- Email: [email protected]
A comparison of rabbit liver and reticulocyte transfer RNA: Evidence of unique species in reticulocytes
366
Biochimica et Biophysica Acta, 349 ( 1 9 7 4 ) 366--375 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m -- Printed in The N e t h e r l a n d s
BBA 9 7 9 9 2
A COMPARISON OF RABBIT LIVER AND RETICULOCYTE TRANSFER RNA: EVIDENCE OF UNIQUE SPECIES IN RETICULOCYTES
D A V I D W.E. SMITH, V I C T O R N. M E L T Z E R
and A N N E L. M C N A M A R A
Department of Pathology, Northwestern UniversityMedical School, 303 East Chicago Avenue, Chicago, Ill.60611 (U.S.A.) (Received December 3rd, 1973)
Summary The comparison of rabbit liver and reticulocyte tRNA has provided evidence that the tRNA content of reticulocytes is specialized for hemoglobin synthesis (Smith, D.W.E. and McNamara, A.L. (1971) Science 171, 577--579 and erratum, 1040). This comparison is extended in the present study to the level of isoaccepting species by reversed-phase chromatography of the tRNAs followed by determination of acceptance of 18 amino acids. Though the elution patterns of isoaccepting species for some amino acids are qualitatively and quantitatively similar, the distribution of tRNA for other amino acids among isoaccepting species is very different in reticulocytes and liver. There are two isoaccepting species each for asparaglne and histidine in reticulocytes compared to only one in liver. These unique species were confirmed by a second reversedphase chromatographic technique, and are seen regardless of whether liver or reticulocyte enzymes are used to aminoacylate the tRNA. The single species for each of these amino acids in liver corresponds to the first of the two species in reticulocytes in order of chromatographic elution. The structural differences between the two species found in reticulocytes are not yet clear. Coding studies were done with the partially purified asparagine and histidine species from liver and reticulocytes using trinucleotide diphosphate stimulated binding of the aminoacylated tRNAs to Escherichia coli ribosomes. Consistent with the "Wobble Hypothesis", all tRNA species responded to both codons for their respective amino acids, though differences in the codon responses were found which may be significant.
Introduction
Reticulocytes, the anucleate precursors of erythrocytes, are uniquely suited to studies of the relationship between tRNA content and protein synthesis. Pre-existing hemoglobin mRNA is translated by pre-existing tRNA, ribo-
367 somes, and factors at a rate which has been well established [1], and hemoglobin accounts for a least 90% of the protein synthesis in these cells [2]. The rate of hemoglobin translation may be limited by the availability of some tRNA species, as originally suggested by Itano [3]. An enumeration of the molecules per rabbit reticulocyte of each tRNA species, as resolved by reversed-phase chromatography, has already been published [4]. The distribution of reticulocyte tRNA between aminoacylated and deacylated conditions and between ribosomes and the cytosol has been determined [5]. The relative abundance of tRNA accepting each amino acid was compared in rabbit reticulocytes and liver [6]. While reticulocytes synthesize a single protein, the liver synthesizes many proteins of a constantly changing variety. If the tRNA content of reticulocytes is specialized for hemoglobin synthesis, there are certain respects in which it should be markedly different from liver tRNA. These include histidine tRNA, which should be relatively more abundant in reticulocytes corresponding to the abundance of histidine in hemoglobin compared to most proteins, and isoleucine tRNA, which might be relatively less abundant in reticulocytes, corresponding to the relative scarcity of isoleucine in hemoglobin. Both of these predictions were fulfilled in the study, with histidine tRNA being relatively three times more abundant in reticulocytes than liver and isoleucine tRNA being two times less abundant. A significant correlation was found between the abundance of tRNAs for most amino acids in reticulocytes and the abundance of these amino acids in rabbit hemoglobin. These results support the concept of tRNA specialization for hemoglobin synthesis. The present communication extends the results by comparing elution patterns of rabbit reticulocyte and liver tRNA, using reversed-phase chromatography. This approach permits comparison of liver and reticulocyte tRNA content at the level of isoaccepting species. Chromatographic profiles of reticulocyte tRNA have already been published [4], and have been confirmed by further experiments in the present study. Well-defined species of asparagine and histidine tRNA have been found in reticulocytes which are n o t found in liver. Coding studies using trinucleotide diphosphates are described for asparag~nyland histidyl-tRNA species from both sources. In addition to the two tRNA species which are unique to reticulocytes, there are major quantitative differences between the relative abundance of tRNA isoaccepting species in liver and reticulocytes. Materials and Methods
Chemicals and radioactive compounds Non-radioactive amino acids and ATP were obtained from Sigma Chemical Company. Uniformly labelled L-[~4C]amino acids of high specific activity were purchased from Amersham/Searle Corporation and New England Nuclear Corporation. Preparation of tRNA and aminoacyl-tRNA synthetases The preparation of tRNA and aminoacyl-tRNA synthetases from reticulocytes of phenylhydrazine-treated rabbits and from the livers of untreated rabbits has been described previously [4--6].
368
Fractionation of tRNA by reversed-phase chromatography: RPC-3 columns tRNA was fractionated by the reversed-phase column chromatographic system type 3 (RPC-3) of Weiss et al. [7], as used previously in our laboratory [4]. Two batches each of liver and reticulocyte tRNA were fractionated for this study, with excellent agreement of the patterns, though the results of only one each of the fractionations are presented. Recovery of absorbance at 260 nm and of amino acid acceptance activity was 80% or more of the column inputs. Assay of column fractions for amino acid acceptance Aliquots of the column fractions containing tRNA were assayed for amino acid acceptance under conditions of limiting tRNA as described previously [4--6]. Acceptance of cysteine and tryptophan was not assayed in this study. Preparation of aminoacyl-tRNA Partially purified species of asparaginyl- and histidyl-tRNA were prepared from RPC-3 column fractions for chromatography and for coding studies. Fractions containing the species were pooled and desalted by precipitation with cold ethanol. 6--12 absorbance units* of the desalted tRNA were aminoacylated in 2.0 ml reaction mixtures containing the same concentrations of components used for the assay of tRNA amino acid acceptance [5,6]. Complete aminoacylation usually required less than 20 rain. The reaction mixture was then chilled and the pH was lowered to 4.5 by the addition of 1 M acetic acid. It was then extracted three times with phenol, with careful maintenance of the aqueous phases at pH 4.5. tRNA was precipitated overnight with cold ethanol, collected on a solvent-resistant membrane filter (Gelman GAS), dissolved in 0.02 M lithium acetate, pH 4.5, and stored at --20°C. Recoveries of incorporated amino acid ranged from 50--80%, and recoveries of material absorbing at 260 nm were approximately 80% of the.pooled column fractions. Of the 1, C-labelled amino acid recovered with the tRNA, at least 80% was precipitable with 5% cold trichloroacetic acid, and little deacylation occurred during storage, as indicated by a constant level of acid-precipitable radioactivity. Fractionation of tRNA and aminoacyl-tRNA : RPC.5 columns An additional fractionation technique, the reversed-phase column chromatographic system type 5 (RPC-5) [8] was used for certain studies in which both deacylated tRNA and partially purified aminoacylated-tRNA species were f~actionated. Aminoacyl-tRNA species, prepared as described above, were placed on these columns along with 14 absorbance units of unfractionated reticulocyte tRNA. In these experiments a maximum of 0.7 absorbance unit and 10000 cpm of esterified labelled amino acid were contributed by the aminoacyl-tRNA preparations. The labelled aminoacyl-tRNA was detected by measuring the radioactivity in 1 ml aliquots of the fractions in Bmy's solution [9]. Correspondence between this tRNA and the species of tRNA in the carrier
*
A n a b s o r b a n e e u n i t is d e f i n e d as t h e q u a n t i t y o f m a t e r i a l i n 1 r n l o f a s o l u t i o n h a v i n g a n a h s o r b a n e e o f 1 . 0 at 2 6 0 n m w h e n m e a s u r e d in a 1 - c m p a t h l e n g t h c e l l .
