Download Transfer RNA Specificity in Mammalian Tissues and Codon

[CANCER RESEARCH 31, 697-700,
May 1971]
Transfer RNA Specificity in Mammalian Tissues and
Codon Responses of Seryl Transfer RNA
Dolph Hatfield,
Franklin H. Portugal, and Mary Caicuts
National Cancer Institute, NIH, Bethesda, Maryland 20014
The possibility that tRNA may play a role in cellular
regulation was first suggested by Itano (6) and subsequently
by Ames and Hartman (2) and by Stent (13). Although no
unique role of tRNA in regulation has thus far been
established, changes in tRNA have been observed during phage
infection, under varying growth conditions, in differentiation,
and in oncogenesis. Presumably, these changes invoke changes
in protein biosynthesis. In multicellular organisms, where
many metabolic processes are unique to individual tissues,
differences in tRNA in different tissues might be expected if
these adaptor molecules play a role in regulation. A study was
therefore undertaken to examine several aminoacyl-tRNA's in
bovine kidney, liver, and brain.
tRNA was isolated by 2 methods. In Method 1, tRNA was
extracted from whole tissues with buffer and phenol, followed
by isolation of the tRNA with diethylaminoethyl
cellulose
chromatography. In Method 2, particulate cellular matter was
first removed, and the tRNA was then extracted with phenol
(5). Isolated tRNA was deacylated in 1.8 M Tris, pH 8.0, for l
hr at 37°.Aminoacyl-tRNA synthetases were prepared from
each tissue (5). Labeled aminoacyl-tRNA's were prepared as
shown above (see Chart 1), with the exception of the 1st
eluting peak of liver seryl-tRNA and the 2nd eluting peak of
brain seryl-tRNA. These peaks have a shoulder in Chart 1 but
no shoulder in Chart 2. Additional studies suggest that these
differences in elution profiles are not due to method of
preparation but to a better resolution obtained in the
experiment shown in Chart 1. In any case, the differences in
methionyl-, arginyl-, and seryl-tRNA's are not due to
mitochondrial tRNA and are present when the tRNA's are
prepared by 2 methods.
The differences are observed when methionyl-, arginyl-, and
seryl-tRNA's of liver are acylated with 3H-labeled amino acid
and those of brain with 14C-labeled amino acid, i.e., when the
isotopes are reversed. The differences are also observed when
these aminoacyl-tRNA's of liver are acylated with an enzyme
previously described (5) and applied to the reversed phase
Chromatographie system of Kelmers et al. (7).
Chromatography
of 3 aminoacyl-tRNA's investigated in
preparation from brain and those of brain with an enzyme
preparation
from liver, i.e., when the aminoacyl-tRNA
synthetases are reversed. These results and those given above
demonstrate that the differences are present in the isolated
tRNA of each tissue and are not due to isotope, to
tissue-specific aminoacyl-tRNA synthetases or to the tRNA of
a subcellular organeile.
To determine whether the tissue specificity observed in
methionyl-, arginyl-, and seryl-tRNA's of bovine liver and
bovine liver and brain are shown in Chart 1. The elution
profiles of methionyl-,
arginyl-, and seryl-tRNA's
are
brain also occur in these tissues of another mammal, the
elution profiles of these aminoacyl-tRNA's from rabbit liver
compared. Significant differences were observed in their
elution profiles, and the most pronounced differences were
found in the seryl-tRNA's of these tissues. The elution profiles
of methionyl-, arginyl-, and seryl-tRNA's of liver and kidney
and of phenylalanyl-, lysyl-, and leucyl-tRNA's of liver,
and brain were compared (Chart 3). The profiles of liver and
brain methionyl-tRNA's
(Chart 3, upper graph) are
kidney, and brain were similar and were not further
investigated.
tRNA in mitochrondria is known tobe different from that in
cytoplasm. Mitochondria were not removed during preparation
of the tRNA used in the above studies. tRNA was therefore
prepared from a postmitochrondrial
fraction of bovine liver
and
brain
(Method
2). Diethylaminoethyl
cellulose
chromatography was not used in this procedure, and thus the
2 methods of preparation differ considerably. The tRNA
prepared by the latter procedure from liver and brain was
acylated
in
separate
experiments
with
radioactive
methionine, arginine, and serine. Their elution profiles are
compared in Chart 2. The upper graph shows the profiles of
liver and brain methionyl-tRNA's,
the middle graph shows
arginyl-tRNA's, and the lower graph shows seryl-tRNA's. The
differences observed in the elution profiles of each of these
aminoacyl-tRNA's are similar to the corresponding profiles
comparable to those of bovine liver and brain. Although
arginyl-tRNA of liver (middle graph) was resolved into 3 major
peaks as compared to 2 in bovine liver, differences between
arginyl-tRNA's of the 2 rabbit tissues are evident. The major
differences which were observed in the seryl-tRNA's of bovine
brain and liver (see Chart 1 and 2) were also present in the
corresponding rabbit tissues (Chart 3, lower graph).
