[CANCER RESEARCH 31, 675-678,
May 1971 ]
Modified Bases and Transfer RNA Function
Alan Peterkofsky,
Marcia Litwack, and Jane Marmor
laboratory of Biochemical Genetics, National Heart and Lung Institute, NIH, Bethesda, Maryland 20014
Summary
We have carried out a variety of comparisons of biological
activities of normal and modification-deficient
tRNA. It
becomes clear that we can make no generalized statement that
methylated bases confer a certain type of property on all
tRNA's, since various effects are observed with different tRNA
species. Methyl-deficient
methionine
tRNA exhibits a
definitely slower acylation rate, while methyl-deficient leucine
tRNA shows a defective recognition by a heterologous
aminoacyl-tRNA synthetase as well as an altered pattern of
codon recognition. It is difficult to assess the importance of
the effects that we have observed in terms of the significance
of the methylation process. The isolation of a viable mutant of
Escherichia coli deficient in ribothymidine in its tRNA (2)
introduces some questions as to whether tRNA methylation is
an absolute requirement for all growth processes. Another area
in which insufficient attention has been focused is control
processes in which tRNA may be involved (5). Perhaps tRNA
methylation is crucial to some of these events.
Introduction
Modified bases account for 10 to 20% of the total
nucleotide context of tRNA. In general, the modified bases are
more prevalent in the tRNA of eukaryotes than in that of
prokaryotic cells. This paper discusses the work from our
laboratory concerned with studies on the importance of
modifed bases for the biological functions of tRNA.
Methylated Bases
A search for the role of methylated bases in the function of
tRNA was launched in several laboratories as the result of a
key observation of Mandel and Borek (10). They found that,
when a methionine auxotroph
of Escherichia coli with
relaxed control over nucleic acid synthesis (20) was subjected
to a period of methionine starvation, tRNA accumulated in an
immature form, devoid of methylated bases. Subsequent
studies demonstrated that the methylated bases in tRNA arose
via the methylation
by 5-adenosylmethionine
of the
unmethylated
polynucleotide
(6, 7). Preparations
of
"methyl-deficient"
tRNA have been prepared by subjecting
the aforementioned methionine auxotroph to a period of
growth in the presence of methionine, followed by a starvation
period in the absence of methionine. tRNA prepared from
cells treated in this way is deficient in at least 50% of its
complement of methylated bases.
Aminoacylation. We (16) and others (9, 19) have carried
out comparisons of the amino acid acceptor capacity of such
methyl-deficient
tRNA preparations compared to tRNA
extracted
from organisms grown under normal amino
acid-supplemented
conditions. With the exception of one
recent report (18), it has been uniformly found that there is
no significant difference in amino acid acceptor capacity
between normal and methyl-deficient E. coli tRNA when the
source of the aminoacyl-tRNA ligases is also E. coli. On the
basis of these data, the conclusion has been drawn that the
major tRNA modification process, namely methylation, does
not play an important part in the recognition of a tRNA by its
corresponding aminoacyl-tRNA
synthetase. However, this
conclusion had to be tempered by the results of another study
carried out in our laboratory (12). It had been found
previously that aminoacyl-tRNA synthetase preparations from
yeast could attach leucine to E. coli tRNA to about 75% the
level that E. coli enzyme preparations could (1,4, 25). When
we tested the capacity of methyl-deficient E. coli tRNA to be
acylated by this yeast enzyme, we found that an appreciably
smaller fraction (35%) of the leucine tRNA could be acylated
(Chart 1). The obvious interpretation of this result was that
the
heterologous
acylation
reaction
detected
a
modification-dependent
recognition
of tRNA
by the
aminoacyl-tRNA synthetase, while the homologous acylation
reaction could not make the distinction between methylated
and unmethylated tRNA. More recently, other studies have
supported the concept that heterologous acylation reactions
are sensitive indices of otherwise undetectable differences in
the nature of the recognition of tRNA by an aminoacyl-tRNA
synthetase. For example, it has been shown (14) that the
heterologous acylation reactions are more sensitive to salt and
buffer effects than are the homologous reactions. More akin to
our studies with methyl-deficient tRNA, Thiebe and Zachau
(22) have made a similar observation with the minor
nucleoside known as Y. The tRNAphe from yeast which
contains Y right next to the anticodon is aminoacylated by
both enzymes derived from yeast and E. coli. However,
treatment of the tRNA with acid, which leads to excision of
the base Y, results in a tRNA preparation which is now
aminoacylated only by the enzyme from yeast. Thus, it can be
shown that base-modified tRNA is an aminoacylation
substrate for a broader range of enzymes than is the
corresponding unmodified tRNA.