369 was determined by assaying amino acid acceptance activity of 0.025 ml aliquots of the column fractions. The small amounts of radioactivity contributed by labelled aminoacyl-tRNA were readily subtracted from the radioactivity accepted by the tRNA in these aliquots.
Coding studies Coding studies were performed by determining the binding of fractionated labelled histidyl- and asparaginyl-tRNA to Escherichia coli ribosomes [10,11] in the presence of trinucleotide diphosphate codons for these amino acids [12--14] as described by Nirenberg and Leder [15] and modified for fractionated mammalian tRNA by Hatfield [10]. The triplet codons for asparagine (AAC, AAU) and histidine (CAC, CAU) were obtained from Dr Marshall Nirenberg through the kindness of Dr Dolph Hatfield. The components of the reaction mixtures are described below, along with the results of the coding studies. Results
Comparison of chromatographic patterns of liver and reticulocyte tRNA As shown in Fig. 1, patterns for aspartic acid, glutamine, glycine, lysine, tyrosine, and valine acceptance are nearly alike, with similar isoaccepting species being resolved, and with the distribution of tRNA among the species being
] ~
o.1"3o
RETICULOCYTE tRNA ' ' "
LIVER tRNA
o2 "7
i
2,
E
o
,
i
Glyclne 4oo
2oo >
,
0 Ifioo
,
=coo
,A ~mslne 4oo
_.
,zoo Voline
0
50
FRACTION NUMBER
IOO
150
200
FRACTION NUMBER
Fig. 1. R e s o l u t i o n o f r e t l c u l o c y t e a n d liver t R N A b y R P C - 3 c o l u m n c h r o m a t o g r a p h y . R e t i c u l o c y t e t R N A (423 absorbance units) and liver tRNA (450 absorbance units) were eluted from an RPC-3 column in s e p a r a t e p r o c e d u r e s . T h e t o p p a n e l s s h o w t h e p a t t e r n s o f e l u t i o n o f m a t e r i a l a b s o r b i n g a t 2 6 0 rum a n d t h e lower panels compare elution patterns of amino acid acceptance act/vity based on assays of 0.05 ml aliquots of the column fractions. Every fraction was assayed through peak regions. The patterns in this f i g u r e a r e t h o s e w h i c h s h o w little d i f f e r e n c e b e t w e e n r e t i c u l o e y t e s a n d liver.
370
RETICULOCYTE tRNA
LIVER tRNA
|i o.~i
~: o 0250 u~cJ t500
'~
Methmnine
,O oo I 900 6O0 500 o
1200
~
600 3o~
J~
600 400
o
o 600 400
~-
S
<
/,t/I ,
~
~
,
<
0
0' 001500500
0
0 1200 800 400
~//~1 Arginine
~ '
' ' [soleucine
,t~
Leucine ,~ , /~ .
i
. . . Phenylalonine
i
'
--' 1 II
/ / ~
.
i
M~ i
,\
,,'
/
600 4OO 20C
~ _ ~
Alpine
°
1200 900 600 500 0 1200 900
~
GLutOm~Acid
,
Proline
, Serine
,
,
~Threonine
i
,
,
/ L
/ ~
,
50 I00 150 200 FRACTION NUMBER
=
,
/
50 t(30 i50 200 FRACTION NUMBER
Fig. 2. C h r o m a t o g r a p h i c r e s o l u t i o n o f r e t i c u l o c y t e a n d liver t R N A . T h e r e s u l t s in this f i g u r e a r e t a k e n f r o m t h e s a m e c h r o m a t o g r a p h i c p r o c e d t ~ e s as t h o s e m Fig. 1. T h e p a t ~ r ~ s h o w n are t h o s e in w h i c h t h e r e are large q u a n t i t a t i v e d i f f e r e n c e s in t h e d i s t r i b u t i o n o f t R N A a m o n g i s o a c c e p t i n g species.
RETICULOCYTE tRNA
w
LIVER tRNA
i| = , L
-.9F, ;
,
Asporagine
'~
~
Histidine
50 IOO laO 2oo o FRACTION NUMBER
FRACTION NUMBER
Fig. 3. Chxomatol~raphic r e s o l u t i o n o f r e t i c u l o c y t e a n d Uver t R N A . T h e r e s u l t s in this ~ are t a k e n f r o m t h e s a m e c b z o m a t o l m p h t c p r o c e d u ~ s as t h o s e s h o w n in F i p 1 a n d 2. T h e p a t t e r n s s h o w n are of asparaifJne a n d h i s t i d i n e t R N A a n d i n d i c a t e in e a c h case a t R N A species c l e a r l y r e s o l v e d f r o m r e t i c u l o c y t e t R N A w h i c h is n o t f o u n d in liver t R N A .
371
similar. In some other comparisons, major quantitative differences are found between reticulocyte and liver tRNA, as shown in Fig. 2. Two peaks of methionine acceptance activity can be fractionated from both liver and reticulocyte tRNA, but the first peak is much larger in reticulocytes, while in liver there is approximately the same amount of acceptance activity in each peak. Preliminary evidence from our laboratory and results from other laboratories [16--18] indicate that the first of the peaks is probably the formylatable methionine tRNA species (methionine tRNA~) involved in initiation of translation. Quantitative differences in the distribution of tRNA among wellresolved isoaccepting species were also found for glutamic acid tRNA. Differences in elution patterns for alanine, arginine, isoleucine, leucine, phenylalanine, proline, serine, and threonine tRNAs, can be explained by the distribution of tRNA among isoaccepting species, but additional chromatographic evidence will be necessary to determine whether this is the case, or whether some of the differences intvolve species unique to one of the sources of the tRNA. Two very clear qualitative differences emerge from the comparison. There are two well resolved species of histidine and asparagine tRNA in reticulocytes compared to only one for each of these amino acids in liver, as shown in Fig. 3. These apparently unique tRNA species provided the basis for additional investigations. The patterns shown in the figures are based on assays with synthetases from the same source as the tRNA, however, the same patterns are seen regardless of the synthetase source, indicating that the differences are due to tRNA.
•r
T
0
b. Acceptance Activity
-,,z
0 3000[ ~.~
~
c. Liver [,4C1 Ash. ,RNA
2000
0
25
50 FRACTION NUMBER
75
Fig. 4. C h r o m a t o g r a p h i c r e s o l u t i o n o f t R N A o n R P C - 5 c o l u m n s . T h e c o l u m n u s e d measttred 1 c m ( d i a m e t e r ) X 3 0 c m , and the p a c k i n g m a t e r i a l w a s o b t a i n e d f r o m Miles L a b o r a t o r i e s , I n c . A l i n e a r gradient o f 2 5 0 m l w a s e m p l o y e d f r o m 0 . 4 - - 0 . 7 M LiC1 c o n t a i n i n g 0 . 0 1 M MgC12, 0 . 0 1 M l i t h i u m acetate, pH 4.5, and 0.002 M ~-mercaptoethanol. The columns were run with a head of 2 M fed by gravity through c a p i l l a r y t u b i n g a t r o o m t e m p e r a t u r e . E i n t i o n u n d e r these c o n d i t i o n s r e q u i r e d a p p r o x . 1 2 h , a n d f r a c t i o n s o f 2 . 5 m l w e r e c o l l e c t e d . A s d e s c r i b e d i n t h e t e x t , t h e r e s o l u t i o n o f a b s o r b a n c e a t 2 6 0 n m is s h o w n in P a n e l a a n d that o f h i s t i d i n e a n d a s p a r a g i n e a c c e p t a n c e b y r e t i c u l o c y t e t R N A is s h o w n i n P a n e l b. P a n e l c s h o w s e l u t i o n o f l i v e r [ 1 4 C ] a s p a r a g i n y l - t R N A c o o e h r o m a t o g r a p h e d w i t h t h e r e t i c u l o c y t e t R N A . Its elut i o n c o r r e s p o n d s t o t h e f i r s t o f t h e t w o r e t i c u l o c y t e a s p a r a g i n e species.