The elution profiles of chicken liver and brain methionyl-,
arginyl-, and seryl-tRNA's
are compared
in Chart 4.
Methionyl-tRNA's (upper graph) were resolved into 4 peaks
and quantitative differences were observed. Differences were
also observed in the elution profiles of arginyl-tRNA's (middle
graph). Differences were observed in the seryl-tRNA's of
chicken liver and brain; but these were not as pronounced as
those in the corresponding tissues of mammals. The peak of
seryl-tRNA in brain which eluted third from the column ran
slightly in front of that in liver, suggesting that differences
were present in this peak. Additionally, a shoulder is present
on Peak III of brain which is not present in liver.
The differences observed in chicken liver and brain
MAY 1971
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697
D. Hatfield, F. H. Portugal, and M. Caicuts
methionyl-,
I4C 3H
2500-r5000
3000
6000
1500+3000
/
(Beefuï-erl
=>
o
o
4000T8000
were also observed
when the tRNA was prepared by Method 1 and when the
isotopes and the aminoacyl-tRNA synthetases of each tissue
were reversed.
Several experiments were carried out to determine whether
the observed differences are due to changes that occur during
isolation. Studies in other laboratories have shown that tRNA
may be modified by action of caustic reagents used in tRNA
perparation (14), dimer formation (1, 10), loss of CCA
terminus (8), and treatment with magnesium which may
activate an inactive tRNA (9). Seryl-tRNA of bovine liver and
brain, which manifested the most pronounced differences in
the present investigation, was selected as a model for these
studies. The differences in seryl-tRNA did not appear to arise
in preparation of brain and liver tRNA as a result of the step
used to deacylate endogenous aminoacyl-tRNA's or as a result
Arg-tRNA
(BeefBrain
[I4C] Arg-tRNA
arginyl-, and seryl-tRNA's
[I4d Ser-tRNA~,
(Beefüver)
2000-1-4000
150
FRACTION NUMBER
Chart 1. Comparison of elution profiles of methionyl-, arginyl-, and
seryl-tRNA's of bovine liver and brain. Liver tRNA was acylated with
liver aminoacyl-tRNA synthetases and l4C-labeled amino acid and
compared to brain tRNA acylated with brain aminoacyl-tRNA
synthetases and 3H-labeled amino acid. tRNA was prepared by Method
of the use of caustic reagents (phenol and ethanol); nor did
they appear to be due to dimer formation or loss of CCA
terminus. Furthermore, the differences did not appear to be
affected by heating brain and liver tRNA at elevated
temperatures in the presence or absence of magnesium or by
prolonged dialysis against EDTA. Slight changes in the elution
profiles of brain and liver seryl-tRNA were observed following
incubation of brain tRNA in liver extract and of liver tRNA in
brain extract, but these changes were not significant enough to
account for the differences observed.
1.
'4C
3H
2000 -ri 0000
[3H] Met-tRNA
(Rabbit Broin)
—,io
2000 -r 4000
1000 - - 5000
o-'t
4000-T-
uj 2000 -- -
3
O
U
O-1
2000 -i-
IOOO - -
o-Jk
150
300
o-1
150
FRACTION NUMBER
Chart 2. Comparison of elution profiles of methionyl-, arginyl-, and
seryl-tRNA's of bovine liver and brain. Conditions were the same as
those in Chart 1 with the exception that tRNA was prepared by
Method 2.
698
3OO
FRACTION NUMBER
Chart 3. Comparison of elution profiles of methionyl-, arginyl-, and
seryl-tRNA's of rabbit liver and brain. Conditions were the same as
those in Chart 2.
CANCER RESEARCH VOL. 31
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tRNA Specificity
Table 1
Codon responses of Peak III from chicken liver
Assay conditions were slightly modified from those of Nirenberg and
Leder (12) as previously described (5). Codons were the gift of Dr.
Marshall W. Nirenberg.
CodonUCUUCCUCAUCG,
UGANonePeak
AGC, UAA, UAG,
III(A
pmoles
bound0)0.5540.0220.1140.005
or less
(0.153)b
0 Amount of seryl-tRNA bound to ribosomes in presence of codon
minus the amount bound in absence of codon.
b Amount of seryl-tRNA bound to ribosomes in absence of codon.
very slight with UCC (see Table 1). The codon responses of
Peaks I to III of bovine liver seryl-tRNA and of Peak III of
chicken liver seryl-tRNA were similar to those of guinea pig
liver reported by Caskey et al. (4) and to those of chicken liver
reported by Bernfield (3) at this symposium.