Another approach that has more recently been fruitful in
showing differences in the recognition properties of normal
and methyl-deficient methionyl-tRNA for its aminoacyl-tRNA
synthetase has been a careful study of acylation kinetics (J.
MAY 1971
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675
Alan Peterkofsky,
Marcia Litwack, and Jane Marmor
Marmor and A. Peterkofsky, unpublished results). We took
advantage of our observation that the methyl-deficient species
of methionyl-tRNA
could be resolved from the normal
methionyl-tRNA
by reversed phase chromatography.
A
comparison of the elution pattern of methyl-deficient tRNA
fully acylated with methionine and the same tRNA acylated to
only a small extent showed a significant difference in the
Table 1
Codon recognition pattern of normal and methyl-deficient leucine tRNA
Data are from the paper of Capra and Peterkofsky (3).
Polynucleotide-directed ribosome-binding assays were performed on
normal and methyl-deficient leucyl-1 "C-tRNA.
% binding
UC6.68.06.1PolyUG6.43.9S.IUC/UG1.032.051.07
tRNANormalMethyl-deficientMethyl-deficient(methylated)0Poly
NORMAL S-RNA
3000
2500
D E coli
2000
0 Methyl-deficient
tRNA
was methylated
in vitro with
S-adenosylmethionine and E. coli tRNA methylase, then acylated with
leucine-' 4C, and tested for ribosome binding.
i -i
Yeosi
feos/ emfine
1500
=8=
1000
Yeast em/me
500
Table 2
Initiation factor-dependent binding of
formylmethionine-tRNA
to ribosomes
/
f co/i emyme
0
10
20
30
40
50 60 0 10 20
TIMEIM.nutes)
30
40
50
60
Chart 1. From the paper of Peterkofsky (12). The time course of
leucine acceptance by normal and methyl-deficient E. coli tRNA with
enzymes from £'.coli or yeast. Conditions for acylation of tRNA with
leucine-14C were as described previously (12).
bound0CodonAUGAUGGUGGUGInitiationMMmoles
Normalfactors
tRNA-0.13+
0.99-0.14+
0.87Methyl-deficienttRNA-0.061.08-0.180.86
" Corrected for binding in absence of codon. Ribosome binding was
as previously described (3). The magnesium concentration was 5 mM.
In addition, each 0.05-ml reaction contained 50 m/jmoles of GTP and
0.005 ml of a purified preparation of initiation factors (/, + /, ) and
unfractionated normal or methyl-deficient tRNA containing 6.7
MMmolesof methionine-3 H-tRNA-formylated with unlabeled formyl
groups.
2r
5000 - 0 L
4000
Completely Acylated
3000
patterns (Chart 2). The order of elution of the 3 peaks are: I,
methyl-deficient methionyl-tRNA; II, normal methionyl-tRNA
(formylatable
species); and III, normal methionyl-tRNA
(nonformylatable). This study shows that, when the enzyme is
presented with a mixture of all species of methionine tRNA, it
acylates the normal nonformylatable species 2 times faster
than the normal formylatable species and 3 times faster than
methyl-deficient tRNA. (See plot of the ratios of 14C/3H in
*%x '
Partially
Acylated
2000
1000
100
70
80
90
FRACTION NUMBER
110
120
Chart 2. Reverse phase cochromatographic (Freon) column elution
profiles of methyl-deficient methionine-tRNA. fully rersus partially
acylated. Methyl-deficient tRNA was fully acylated under standard
conditions
with
methionine-'4C.