372
Confirmation of chromatographic differences for histidine and asparagine tRNA Elution profiles for histidine and asparagine tRNA from liver and reticulocytes were compared further by chromatography on a different reversed-phase column system, RPC-5 [8]. Fig. 4 shows a typical experiment. Panels a and b show the resolution of absorbance at 260 nm and of histidine and asparagine acceptance activity of reticulocyte tRNA. The results confirm the existence of two well-separated isoaccepting species for each of these amino acids in reticulocytes. Chromatography of liver tRNA (not shown in the figure) resolved only a single species each of histidine and asparagine tRNA. Panel c shows the elution pattern of the aminoacylated asparagine tRNA species from liver, chromatographed along with carrier reticulocyte tRNA. In this experiment, it was determined both that the liver asparagine tRNA species moves as a single peak on rechromatography and that it corresponds to the first of the two asparagine tRNA species in reticulocytes. In column experiments which are not shown, both reticulocyte tRNA species for asparagine and histidine and the liver histidine tRNA species rechromatographed as single peaks, without evidence of heterogeneity. The two species each of histidine and asparagine t R N A from reticulocytes are eluted in the same sequence from RPC-5 columns as RPC-3 columns. The single histidine tRNA species in liver corresponds to the first of the two reticulocyte histidine tRNA peaks.
Coding studies with histidyl- and asparaginyl-tRNA To our knowledge, the present work is the first time coding studies have been done on isolated animal t R N A isoaccepting species for histidine and asparagine. The amounts of aminoacyl-tRNA, trinucleotide diphosphates, and other elements in the assays are described in Table I, and the results are shown
TABLE I C O M P O N E N T S A D D E D TO C O D I N G R E A C T I O N S * Species of a m i n o a c y l t R N A
Aminoacyl Added tRNA pmoles (A260 nm units)
cpm/ pmole
Codon
A m o u n t of c o d o n added (A260 nm units)
AAU AAC
0.292 0.326
CAU CAC
0.183 0.180
I. Asparagine e x p e r i m e n t s
R e t i c u l o c y t e [14C] A s n - t R N A I R e t i c u l o c y t e [14C] A s n - t R N A II Liver [ 14C] A s n - t R N A
0.041 0.043 0.051
2.71 2.85 3.71
276 276 276
2. H i s t i d i n e e x p e r i m e n t s
R e t i c u l o c y t e [ 14C] H i s - t R N A I R e t i c u l o c y t e [ 1 4 C ] H I s - t R N A II Liver [ 14C] H I s - t R N A
0.059 0.032 0.072
2.11 2.48 2.13
590 590 590
* I n a d d i t i o n t o the above c o m p o n e n t s which varied, each reaction contained 0 . 0 5 M ~ acetate, p H 7.3; 0 . 0 5 M p o t a s s i u m a c e t a t e ; 0.01 or 0 . 0 2 M m a g n e s i u m acetate, as s h o w n above; and E. colt ribosomes, 2.0 absorbance u n i t s , i n a t o t a l v o l u m e o f 0 . 0 5 5 ml.
373 TABLE II RESPONSES OF FRACTIONATED tRNAs TO CODONS Triplet c o d o n and r e s p o n s e (A p m o l e b o u n d to r i b o s o m e s ) *
E x p e r i m e n t s in 10 mM Mg 2+ R e t i c u l o c y t e [14C] Asn-tRNA I R e t i c u l o c y t e [ 14C] Asn-tRNA II Liver [ 14C] Asn-tRNA R e t i c u l o c y t e [14C] His-tRNA I R e t i c u l o c y t e [ 14C] His-tRNA II Liver [14C] His-tRNA E x p e r i m e n t s in 20 mM Mg 2+ R e t i c u l o c y t e [ 14C] Asn-tRNA I R e t i c u l o c y t e [ 14C] Asn-tRNA II Liver [ 14C] Asn-tRNA R e t i c u l o c y t e [ 14C] His-tRNA I R e t i c u l o c y t e [14C] His-tRNA II Liver [ 14C] His-tRNA
Asparagine c o d o n s
Histidine c o d o n s
AAC
AAU
CAC
0.043 0.043 0.072
0.094 0.033 0.072 0.025 0.017 0.008
1.163 1.225 1.087
CAU
0.169 0.051 0.117
(0.011) (0.022) (0.029) (0.012) (0.017) (0.019)
0.688 0.668 0.407
(0.170) (0.185) (0.130) (0.061) (0.129) (0.073)
1.270 0.949 0.840 0.134 0.266 0.124
None**
* A m o u n t of a m i n o a c y l - t R N A b o u n d to r i b o s o m e s in presence o f c o d o n m i n u s the a m o u n t b o u n d in absence o f codon. ** A m o u n t o f a m i n o a c y l - t R N A b o u n d to r i b o s o m e s in absence of c o d o n ( s h o w n in parentheses because these a m o u n t s have already b e e n subtracted f~om the values s h o w n for c o d o n responses).