The peak of seryl-tRNA that eluted in the salt wash (Peak
IV of liver and Peak VI of brain) responded to UGA and UCU.
For resolution of the UGA and UCU response, brain
seryl-tRNA was fractionated at a different salt gradient (5),
-0.5
and the terminal peak was assayed with UGA, UCU, UCA,
UGU, UGC, UGG, UUA, AGA, CGA, and GGA. This peak
responded only to UGA. Therefore, a species of seryl-tRNA is
present in isolated tRNA of bovine tissues which recognizes
specifically the codon UGA. Furthermore,
a species of
150
300
seryl-tRNA obtained from rabbit liver and from chicken liver
FRACTIONNUMBER
Chart 4. Comparison of elution profiles of methionyl-, arginyl-, and recognized UGA (5).
seryl-tRNA's of chicken liver and brain. Conditions were the same as
Bernfield
(3)
reported
the
occurrence
of
0-phosphorylseryl-tRNA
in rooster liver (see also Ref. l 1);
those in Chart 2.
this species eluted in a terminal position from benzoylated
diethylaminoethyl cellulose and did not respond to any of the
Seryl-tRNA of bovine liver and brain was fractionated on a known serine codons. It seems likely that the seryl-tRNA
reversed phase Chromatographie column, and the individual which recognizes UGA (seryl-tRNAyGA) is the same species
Studies
peaks were assayed for codon recognition (5). Four peaks of reported by Bernfield to be 0-phospnorylseryl-tRNA.
to elucidate the role of seryl-tRNAUGA in higher organisms,
liver seryl-tRNA eluted from the column and were designated,
in their order of elution, Peaks I to IV (Peak IV eluted in l M as well as the possibility the serine moiety may undergo
further modification, are in progress.
NaCl). In a separate experiment, 6 peaks of brain seryl-tRNA
eluted from the column and were designated I to VI. Peaks I,
III, and IV in liver correspond to Peaks I, IV, and VI,
respectively, in brain; Peaks II and V in brain constitute peaks
REFERENCES
which are not evident in liver. The results of the codon studies
have been reported elsewhere (5) and are summarized below.
1. Adams, A., and Zachau, H. G. Serine Specific Transfer Ribonucleic
All fractionated peaks were assayed at 0.01 M Mg4*with
known serine codons, UCU, UCC, UCA, UCG, AGU, and
AGC, and with terminator codons, UGA, UAA, and UAG.
Peak I of liver and Peaks I and II of brain responded to AGU
and AGC. Peak II of liver and Peak III of brain responded to
UCG. Peak III of liver and Peaks IV and V of brain responded
to UCU, UCA, and UCC; the level of response was most
pronounced with UCU, less with UCA, and very slight with
UCC. The corresponding peak (Peak III) of seryl-tRNA in
chicken liver responded similarly to these codons; the level of
response was most pronounced with UCU, less with UCA, and
Acids 15. Some Properties of the Aggregates from Serine Specific
Transfer Ribonucleic Acids. European J. Biochem., 5: 556-558,
1968.
2. Ames, B. N., and Hartman, P. E. The Histidine Operon. Cold
Spring Harbor Symp. Quant. Biol., 28: 349-356, 1963.
3. Bernfield, M. R., and Mäenpää,
P. H. Seryl Transfer RNA Changes
during Estrogen-induced Synthesis and a Unique Seryl Transfer
RNA Modification. Cancer Res., 31: 684-687, 1971.
4. Caskey, C. T., Beaudet, A., and Nirenberg, M. RNA Codons and
Protein Synthesis 15. Dissimilar Responses of Mammalian and
Bacterial Transfer RNA Fractions to Messenger RNA Codons. J.
Mol. Biol., 37: 99-118, 1968.
MAY 1971
Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1971 American Association for Cancer Research.
699
D. Hatfield, F. H. Portugal, and M. Caicuts
5. Hatfield, D., and Portugal, F. H. Seryl-tRNA in Mammalian
Tissues: Chromatographie Differences in Brain and Liver and a
Specific Response to the Codon, UGA. Proc. Nati. Acad. Sei. U. S.,
67: 1200-1206, 1970.
6. llano, H. A. The Synthesis and Structure of Abnormal
Hemoglobins. In: J. H. P. Jonxis (ed.). Abnormal Hemoglobins in
Africa. A Symposium, pp. 3-16. Philadelphia: F. A. Davis Co.,
1965.