In a separate
reaction,
methyl-deficient tRNA was acylated with methionine-3 H to only 7% of
the maximum acylation. The 2 samples of methionine tRNA were
mixed and fractionated on the reversed phase column (23). tRNA in
column fractions was precipitated with trichloracetic acid and collected
on Millipore filters. The 3H and 14C counts were detected by
scintillation counting. The upper part of the figure shows the ratio of
counts in the fully acylated versus partially acylated fractions.
676
the column fractions.) Thus while measurements of the
capacity of the aminoacyl-tRNA synthetase to acylate tRNA
may not distinguish between normal and methyl-deficient
tRNA, careful kinetic studies have shown that methyldeficient methionine tRNA is a poorer substrate than is the
normally methylated tRNA.
Codon Recognition. The other major activity of tRNA
related to protein synthesis is the codon recognition function.
Studies in our laboratory
(3) have demonstrated
that
methyl-deficient
leucine tRNA presents a different codon
recognition pattern from normal leucine tRNA (Table 1).
Under the conditions of our experiments, normal leucine
tRNA was bound to ribosomes equally well in response to
either copolymers containing U and C or those containing U
and G. On the other hand, methyl-deficient leucine tRNA
responded much better in this test to the UC copolymer than
CANCER RESEARCH VOL. 31
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Modified Bases and tRNA Function
to the UG copolymer. It was evident that the effect was
specifically due to a lack of methylated bases since in vitro
methylation of the methyl-deficient tRNA restored its codon
recognition pattern to that of the normal tRNA. The studies
of Stern et al. (21) demonstrated
that methyl-deficient
phenylalanine tRNA has an unchanged pattern of codon
recognition but a lower efficiency of codon-induced binding to
ribosomes. In more recent studies of methionine tRNA (J.
Marmor and A. Peterkofsky, unpublished results), it was found
that the methyl-deficient tRNA showed essentially the same
codon-dependent
ribosome-binding
properties as did the
normal tRNA (Table 2). Therefore, in codon recognition tests,
methyl-deficient
tRNA shows a wide range of activities
compared to the normal tRNA; leucine tRNA shows a changed
pattern of codon response, phenylalanine tRNA shows a
changed efficiency of response, and methionine tRNA is
indistinguishable from normal.
Specific Properties of Methionine tRNA
Of the multitude
of amino acid-specific tRNA's in E. coli,
that for methionine
is unique. The special properties of the
Table 3
Test for interaction of methyl-deficient formylmethionine-tRNA
with elongation factor-GTP complex
tRNA added
% of control binding
Methyl-deficient formylmethionine tRNA"
Normal formylmethionine tRNA
Methyl-deficient methionine tRNA
Deacylated tRNA
112
105
45
104
a tRNA was acylated with methionine and transformylated with
leucovorin. The residual unformylated methionine was removed by
enzymatic deacylation. The capacity of normal or methyl-deficient
formylmethionine tRNA to interact with the elongation factor-GTP
complex was measured essentially as described by Weissbach et al. (24).
After formation of the GTP-3H-T factor complex by incubation for 5
min at 37°,individual tRNA preparations were added, and incubation
was continued for an additional 5 min at 0°.The reactions were then
filtered on Millipore filters, and the 3H counts retained on the filters
were determined by scintillation counting; 100% binding corresponds
to 85,000 cpm. The amounts of tRNA added were: methyl-deficient
formylmethionine
tRNA, 98 /jamóles; normal formylmethionine
tRNA, 83 ji/umoles; methyl-deficient methionine tRNA, 100 MMmoles;
deacylated tRNA, 168 MMmolesof methionine acceptor activity.
methionine tRNA are connected with its role as the initiator
tRNA for bacterial protein synthesis. Thus, only methionine
tRNA is a substrate for the transformylating
enzyme
converting methionine to jV-formylmethionine. We were able
to show that a deficiency in methylated bases had no effect on
the kinetics or the extent of the transformylation reaction.