in Table II. The results are readily reproducible, and there is excellent binding at several times blank values (without codon) at 20 mM Mg 2÷ with the appropriate codons. The studies at 10 mM Mg2÷ revealed similar binding, but at much lower levels and without increased specificity. For both asparagine and histidine, two codons have been established [ 14] which differ in each case only by a pyrimidine in the third letter of the triplet. As expected from the "Wobble Hypothesis" [19], each tRNA species was bound to ribosomes in the presence of both code words. Consistent quantitative differences were observed with asparaginyl-tRNA which may be significant. While the first peak of asparaginyl-tRNA from reticulocytes was bound equally to ribosomes in the presence of both triplets, the second peak and the peak of liver asparaginyl-tRNA were bound better in the presence of AAC than AAU. It may also be significant that the binding of the second reticulocyte histidyltRNA species as stimulated by CAC, is substantially higher than CAC-stimulated binding of other species. Discussion The study reported here has been done differently from other studies comparing tRNA from different cells. The RPC-3 column, though it has not been widely used, is very reliable and permits a nearly tube-by-tube comparison o f the results of different fractionations. The assay of amino acid acceptance by tRNA after chromatography, rather than the use of labelled aminoacyl-
374
tRNA in the column inputs, permits quantitative assessment of tRNA after elution and is not dependent on the stability of aminoacyl bonds during elution, which is known to vary for different aminoacyl-tRNAs [20,21]. Other workers using RPC-2 columns [22] and labelled aminoacyl-tRNA have also found various numbers of isoaccepting species for asparagine [23--26] and histidine [23--28], as well as other amino acids from several kinds of cells. The nature of the multiple isoaccepting tRNA species found in these studies remains uncertain. When isoaccepting species resolved chromatographically have been examined, some have represented different gene products with differing base sequences (see refs 29--31), others have represented different post-synthetic modifications of tRNA (see refs 32 and 33), and yet others have represented different structural configurations (see ref. 34). The failure of coding experiments to permit certain assignment of anticodon sequences to the isoaccepting species for histidine and asparagine in the present study, is consistent with the "Wobble Hypothesis" [19] and does not rule out the possibility that the isoaccepting species may have different base sequences in the anticodon as well as elsewhere. The recent studies of Farkas et al. [35] suggest that one of the reticulocyte histidine tRNA species has a guanine group attached to some internal nucleotide. It is possible that the difference between the two histidine tRNA species in reticulocytes is the presence of this guanine group, and that the guanylation reaction is specific to reticulocytes so that the guanylated species is not found in some other kinds of cells such as liver. The present work extends the comparison between reticulocyte and liver tRNA, as previously published [6] to the level of isoaccepting species. In the case of histidine, part of the difference in the relative amounts of acceptance activity can be attributed to a tRNA species found only in reticulocytes. With isoleucine, however, the differences in the relative abundance of tRNA in liver and reticulocytes cannot be related to a unique tRNA species or even to large differences in the distribution of tRNA among the same isoaccepting species. There is little difference in the relative abundance of asparagine tRNA between liver and reticulocytes, but an isoaccepting species is found in reticulocytes which is not present in liver. There are no results that can be clearly related to the abundance of mitochondria in liver compared to reticulocytes [36], although studies from other laboratories (see ref. 37) show that there are unique mitochondrial tRNA species for some amino acids that are encoded by mitochondrial DNA. The present study comparing the isoaccepting species of liver and reticulocyte tRNA provides additional data concerning the nature of the specialization of the reticulocyte tRNA content for hemoglobin synthesis. In a few cells and tissues, in addition to reticulocytes, it has been possible to develop evidence of tRNA specialization for protein synthesis. These include the silk glands of silk worms [38], mammary glands at the time of lactation [39], the fibroblasts of healing wounds [40], and the livers of roosters treated with estrogens [41]. There is a study by Litt and Kabat [42] comparing the tRNA of reticulocytes of anemic and normal sheep, which make hemoglobins of different amino acid sequence, which gives evidence, in addition to that from our laboratory, that reticulocyte tRNA is specialized for hemoglobin synthesis.
375
Acknowledgements This work was supported by Research Grant AM-15467 from the National Institutes of Health. One of the authors (V.N.M.) was supported by a fellowship from the American Cancer Society, Illinois Division during part of the time he worked on this project. The authors are very grateful to Dr Dolph L. Hatfield of the National Institutes of Health for the help and useful discussion he provided.
References 1 Lingrel, J.B. and Borsook, H. (1963) Biochemistry 2, 309--314 2 Lodish, H.F. and Desalu O. (1973) J. Biol. Chem. 248, 3520--3527 3 Itano, H.A. (1965) in Abnormal Hemoglobins in Africa (J.H.P. Jonxis, ed.), pp. 3--16, BlackweU, Oxford 4 Smith, D.W.E. and McNamara, A.L. (1972) Biochim. Biophys. Acta 269, 66--77 5 Smith, D.W.E. and McNamara, A.L. (1974) J. Biol. Chem. 249, 1330--1334 6 Smith, D.W.E. and McNamara, A.L. (1971) Science 171,577--579 and erratum, 1040 7 Weiss, J.F., Pearson, R.L. and Kelmers, A.D. (1968) Biochemistry 7, 3479--3487 8 Kelmers, A.D. and Heatherly, D.E. (1971) Anal. Biochem. 44, 486--495 9 Bray, G.A. (1960) Anal. Biochem. 1,279--285 10 Hatfield, D. (1972) Proc. Natl. Acad. Sci. U.S. 69, 3014--3018 11 Pestka, S. (1968) J. Biol. Chem. 243, 2810--2820 12 Trupin, J.S., Rottman, F . M , Brimacombe, R.L.C., Leder, P., Bernfield, M.R. and Nirenberg, M.W. (1965) Proc. Natl. Acad. Sci. U.S. 53, 807--811 13 Nirenberg, M., Leder, P., Bernfield, M., Brimacombe, R., Trupin, J., Rottman, F., and O'Neal, L. (1965) Proc. Natl. Acad. Sci. U.S. 53, 1161--1168 14 Marshall, R.E., Caskey, C.T. and Nirenberg, M. (1967) Science 153,820--826 15 Nixenberg, M. and Ledar, P. (1964) Science 145, 1399---1407 16 Caskey, C.T., Beaudet, A. and Nirenberg, M. (1968) J. Mol. Biol. 37, 99--118 17 Shafritz, D.A. and Anderson, W.F. (1970) Nature 227, 918--920 18 Yang, W.-K. (1971) Cancer Res. 31,639---643 19 Crick, F.H.C. (1966) J. Mol. Biol. 19, 548--555 20 Satin, P.S. and Zamecnik, P.C. (1964) Biochim. Biophys. Acta 91,653---655 21 Gatica, M., Allende, C.C., Mora, G., AUende, J.E. and Medina J. (1966) Biochim. Biophys. Acta 129, 201--203
22 23 24 25 26 27 28 29 30 31 32. 33 34 35 36 37 38 39 40 41 42
Weis, J,F. and Kelmers, A.D. (1967) Biochemistry 6, 2507--2513 Gallo, R.C. and Pestka, S. (1970) J, Mol. Biol. 52, 195---219 Yang, W.-K. and Novelli, G.D. (1968) Proc. Natl. Acad. Sci. U.S. 59, 208--215 Ortwerth, B.J. (1971) Biochemistry 10, 4 1 9 0 - 4 1 9 7 Gonano, F., Chiarugi, V.P., Pirro, G. and Marlni, M. (1971) Biochemistry 10, 900--908 Yang, W.-K., Hellman, A., Martin, D.H., Hellman, K.B. and Novellt G.D. (1969) Proc. Natl. Acad. Sci. U.S. 64, 1411--1418 Volkers, S.A.S. and Taylor, M.W. (1971) Biochemistry 10, 488--497 Cory, S., Marcker, K.A., Dube, S.A. and Clark, B.F.C. (1968) Nature 220, 1039--1040 Staehelin, M. (1971) Experlentia 27, 1--11 Folk, W.R. and Yaniv, M. (!972) Nat. New Biol. 237, 165--166 Shugart, L., Nove]li, G.D. and Stulberg, M.P. (1968) Biochim. Biophys. Acta 157, 83--90 Gefter, M.L. and Russell, R.L. (1969) J. Mol. Biol. 39, 145--157 Ishida, T. and Sueoka, N. (1968) J. Mol. Biol. 37, 313--316 Farkas, W.R., Hanklns, W.D. and Singh, R. (1973) Biochim. Biophys. Acta 294, 94--105 Jensen, W.N., Moreno, G.D. and Bessis, M. (1965) Blood 25, 933--943 Nass, M.M.K. and Buck, C.A. (1970) J. Mol. Biol. 54, 187--198 Garel, J.-P., Mandel, P., Chavancy, G. and Dalllie, J. (1971) Biochemie 53, 1195--1200 Elska, A., Matsuka, G. and Matiash, U. (1971) Biochim. Biophys. Acta 247, 430--440 Lanks, K.W. and Weinstein, I.B. (1979) Biochem. Biophys. Res. Commun. 40, 708--715 M~ienp~i, P.H. (1972) Biochem. Biophys. Res. Commun. 4 7 , 9 7 1 - - 9 7 4 Litt, M. and Kabat, D. (1972) J. Biol. Chem. 247, 6659---6664
Biochimica et Biophysica Acta, 349 ( 1 9 7 4 ) 366--375 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m -- Printed in The N e t h e r l a n d s
BBA 9 7 9 9 2
A COMPARISON OF RABBIT LIVER AND RETICULOCYTE TRANSFER RNA: EVIDENCE OF UNIQUE SPECIES IN RETICULOCYTES
D A V I D W.E. SMITH, V I C T O R N. M E L T Z E R
and A N N E L. M C N A M A R A
Department of Pathology, Northwestern UniversityMedical School, 303 East Chicago Avenue, Chicago, Ill.60611 (U.S.A.) (Received December 3rd, 1973)
Summary The comparison of rabbit liver and reticulocyte tRNA has provided evidence that the tRNA content of reticulocytes is specialized for hemoglobin synthesis (Smith, D.W.E. and McNamara, A.L. (1971) Science 171, 577--579 and erratum, 1040). This comparison is extended in the present study to the level of isoaccepting species by reversed-phase chromatography of the tRNAs followed by determination of acceptance of 18 amino acids. Though the elution patterns of isoaccepting species for some amino acids are qualitatively and quantitatively similar, the distribution of tRNA for other amino acids among isoaccepting species is very different in reticulocytes and liver. There are two isoaccepting species each for asparaglne and histidine in reticulocytes compared to only one in liver. These unique species were confirmed by a second reversedphase chromatographic technique, and are seen regardless of whether liver or reticulocyte enzymes are used to aminoacylate the tRNA. The single species for each of these amino acids in liver corresponds to the first of the two species in reticulocytes in order of chromatographic elution. The structural differences between the two species found in reticulocytes are not yet clear. Coding studies were done with the partially purified asparagine and histidine species from liver and reticulocytes using trinucleotide diphosphate stimulated binding of the aminoacylated tRNAs to Escherichia coli ribosomes. Consistent with the "Wobble Hypothesis", all tRNA species responded to both codons for their respective amino acids, though differences in the codon responses were found which may be significant.
