterminal nucleotide sequence. Furthermore, it will be shown that the difference in the chromatographic mobilities of the two species of t-RNA serine can be erased by addition of an adenosine nucleoside to the species terminating in CpC. Materials and Methods Preparation of Seryl-&RNA Synthetase and CMPAMP-Incorporating Enzyme. Crystalline seryl-t-RNA synthetase was prepared according to the method of Makman and Cantoni (1965). A partially purified CMP-AMP-incorporating enzyme was prepared at 2-4” as follows.
ADENOSINE-TERMINAL
TRANSFER RIBONUCLEIC
3617 ACIDS
BIOCHEMISTRY IGG.00G~
.
I
,
,
,
, I
16o
'
g
80,000-
"
Mi,OOO-
8
aa
:
40,000-
0
2 I
>
150
FIGURE 1: Partition chromatography of bulk yeast t-RNA on Sephadex G-25 according to the method of Tanaka e t al. (1962). Only the early portion of the chromatogram, containing peaks of acceptor activity for serine and tyrosine is shown.
ZOO
220
240
260
-
FRACTION N U M B E R
FIGURE 2: Separation of cytidine- and adenosineterminal species of serine-specific t-RNA of yeast by countercurrent distribution in the modified Zachau units of the butylamine salt solvent system; 23,000 OD26o of t-RNA serine (Figure 1, fractions 20-55) was dissolved in 10 ml each of upper and lower phase, loaded into the first countercurrent tube, and transferred through 400 turns. Curve A: serine-acceptor activity with crude seryl-t-RNA synthetase. Curve B: activity with purified synthetase, indicating adenosine-terminal t-RNA. Curve C: the difference between the areas under curves A and B, indicating the amount of cytidine-terminal t-RNA present.
3618
Each of four batches of 100 g of Fleishman's pressed baker's yeast was homogenized with 300 g of glass beads (Minnesota Mining and Manufacturing Co., No. 150, 75 p, prewashed in 30% nitric acid in a steam
I70
190 FRACTION NUMBER
210
230
FIGURE 3 : Separation of cytidine- and adenosineterminal species of t-RNA serine (Baker's yeast) by countercurrent distribution in the system of Doctor and Connelly (1961); 1530 ODz6,,units of the potassium salt of t-RNA serine obtained by partition chromatography (Figure 1) were solubilized in 50 ml each of upper and lower phase, charged into the first five countercurrent tubes, and transferred for 300 turns. Curve A: serineacceptor activity with pure seryl-t-RNA synthetase plus partially purified CMP-AMP-incorporating enzyme. Curve B : activity with purified synthetase alone, indicating adenosine-terminal t-RNA. Curve C : the difference between the areas under curves A and B, indicating the amount of cytidine-terminal t-RNA.
bath until free of material absorbing at 260 mp), 103 ml of 0.02 M POc buffer (K), pH 7.6, containing 0.002 M MgC12, and 0.4 ml of n-octyl alcohol in a Waring Blendor for five 3-min periods with 6-min intervals on ice between successive homogenizations. Each final homogenate was mixed with 200 ml of the POr MgClz buffer and decanted after the beads had settled. The beads were washed three times with 125 ml of buffer and the washings combined with the concentrated homogenate. The pooled homogenate was then centrifuged for 1 hr at 78,OOOg in the No. 30 rotor of a Spinco Model L centrifuge. To 1000 ml of the resultant supernatant fluid was added 20 g of crystalline streptomycin sulfate (Calbiochem) over a 30-min period with constant stirring. After 20 min of further stirring the extract was centrifuged for 30 min at 20,000g in the VRA head of the Lourdes LRA centrifuge. To the supernatant fluid (980 ml), 254.8 g of solid ammonium sulfate (Merck Co.) was added over a 15-min period with constant stirring. The mixture (42.5% saturation) was allowed to stir an additional 20 min and then centrifuged in the Lourdes VRA head at 20,000g for 20 min. The supernatant fluid (1080 ml) was brought to 53% saturation by the slow addition of 70.2 g of ammonium sulfate. After 20 min of further stirring, the mixture was again centrifuged
LEBOWITZ, IPATA, MAKMAN, RICHARDS, A N D C A N T O N 1
140
160
180
200
220
FRACTION NUMBER
FRACTION NUL:BER
15
C -I
0 -4
D
r
z 15
0 t-
0
U
i
-0
u U
2r
a
m
0