Another property of jV-formylmethionine tRNA is its capacity
specifically to recognize protein synthesis initiation factors but
not elongation factors. The experiment described in Table 2
showed that there was no abnormality in the ability of
methyl-deficient methionine tRNA to interact with initiation
factors. Table 3 shows the results of a test to determine
whether methylation of tRNA is necessary to confer on
methionine tRNA the property of not interacting with
elongation factors. The complex formed by GTP and
elongation factors (Complex I) is bound by Millipore filters.
All tRNA's except formylmethionine
tRNA interact with
Complex I to form a new complex (II) which is not bound to
filters. We asked whether methyl-deficient formylmethionine
tRNA might be recognized by elongation factors in the same
way that the nonformylatable species of methionine tRNA is.
The data indicate that neither normal nor methyl-deficient
formylmethionine tRNA can convert Complex I to Complex
II. Thus, methylation of tRNA is not a prerequisite for the
exclusion of recognition of formylmethionine
tRNA by
elongation factors. A further set of experiments was carried
Table 4
Variability ofiPA content of lactobacillus tRNA
Cultures of L. acidophilus 4963 were grown as previously described
(15), except that the concentration of mevalonic acid in the culture
medium was varied. Cell growth was determined by absorbance at 650
mu in a 1-cm light path. tRNA was prepared as described (15).
4Cconcentration
Mevalonic acid-'
activityof
yield(A650)0.230.340.380.410.440.450.440.45Specific
tRNA(cpm/A260)408459516652885
culture(Min
IO"7)3.96.710.417.740.277.4114.5137Cell
X
Table 5
Message-dependent peptide bond formation by normal and iPA-deficient aminoacyl-tRNA
Normal and iPA-deficient (50% deficient in iPA) Lactobacillus tRNA was acylated with the amino acids in column 1. The acylated tRNA's were
tested for template-dependent peptide bond formation with an E. coli S-30 preparation as described by Nirenberg (11). The hemoglobin message
experiments used rabbit reticulocyte lysate as described by Gonano (8).
% incorporation
poly UG
poly UA
poly UC
Hb Message
AminoacidLeucineSerineTryptophanTyrosineNormal555733iPA-deficient553704Normal332043iPA-deficient312040Normal354101iPA-deficient344501Normal7161717iP
MAY
1971
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677
Alan Peterkofsky,
Marcia Litwack, and Jane Marmor
out with the assistance of Dr. Philip Leder to test the function
of methyl-deficient formylmethionine tRNA as a substrate for
the ribosomal-bound peptide synthetase. Bacterial ribosomes
programmed with RNA phage message will translate only the
initial dipeptide sequence of the coat protein in the presence
of fusidic acid (17). We found that normal and
methyl-deficient
formylmethionine
tRNA were indistin
guishable in this test system. These studies showed that while
tRNA methylation affects some properties of various tRNA
species,
the special initiation
functions
of formyl
methionine tRNA are unchanged by a lack of methylated
bases.
¡PA1
In contrast
to methylated
bases, which are found
distributed throughout the tRNA chain, iPA occurs uniquely
adjacent to the 3' end of the anticodon of only those tRNA's
for which the codons begin with U (15). Because of this
specificity of location, we thought that it would be fruitful to
investigate the biological properties of tRNA with and without
this base. Following up the observation that mevalonic acid
was a precursor of iPA in Lactobacillus acidophilus (13), we
studied the nutritional aspects of mevalonic acid utilization in
this organism. Surprisingly, we found (M. Litwack and A.
Peterkofsky, unpublished experiments) that the amount of
iPA in tRNA was dependent on the level of mevalonic acid in
the culture medium (Table 4). Concentrations of mevalonic
acid that led to low iPA levels in tRNA did not, however, limit
growth. In this way, we were able to isolate tRNA
preparations from L. acidophilus that differed in iPA content
by at least a factor of 2, a situation comparable to the
formation of methyl-deficient
tRNA by relaxed control
organisms. Considering the unique localization of iPA in
tRNA, we were not surprised to find that iPA-deficient tRNA
was equally as active as normal tRNA for the aminoacylation
reaction. We then compared normal and iPA-deficient
aminoacyl-tRNA for peptide bond-forming activity in several
systems (Table 5). E. coli S-30's programmed with poly UG,
UA.and UC were tested with leucine, serine, tryptophan, and
tyrosine tRNA's. The normal and iPA-deficient species are
indistinguishable.