Introduction
Reticulocytes, the anucleate precursors of erythrocytes, are uniquely suited to studies of the relationship between tRNA content and protein synthesis. Pre-existing hemoglobin mRNA is translated by pre-existing tRNA, ribo-
367 somes, and factors at a rate which has been well established [1], and hemoglobin accounts for a least 90% of the protein synthesis in these cells [2]. The rate of hemoglobin translation may be limited by the availability of some tRNA species, as originally suggested by Itano [3]. An enumeration of the molecules per rabbit reticulocyte of each tRNA species, as resolved by reversed-phase chromatography, has already been published [4]. The distribution of reticulocyte tRNA between aminoacylated and deacylated conditions and between ribosomes and the cytosol has been determined [5]. The relative abundance of tRNA accepting each amino acid was compared in rabbit reticulocytes and liver [6]. While reticulocytes synthesize a single protein, the liver synthesizes many proteins of a constantly changing variety. If the tRNA content of reticulocytes is specialized for hemoglobin synthesis, there are certain respects in which it should be markedly different from liver tRNA. These include histidine tRNA, which should be relatively more abundant in reticulocytes corresponding to the abundance of histidine in hemoglobin compared to most proteins, and isoleucine tRNA, which might be relatively less abundant in reticulocytes, corresponding to the relative scarcity of isoleucine in hemoglobin. Both of these predictions were fulfilled in the study, with histidine tRNA being relatively three times more abundant in reticulocytes than liver and isoleucine tRNA being two times less abundant. A significant correlation was found between the abundance of tRNAs for most amino acids in reticulocytes and the abundance of these amino acids in rabbit hemoglobin. These results support the concept of tRNA specialization for hemoglobin synthesis. The present communication extends the results by comparing elution patterns of rabbit reticulocyte and liver tRNA, using reversed-phase chromatography. This approach permits comparison of liver and reticulocyte tRNA content at the level of isoaccepting species. Chromatographic profiles of reticulocyte tRNA have already been published [4], and have been confirmed by further experiments in the present study. Well-defined species of asparagine and histidine tRNA have been found in reticulocytes which are n o t found in liver. Coding studies using trinucleotide diphosphates are described for asparag~nyland histidyl-tRNA species from both sources. In addition to the two tRNA species which are unique to reticulocytes, there are major quantitative differences between the relative abundance of tRNA isoaccepting species in liver and reticulocytes. Materials and Methods
Chemicals and radioactive compounds Non-radioactive amino acids and ATP were obtained from Sigma Chemical Company. Uniformly labelled L-[~4C]amino acids of high specific activity were purchased from Amersham/Searle Corporation and New England Nuclear Corporation. Preparation of tRNA and aminoacyl-tRNA synthetases The preparation of tRNA and aminoacyl-tRNA synthetases from reticulocytes of phenylhydrazine-treated rabbits and from the livers of untreated rabbits has been described previously [4--6].
368
Fractionation of tRNA by reversed-phase chromatography: RPC-3 columns tRNA was fractionated by the reversed-phase column chromatographic system type 3 (RPC-3) of Weiss et al. [7], as used previously in our laboratory [4]. Two batches each of liver and reticulocyte tRNA were fractionated for this study, with excellent agreement of the patterns, though the results of only one each of the fractionations are presented. Recovery of absorbance at 260 nm and of amino acid acceptance activity was 80% or more of the column inputs. Assay of column fractions for amino acid acceptance Aliquots of the column fractions containing tRNA were assayed for amino acid acceptance under conditions of limiting tRNA as described previously [4--6]. Acceptance of cysteine and tryptophan was not assayed in this study. Preparation of aminoacyl-tRNA Partially purified species of asparaginyl- and histidyl-tRNA were prepared from RPC-3 column fractions for chromatography and for coding studies. Fractions containing the species were pooled and desalted by precipitation with cold ethanol. 6--12 absorbance units* of the desalted tRNA were aminoacylated in 2.0 ml reaction mixtures containing the same concentrations of components used for the assay of tRNA amino acid acceptance [5,6]. Complete aminoacylation usually required less than 20 rain. The reaction mixture was then chilled and the pH was lowered to 4.5 by the addition of 1 M acetic acid. It was then extracted three times with phenol, with careful maintenance of the aqueous phases at pH 4.5. tRNA was precipitated overnight with cold ethanol, collected on a solvent-resistant membrane filter (Gelman GAS), dissolved in 0.02 M lithium acetate, pH 4.5, and stored at --20°C. Recoveries of incorporated amino acid ranged from 50--80%, and recoveries of material absorbing at 260 nm were approximately 80% of the.pooled column fractions. Of the 1, C-labelled amino acid recovered with the tRNA, at least 80% was precipitable with 5% cold trichloroacetic acid, and little deacylation occurred during storage, as indicated by a constant level of acid-precipitable radioactivity. Fractionation of tRNA and aminoacyl-tRNA : RPC.5 columns An additional fractionation technique, the reversed-phase column chromatographic system type 5 (RPC-5) [8] was used for certain studies in which both deacylated tRNA and partially purified aminoacylated-tRNA species were f~actionated. Aminoacyl-tRNA species, prepared as described above, were placed on these columns along with 14 absorbance units of unfractionated reticulocyte tRNA. In these experiments a maximum of 0.7 absorbance unit and 10000 cpm of esterified labelled amino acid were contributed by the aminoacyl-tRNA preparations. The labelled aminoacyl-tRNA was detected by measuring the radioactivity in 1 ml aliquots of the fractions in Bmy's solution [9]. Correspondence between this tRNA and the species of tRNA in the carrier
*
A n a b s o r b a n e e u n i t is d e f i n e d as t h e q u a n t i t y o f m a t e r i a l i n 1 r n l o f a s o l u t i o n h a v i n g a n a h s o r b a n e e o f 1 . 0 at 2 6 0 n m w h e n m e a s u r e d in a 1 - c m p a t h l e n g t h c e l l .