7. Kelmers, A. D., Novelli, G. D., and Stulberg, M. P. Separation of
Transfer Ribonucleic Acids by Reverse Phase Chromatography. J.
Biol. Chem., 240: 3979-3983, 1965.
8. Lebowitz, P., Ipata, P. L., Makman, M. H., Richards, H. H., and
Cantoni, G. L. Resolution of Cytidine- and Adenosine-terminal
Transfer Ribonucleic Acids. Biochemistry, 5: 3617-3625, 1966.
9. Lindahl, T., Adams, A., and Fresco, J. R. Renaturation of Transfer
Ribonucleic Acids through Site Binding of Magnesium. Proc. Nati.
Acad. Sei. U. S., 55: 941-948, 1966.
10. Loehr, J. S., and Keller, E. B. Dimers of Alanine Transfer RNA
with Acceptor Activity. Proc. Nati. Acad. Sci. U. S., 61:
1115-1122, 1968.
11. Mäenpää,
P. H., and Bernfield, M. R. A Specific Rooster Liver
tRNA Containing Phosphoserine. Federation Proc.,29: 468, 1970.
12. Nirenberg, M., and Leder, P. RNA Codewords and Protein
Synthesis. The Effect of Trinucleotides upon the Binding of sRNA
to Ribosomes. Science, 145: 1399-1407, 1964.
13. Stent, G. S. The Operon: On its Third Anniversary. Science, 144:
816-820, 1964.
14. Sueoka, N., and Hardy, J. Deproteinization of Cell Extract with
Silicic Acid. Arch. Biochem. Biophys., 125: 558-566, 1968.
evidence for phosphoseryl-tRNA in yeast or bacterial tRNA
acylated either with homologous or with the rat or rooster
liver enzyme.
Dr. Hatfield: With rat liver, have you been able to obtain a
phosphorylating seryl-tRNA enzyme?
Dr. Bernfield: We have some evidence for that, but I should
mention that it is very difficult to do unless you get
reasonably pure tRNA from that peak. It is very difficult to
pick up unless you further fractionate that last tRNA peak.
Dr. Weinstein: I would like to make a comment on
redundancy which I think your data indicate very nicely. As
the brain goes through the trouble of making an extra serine
tRNA, Peak II which has codon recognition identical to Peak
I, and there are, of course, many such examples, normal liver
has 3 tyrosyl-tRNA's which have identical codon recognition. I
think we are stuck with the problem of redundancy. I wonder
what your comments are?
Dr. Hatfield: One of the problems here may be that this
species really needs another methyl group or something of this
nature to become identical to Peak I, and that may be what we
are actually separating. Peak II may be a precursor to Peak I.
I don't know what to say about tRNA differences at the
moment. I don't think anyone knows what to make of them.
Dr. Borek: I would just like to be redundant to what Dr.
Weinstein said. There are all of these changes in the
seryl-tRNA's. Dr. Bernfield finds it in the rooster with
enormous doses of hormone. We find it with normal
administration of hormone in the ovariectomized uterus.
Unfortunately, all we can measure is codon response. Isn't
Discussion
Dr. Bemfield: Have you looked for phosphoserine?
Dr. Hatfield: Yes, we have. We have encountered problems
in looking for derivatives of serine, and I don't know why.
In the case of JV-acetylserine, we spent a lot of time on this
problem. We are able to acetylate the serine but not
enzymatically. I think this is just one problem we have
encountered there. With regard to phosphoserine, what we
have done is to isolate the last peak, and we have tried to
phosphorylate the serine. We have not thus far been successful.
Dr. Bernfield: I could mention in this regard we have not
found any evidence of an acetylserine in the rat and rooster. I
should also mention that we were not able to find any
700
this essentially our problem? The codon response is rather
limited. There are only 64 of them. Once we could find some
in vitro assays which would give us greater latitude than the
codon response, I think we might be finding differences in
function.
Dr. Bock: I would like to comment on the codon
recognition pattern where you find that, unlike the normal
"wobble hypothesis" patterns, Base A is the 3rd letter of the
codon. Ukita has found that type of pattern for a glutamate
acceptor tRNA and has shown that a modified 2-thiouridine is
the base which recognizes the A. It is possible selectively to
destroy the thiouridine bases without harming other RNA
components. Thus it may be possible to test whether the
tRNA you studied also has a mio base in the anticodon.
CANCER RESEARCH VOL. 31
Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1971 American Association for Cancer Research.
Transfer RNA Specificity in Mammalian Tissues and Codon
Responses of Seryl Transfer RNA
Dolph Hatfield, Franklin H. Portugal and Mary Caicuts
Cancer Res 1971;31:697-700.
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