Similar
results
were
obtained
1The abbreviation used is: iPA, isopentenyladenosine.
678
when
hemoglobin synthesis was measured in reticulocyte lysates.
Thus iPA deficiency in Lactobacillus tRNA appears not to
affect either aminoacylation or codon recognition functions.
REFERENCES
1. Bennett, T. P., Goldstein, J., and Lipmann, F. Proc. Nati. Acad.
Sci. U. S.,49: 850, 1963.
2. Bjork, G., and Isaksson, L. J. Mol. Biol., 51: 83, 1970.
3. Capra, J. D., and Peterkofsky, A. J. Mol. Biol., 33: 591, 1968.
4. Doctor, B. P., and Mudd, J. A. J. Biol. Chem., 238: 3677, 1963.
5. Eidlic, L., and Neidhardt, F. C. Proc. Nati. Acad. Sei. U. S., 53:
539, 1965.
6. Fleissner, E., and Borek, E. Proc. Nati. Acad. Sci. U. S., 48: 1199,
1962.
7. Gold, M., and Hurwitz, J. Federation Proc., 22: 230, 1963.
8. Gonano, F. Biochemistry, 6: 977, 1967.
9. Littauer, U. Z., Muench, K., Berg, P., Gilbert, W., and Spahr, P. F.
Cold Spring Harbor Symp. Quant. Biol., 28: 157, 1963.
10. Mandel, L. R., and Borek, E. Biochem. Biophys. Res. Commun., 4:
14, 1961.
11. Nirenberg, M. In: S. P. Colowick and N. O. Kaplan (eds.), Methods
in Enzymology, Vol. 6, p. 17. New York: Academic Press, Inc.,
1963.
12. Peterkofsky, A. Proc. Nati. Acad. Sci. U.S., 52: 1233, 1964.
13. Peterkofsky, A. Biochemistry, 7: 472, 1968.
14. Peterkofsky, A., Gee, S. J., and Jesensky, C. Biochemistry, 5:
2789, 1966.
15. Peterkofsky, A., and Jesensky, C. Biochemistry, 8: 3798, 1969.
16. Peterkofsky, A., Jesensky, C., Bank, A., and Mehler, A. H. J. Biol.
Chem., 239: 2918, 1964.
17. Roufa, D. J., Doctor, B. P., and Leder, P. Biochem. Biophys. Res.
Commun.,39: 231,1970.
18. Shugart, L., Chastain, B. H., Novelli, G. D., and Stulberg, M. P.
Biochem. Biophys. Res. Commun., 31: 404, 1968.
19. Starr, J. L. Biochem. Biophys. Res. Commun., 10: 181 (1963).
20. Stent, G. S., and Brenner, S. Proc. Nati. Acad. Sei. U. S., 47: 2005,
1961.
21. Stern, R., Gonano, F., Fleissner, E., and Littauer, U. Z.
Biochemistry, 9: 10, 1969.
22. Thiebe, R., and Zachau, H. G. Biochem. Biophys, Res. Commun.,
33: 260, 1968.
23. Weiss, J. F., and Keimers, A. D. Biochemistry, 6: 2507, 1967.
24. Weissbach, H., Miller, D. L., and Hackmann, J. Arch. Biochem.
Biophys., 137: 262, 1970.
25. Yamane, T., and Sueoka, N. Proc. Nati. Acad. Sei. U. S., 50: 1093,
1963.
CANCER
RESEARCH
VOL.
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31
Modified Bases and Transfer RNA Function
Alan Peterkofsky, Marcia Litwack and Jane Marmor
Cancer Res 1971;31:675-678.
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