369 was determined by assaying amino acid acceptance activity of 0.025 ml aliquots of the column fractions. The small amounts of radioactivity contributed by labelled aminoacyl-tRNA were readily subtracted from the radioactivity accepted by the tRNA in these aliquots.
Coding studies Coding studies were performed by determining the binding of fractionated labelled histidyl- and asparaginyl-tRNA to Escherichia coli ribosomes [10,11] in the presence of trinucleotide diphosphate codons for these amino acids [12--14] as described by Nirenberg and Leder [15] and modified for fractionated mammalian tRNA by Hatfield [10]. The triplet codons for asparagine (AAC, AAU) and histidine (CAC, CAU) were obtained from Dr Marshall Nirenberg through the kindness of Dr Dolph Hatfield. The components of the reaction mixtures are described below, along with the results of the coding studies. Results
Comparison of chromatographic patterns of liver and reticulocyte tRNA As shown in Fig. 1, patterns for aspartic acid, glutamine, glycine, lysine, tyrosine, and valine acceptance are nearly alike, with similar isoaccepting species being resolved, and with the distribution of tRNA among the species being
] ~
o.1"3o
RETICULOCYTE tRNA ' ' "
LIVER tRNA
o2 "7
i
2,
E
o
,
i
Glyclne 4oo
2oo >
,
0 Ifioo
,
=coo
,A ~mslne 4oo
_.
,zoo Voline
0
50
FRACTION NUMBER
IOO
150
200
FRACTION NUMBER
Fig. 1. R e s o l u t i o n o f r e t l c u l o c y t e a n d liver t R N A b y R P C - 3 c o l u m n c h r o m a t o g r a p h y . R e t i c u l o c y t e t R N A (423 absorbance units) and liver tRNA (450 absorbance units) were eluted from an RPC-3 column in s e p a r a t e p r o c e d u r e s . T h e t o p p a n e l s s h o w t h e p a t t e r n s o f e l u t i o n o f m a t e r i a l a b s o r b i n g a t 2 6 0 rum a n d t h e lower panels compare elution patterns of amino acid acceptance act/vity based on assays of 0.05 ml aliquots of the column fractions. Every fraction was assayed through peak regions. The patterns in this f i g u r e a r e t h o s e w h i c h s h o w little d i f f e r e n c e b e t w e e n r e t i c u l o e y t e s a n d liver.
370
RETICULOCYTE tRNA
LIVER tRNA
|i o.~i
~: o 0250 u~cJ t500
'~
Methmnine
,O oo I 900 6O0 500 o
1200
~
600 3o~
J~
600 400
o
o 600 400
~-
S
<
/,t/I ,
~
~
,
<
0
0' 001500500
0
0 1200 800 400
~//~1 Arginine
~ '
' ' [soleucine
,t~
Leucine ,~ , /~ .
i
. . . Phenylalonine
i
'
--' 1 II
/ / ~
.
i
M~ i
,\
,,'
/
600 4OO 20C
~ _ ~
Alpine
°
1200 900 600 500 0 1200 900
~
GLutOm~Acid
,
Proline
, Serine
,
,
~Threonine
i
,
,
/ L
/ ~
,
50 I00 150 200 FRACTION NUMBER
=
,
/
50 t(30 i50 200 FRACTION NUMBER
Fig. 2. C h r o m a t o g r a p h i c r e s o l u t i o n o f r e t i c u l o c y t e a n d liver t R N A . T h e r e s u l t s in this f i g u r e a r e t a k e n f r o m t h e s a m e c h r o m a t o g r a p h i c p r o c e d t ~ e s as t h o s e m Fig. 1. T h e p a t ~ r ~ s h o w n are t h o s e in w h i c h t h e r e are large q u a n t i t a t i v e d i f f e r e n c e s in t h e d i s t r i b u t i o n o f t R N A a m o n g i s o a c c e p t i n g species.
RETICULOCYTE tRNA
w
LIVER tRNA
i| = , L
-.9F, ;
,
Asporagine
'~
~
Histidine
50 IOO laO 2oo o FRACTION NUMBER
FRACTION NUMBER
Fig. 3. Chxomatol~raphic r e s o l u t i o n o f r e t i c u l o c y t e a n d Uver t R N A . T h e r e s u l t s in this ~ are t a k e n f r o m t h e s a m e c b z o m a t o l m p h t c p r o c e d u ~ s as t h o s e s h o w n in F i p 1 a n d 2. T h e p a t t e r n s s h o w n are of asparaifJne a n d h i s t i d i n e t R N A a n d i n d i c a t e in e a c h case a t R N A species c l e a r l y r e s o l v e d f r o m r e t i c u l o c y t e t R N A w h i c h is n o t f o u n d in liver t R N A .
371
similar. In some other comparisons, major quantitative differences are found between reticulocyte and liver tRNA, as shown in Fig. 2. Two peaks of methionine acceptance activity can be fractionated from both liver and reticulocyte tRNA, but the first peak is much larger in reticulocytes, while in liver there is approximately the same amount of acceptance activity in each peak. Preliminary evidence from our laboratory and results from other laboratories [16--18] indicate that the first of the peaks is probably the formylatable methionine tRNA species (methionine tRNA~) involved in initiation of translation. Quantitative differences in the distribution of tRNA among wellresolved isoaccepting species were also found for glutamic acid tRNA. Differences in elution patterns for alanine, arginine, isoleucine, leucine, phenylalanine, proline, serine, and threonine tRNAs, can be explained by the distribution of tRNA among isoaccepting species, but additional chromatographic evidence will be necessary to determine whether this is the case, or whether some of the differences intvolve species unique to one of the sources of the tRNA. Two very clear qualitative differences emerge from the comparison. There are two well resolved species of histidine and asparagine tRNA in reticulocytes compared to only one for each of these amino acids in liver, as shown in Fig. 3. These apparently unique tRNA species provided the basis for additional investigations. The patterns shown in the figures are based on assays with synthetases from the same source as the tRNA, however, the same patterns are seen regardless of the synthetase source, indicating that the differences are due to tRNA.
•r
T
0
b. Acceptance Activity
-,,z
0 3000[ ~.~
~
c. Liver [,4C1 Ash. ,RNA
2000
0
25
50 FRACTION NUMBER
75
Fig. 4. C h r o m a t o g r a p h i c r e s o l u t i o n o f t R N A o n R P C - 5 c o l u m n s . T h e c o l u m n u s e d measttred 1 c m ( d i a m e t e r ) X 3 0 c m , and the p a c k i n g m a t e r i a l w a s o b t a i n e d f r o m Miles L a b o r a t o r i e s , I n c . A l i n e a r gradient o f 2 5 0 m l w a s e m p l o y e d f r o m 0 . 4 - - 0 . 7 M LiC1 c o n t a i n i n g 0 . 0 1 M MgC12, 0 . 0 1 M l i t h i u m acetate, pH 4.5, and 0.002 M ~-mercaptoethanol. The columns were run with a head of 2 M fed by gravity through c a p i l l a r y t u b i n g a t r o o m t e m p e r a t u r e . E i n t i o n u n d e r these c o n d i t i o n s r e q u i r e d a p p r o x . 1 2 h , a n d f r a c t i o n s o f 2 . 5 m l w e r e c o l l e c t e d . A s d e s c r i b e d i n t h e t e x t , t h e r e s o l u t i o n o f a b s o r b a n c e a t 2 6 0 n m is s h o w n in P a n e l a a n d that o f h i s t i d i n e a n d a s p a r a g i n e a c c e p t a n c e b y r e t i c u l o c y t e t R N A is s h o w n i n P a n e l b. P a n e l c s h o w s e l u t i o n o f l i v e r [ 1 4 C ] a s p a r a g i n y l - t R N A c o o e h r o m a t o g r a p h e d w i t h t h e r e t i c u l o c y t e t R N A . Its elut i o n c o r r e s p o n d s t o t h e f i r s t o f t h e t w o r e t i c u l o c y t e a s p a r a g i n e species.
372
Confirmation of chromatographic differences for histidine and asparagine tRNA Elution profiles for histidine and asparagine tRNA from liver and reticulocytes were compared further by chromatography on a different reversed-phase column system, RPC-5 [8]. Fig. 4 shows a typical experiment. Panels a and b show the resolution of absorbance at 260 nm and of histidine and asparagine acceptance activity of reticulocyte tRNA. The results confirm the existence of two well-separated isoaccepting species for each of these amino acids in reticulocytes. Chromatography of liver tRNA (not shown in the figure) resolved only a single species each of histidine and asparagine tRNA. Panel c shows the elution pattern of the aminoacylated asparagine tRNA species from liver, chromatographed along with carrier reticulocyte tRNA. In this experiment, it was determined both that the liver asparagine tRNA species moves as a single peak on rechromatography and that it corresponds to the first of the two asparagine tRNA species in reticulocytes. In column experiments which are not shown, both reticulocyte tRNA species for asparagine and histidine and the liver histidine tRNA species rechromatographed as single peaks, without evidence of heterogeneity. The two species each of histidine and asparagine t R N A from reticulocytes are eluted in the same sequence from RPC-5 columns as RPC-3 columns. The single histidine tRNA species in liver corresponds to the first of the two reticulocyte histidine tRNA peaks.
Coding studies with histidyl- and asparaginyl-tRNA To our knowledge, the present work is the first time coding studies have been done on isolated animal t R N A isoaccepting species for histidine and asparagine. The amounts of aminoacyl-tRNA, trinucleotide diphosphates, and other elements in the assays are described in Table I, and the results are shown
TABLE I C O M P O N E N T S A D D E D TO C O D I N G R E A C T I O N S * Species of a m i n o a c y l t R N A
Aminoacyl Added tRNA pmoles (A260 nm units)
cpm/ pmole
Codon
A m o u n t of c o d o n added (A260 nm units)
AAU AAC
0.292 0.326
CAU CAC
0.183 0.180
I. Asparagine e x p e r i m e n t s
R e t i c u l o c y t e [14C] A s n - t R N A I R e t i c u l o c y t e [14C] A s n - t R N A II Liver [ 14C] A s n - t R N A
0.041 0.043 0.051
2.71 2.85 3.71
276 276 276
2. H i s t i d i n e e x p e r i m e n t s
R e t i c u l o c y t e [ 14C] H i s - t R N A I R e t i c u l o c y t e [ 1 4 C ] H I s - t R N A II Liver [ 14C] H I s - t R N A
0.059 0.032 0.072
2.11 2.48 2.13
590 590 590
* I n a d d i t i o n t o the above c o m p o n e n t s which varied, each reaction contained 0 . 0 5 M ~ acetate, p H 7.3; 0 . 0 5 M p o t a s s i u m a c e t a t e ; 0.01 or 0 . 0 2 M m a g n e s i u m acetate, as s h o w n above; and E. colt ribosomes, 2.0 absorbance u n i t s , i n a t o t a l v o l u m e o f 0 . 0 5 5 ml.
373 TABLE II RESPONSES OF FRACTIONATED tRNAs TO CODONS Triplet c o d o n and r e s p o n s e (A p m o l e b o u n d to r i b o s o m e s ) *
E x p e r i m e n t s in 10 mM Mg 2+ R e t i c u l o c y t e [14C] Asn-tRNA I R e t i c u l o c y t e [ 14C] Asn-tRNA II Liver [ 14C] Asn-tRNA R e t i c u l o c y t e [14C] His-tRNA I R e t i c u l o c y t e [ 14C] His-tRNA II Liver [14C] His-tRNA E x p e r i m e n t s in 20 mM Mg 2+ R e t i c u l o c y t e [ 14C] Asn-tRNA I R e t i c u l o c y t e [ 14C] Asn-tRNA II Liver [ 14C] Asn-tRNA R e t i c u l o c y t e [ 14C] His-tRNA I R e t i c u l o c y t e [14C] His-tRNA II Liver [ 14C] His-tRNA
Asparagine c o d o n s
Histidine c o d o n s
AAC
AAU
CAC
0.043 0.043 0.072
0.094 0.033 0.072 0.025 0.017 0.008
1.163 1.225 1.087
CAU
0.169 0.051 0.117
(0.011) (0.022) (0.029) (0.012) (0.017) (0.019)
0.688 0.668 0.407
(0.170) (0.185) (0.130) (0.061) (0.129) (0.073)
1.270 0.949 0.840 0.134 0.266 0.124
None**
* A m o u n t of a m i n o a c y l - t R N A b o u n d to r i b o s o m e s in presence o f c o d o n m i n u s the a m o u n t b o u n d in absence o f codon. ** A m o u n t o f a m i n o a c y l - t R N A b o u n d to r i b o s o m e s in absence of c o d o n ( s h o w n in parentheses because these a m o u n t s have already b e e n subtracted f~om the values s h o w n for c o d o n responses).
in Table II. The results are readily reproducible, and there is excellent binding at several times blank values (without codon) at 20 mM Mg 2÷ with the appropriate codons. The studies at 10 mM Mg2÷ revealed similar binding, but at much lower levels and without increased specificity. For both asparagine and histidine, two codons have been established [ 14] which differ in each case only by a pyrimidine in the third letter of the triplet. As expected from the "Wobble Hypothesis" [19], each tRNA species was bound to ribosomes in the presence of both code words. Consistent quantitative differences were observed with asparaginyl-tRNA which may be significant. While the first peak of asparaginyl-tRNA from reticulocytes was bound equally to ribosomes in the presence of both triplets, the second peak and the peak of liver asparaginyl-tRNA were bound better in the presence of AAC than AAU. It may also be significant that the binding of the second reticulocyte histidyltRNA species as stimulated by CAC, is substantially higher than CAC-stimulated binding of other species. Discussion The study reported here has been done differently from other studies comparing tRNA from different cells. The RPC-3 column, though it has not been widely used, is very reliable and permits a nearly tube-by-tube comparison o f the results of different fractionations. The assay of amino acid acceptance by tRNA after chromatography, rather than the use of labelled aminoacyl-
374
tRNA in the column inputs, permits quantitative assessment of tRNA after elution and is not dependent on the stability of aminoacyl bonds during elution, which is known to vary for different aminoacyl-tRNAs [20,21]. Other workers using RPC-2 columns [22] and labelled aminoacyl-tRNA have also found various numbers of isoaccepting species for asparagine [23--26] and histidine [23--28], as well as other amino acids from several kinds of cells. The nature of the multiple isoaccepting tRNA species found in these studies remains uncertain. When isoaccepting species resolved chromatographically have been examined, some have represented different gene products with differing base sequences (see refs 29--31), others have represented different post-synthetic modifications of tRNA (see refs 32 and 33), and yet others have represented different structural configurations (see ref. 34). The failure of coding experiments to permit certain assignment of anticodon sequences to the isoaccepting species for histidine and asparagine in the present study, is consistent with the "Wobble Hypothesis" [19] and does not rule out the possibility that the isoaccepting species may have different base sequences in the anticodon as well as elsewhere. The recent studies of Farkas et al. [35] suggest that one of the reticulocyte histidine tRNA species has a guanine group attached to some internal nucleotide. It is possible that the difference between the two histidine tRNA species in reticulocytes is the presence of this guanine group, and that the guanylation reaction is specific to reticulocytes so that the guanylated species is not found in some other kinds of cells such as liver. The present work extends the comparison between reticulocyte and liver tRNA, as previously published [6] to the level of isoaccepting species. In the case of histidine, part of the difference in the relative amounts of acceptance activity can be attributed to a tRNA species found only in reticulocytes. With isoleucine, however, the differences in the relative abundance of tRNA in liver and reticulocytes cannot be related to a unique tRNA species or even to large differences in the distribution of tRNA among the same isoaccepting species. There is little difference in the relative abundance of asparagine tRNA between liver and reticulocytes, but an isoaccepting species is found in reticulocytes which is not present in liver. There are no results that can be clearly related to the abundance of mitochondria in liver compared to reticulocytes [36], although studies from other laboratories (see ref. 37) show that there are unique mitochondrial tRNA species for some amino acids that are encoded by mitochondrial DNA. The present study comparing the isoaccepting species of liver and reticulocyte tRNA provides additional data concerning the nature of the specialization of the reticulocyte tRNA content for hemoglobin synthesis. In a few cells and tissues, in addition to reticulocytes, it has been possible to develop evidence of tRNA specialization for protein synthesis. These include the silk glands of silk worms [38], mammary glands at the time of lactation [39], the fibroblasts of healing wounds [40], and the livers of roosters treated with estrogens [41]. There is a study by Litt and Kabat [42] comparing the tRNA of reticulocytes of anemic and normal sheep, which make hemoglobins of different amino acid sequence, which gives evidence, in addition to that from our laboratory, that reticulocyte tRNA is specialized for hemoglobin synthesis.
375
Acknowledgements This work was supported by Research Grant AM-15467 from the National Institutes of Health. One of the authors (V.N.M.) was supported by a fellowship from the American Cancer Society, Illinois Division during part of the time he worked on this project. The authors are very grateful to Dr Dolph L. Hatfield of the National Institutes of Health for the help and useful discussion he provided.
References 1 Lingrel, J.B. and Borsook, H. (1963) Biochemistry 2, 309--314 2 Lodish, H.F. and Desalu O. (1973) J. Biol. Chem. 248, 3520--3527 3 Itano, H.A. (1965) in Abnormal Hemoglobins in Africa (J.H.P. Jonxis, ed.), pp. 3--16, BlackweU, Oxford 4 Smith, D.W.E. and McNamara, A.L. (1972) Biochim. Biophys. Acta 269, 66--77 5 Smith, D.W.E. and McNamara, A.L. (1974) J. Biol. Chem. 249, 1330--1334 6 Smith, D.W.E. and McNamara, A.L. (1971) Science 171,577--579 and erratum, 1040 7 Weiss, J.F., Pearson, R.L. and Kelmers, A.D. (1968) Biochemistry 7, 3479--3487 8 Kelmers, A.D. and Heatherly, D.E. (1971) Anal. Biochem. 44, 486--495 9 Bray, G.A. (1960) Anal. Biochem. 1,279--285 10 Hatfield, D. (1972) Proc. Natl. Acad. Sci. U.S. 69, 3014--3018 11 Pestka, S. (1968) J. Biol. Chem. 243, 2810--2820 12 Trupin, J.S., Rottman, F . M , Brimacombe, R.L.C., Leder, P., Bernfield, M.R. and Nirenberg, M.W. (1965) Proc. Natl. Acad. Sci. U.S. 53, 807--811 13 Nirenberg, M., Leder, P., Bernfield, M., Brimacombe, R., Trupin, J., Rottman, F., and O'Neal, L. (1965) Proc. Natl. Acad. Sci. U.S. 53, 1161--1168 14 Marshall, R.E., Caskey, C.T. and Nirenberg, M. (1967) Science 153,820--826 15 Nixenberg, M. and Ledar, P. (1964) Science 145, 1399---1407 16 Caskey, C.T., Beaudet, A. and Nirenberg, M. (1968) J. Mol. Biol. 37, 99--118 17 Shafritz, D.A. and Anderson, W.F. (1970) Nature 227, 918--920 18 Yang, W.-K. (1971) Cancer Res. 31,639---643 19 Crick, F.H.C. (1966) J. Mol. Biol. 19, 548--555 20 Satin, P.S. and Zamecnik, P.C. (1964) Biochim. Biophys. Acta 91,653---655 21 Gatica, M., Allende, C.C., Mora, G., AUende, J.E. and Medina J. (1966) Biochim. Biophys. Acta 129, 201--203
22 23 24 25 26 27 28 29 30 31 32. 33 34 35 36 37 38 39 40 41 42
Weis, J,F. and Kelmers, A.D. (1967) Biochemistry 6, 2507--2513 Gallo, R.C. and Pestka, S. (1970) J, Mol. Biol. 52, 195---219 Yang, W.-K. and Novelli, G.D. (1968) Proc. Natl. Acad. Sci. U.S. 59, 208--215 Ortwerth, B.J. (1971) Biochemistry 10, 4 1 9 0 - 4 1 9 7 Gonano, F., Chiarugi, V.P., Pirro, G. and Marlni, M. (1971) Biochemistry 10, 900--908 Yang, W.-K., Hellman, A., Martin, D.H., Hellman, K.B. and Novellt G.D. (1969) Proc. Natl. Acad. Sci. U.S. 64, 1411--1418 Volkers, S.A.S. and Taylor, M.W. (1971) Biochemistry 10, 488--497 Cory, S., Marcker, K.A., Dube, S.A. and Clark, B.F.C. (1968) Nature 220, 1039--1040 Staehelin, M. (1971) Experlentia 27, 1--11 Folk, W.R. and Yaniv, M. (!972) Nat. New Biol. 237, 165--166 Shugart, L., Nove]li, G.D. and Stulberg, M.P. (1968) Biochim. Biophys. Acta 157, 83--90 Gefter, M.L. and Russell, R.L. (1969) J. Mol. Biol. 39, 145--157 Ishida, T. and Sueoka, N. (1968) J. Mol. Biol. 37, 313--316 Farkas, W.R., Hanklns, W.D. and Singh, R. (1973) Biochim. Biophys. Acta 294, 94--105 Jensen, W.N., Moreno, G.D. and Bessis, M. (1965) Blood 25, 933--943 Nass, M.M.K. and Buck, C.A. (1970) J. Mol. Biol. 54, 187--198 Garel, J.-P., Mandel, P., Chavancy, G. and Dalllie, J. (1971) Biochemie 53, 1195--1200 Elska, A., Matsuka, G. and Matiash, U. (1971) Biochim. Biophys. Acta 247, 430--440 Lanks, K.W. and Weinstein, I.B. (1979) Biochem. Biophys. Res. Commun. 40, 708--715 M~ienp~i, P.H. (1972) Biochem. Biophys. Res. Commun. 4 7 , 9 7 1 - - 9 7 4 Litt, M. and Kabat, D. (1972) J. Biol. Chem. 247, 6659---6664