volume 3 no.5 May 1976
Nucleic Acids Research
Changes in transfer ribonucleic acids of Bacillus subtilis during different growth
phases.
Ram P. Singhal* and Barbara Void
•Department of Chemistry, Wichita State University, Wichita, KS 67208, USA.
Received 24 February 1976
ABSTRACT
The transfer ribonucleic acids (tRNAs) of B. subtilis at different
growth phases are examined for changes in the composition and the methylation of minor constituents. The composition of the tRNAs indicates about
equal amounts of adenosine and uridine, and of guanosine and cytidine.
About 3-4 residues are present as modified bases in the average tRNA molecule. The net composition of tRNAs appears to remain unaltered during different growth phases. In vitro methylation of tRNAs indicates lack of
methyl groups in both exponentially growing cells and spores. In vivo
methylation studies show tRNA methylation occurs during the stationary
phase in the absence of net tRNA synthesis. Thus, both in vitro and in
vivo methylation indicate that the tRNAs in exponentially growing cells
do not contain their full complement of modified bases. More complete
modification is noted in tRNAs from stationary cells or spores. Henc.e,
tRNA modifications in general are preserved with fidelity even in the dormant spore but the possibility is left open that specific modifications of
selected isoacceptors of tRNAs may occur.
INTRODUCTION
The developmental cycle of 3. subtilis offers the extremes of growth
phases from actively dividing cells to dormant spores. Spore tRNAs may
represent a unique situation with respect to tRNA modification. The quantities of certain isoacceptor tRNAs have been reported to vary between the
exponential growth phase and the spore phase
. Also, 30 per cent of
spore tRNAs, compared to 2 per cent of exponential tRNAs, have been shown
Q Q
to lack adenosine at their 3' ends ' . Although a partial or total cessation of net tRNA synthesis occurs at the end of the exponential growth
phase, turnover and varied transcriptions during the stationary phase
have been reported. A DNA methylase occurs principally in the early exponential phase, but declines rapidly thereafter 12 . Like DNA methylases,
RNA methylases can be expected to differ with changes in the developmental
cycle. However, no tRNA-modifying enzymes have been studied as a function
of cell development.
© Information Retrieval Limited 1 Falconberg Court London W1V5FG England
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Transfer RNAs from B. subtilis are examined in this report. Since
very l i t t l e i s known about the chemistry of tRNAs from this spore-forming
organism, chemical composition, nature of minor, modified constituents and
in vivo methylation at different developmental stages are studied.
METHODS
Growth of cells.
Cells were grown in either a fully-supplemented normal medium, called Tryptone-yeast extract medium or in the minimal salts
medium . Cultures were started at an absorbance of 0.1 optical density
unit measured at 660 nm and were grown with shaking at 37°.
Isolation of tRNAs. Cells were harvested by centrifugation; washed
twice with 10 mM Tris pH 8.0, 0.15 M NaCl, 1 mM EDTA, 20% sucrose, and suspended in 50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM EDTA, 20 mM 2-mercaptoethanol. Cultures not having a radioactive label were broken by sonication.
After sonication, electrophoretically purified deoxyribonuclease was added
(10 pg per ml) and the lysate centrifuged. The supernatant fluid was removed and made 0.2% with respect to sodium dodecyl sulfate and 0.5 M to
NaCl. An equal volume of water saturated phenol was added and the phases
were mixed and separated by centrifugation. The aqueous phase was collected. The phenol phase was washed once with a small volume of buffer. The
aqueous phases were combined and extracted once with chloroform/isoamyl
alcohol (24:1 v / v ) . After ethanol precipitation, the RNA was fractionated
on a Sephadex G-100 column . The tRNA peak was pooled and precipitated
with ethanol. The tRNA was freed of any contaminant DNA by treating once
again with pure deoxyribonuclease, followed either by chromatography on a
small DEAE-cellulose column or by dialysis and precipitation with ethanol.
Isotopically-labeled tRNAs were prepared in the same manner except
that the c e l l s were broken by lysis with lysozyme (1 mg per ml) in the
presence of deoxyribonuclease (10 ug per ml). The final treatment with deoxyribonuclease and dialysis were omitted in this case.
Methylation of tRNAs in vivo. Transfer RNAs were methylated in vivo
by growing c e l l s in the minimal s a l t s medium in the presence of L[metfe/l-^Cjmethionine. Precultures were grown in Spizizen's minimal s a l t s ,
0.5% glucose, 0.1% casein hydrolysate, and 40 ug L-tryptophan per ml. Cells
from the preculture were washed twice with sterile minimal medium and inoculated into a final culture containing Spizizen's minimal s a l t s , 0.5% glucose, 1 mM adenosine, 1 mM guanosine, 1 mM thymidine, 1 mM cysteine, 40 ug
L-tryptophan per ml, 2 pg L-tyrosine per ml, and 15 other L-amino acids at
a concentration of 20 ug per ml. The amount of nonradioactive L-methionine
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Nucleic Acids Research
or L-[methyl-11* C]methionine and the period of labeling are given in the
Tables.
Methylation of tBNAs in vitro.
The methyl donor for in vitro studies
1
was S-adenosyl-L-[metfa/Z- '*C]methionine. A typical reaction mixture of 200
ul contained 10% glycerol, 40 mM Tris pH 8.9, 8 mM 2-mercaptoethanol, 4 mM
MgCl2, 6 umol L-methionine, 1.4 vg (0.2 uCi) S-adenosyl-L-[met%Z-ll(C]methionine, various concentrations of tRNAs (see Table I I I ) , and either 2
mg protein from a partially purified tRNA methylase preparation or 2.9 mg
protein from a crude extract (see below). Reaction mixtures were incubated
at 30° for 1 h. Samples of 40 ul were withdrawn in duplicate and spotted
on paper discs, which were washed twice with cold 5% trichloroacetic acid
and once with methanol-ether mixture (1:1 v/v) , put into scintillation
fluid, and quantitated for radioactivity in a scintillation spectrometer.
The radioactivity thus assayed represents net incorporation of the radiocarbon in tRNAs. After removal of the first samples, 10 yg pancreatic ribonuclease was added to the reactions and the incubation continued for another 30 min at 37°. A second set of samples was withdrawn and treated
as described. The radioactivity in these samples represents the activity
of the nucleic acid preparation that can not be removed by the ribonuclease. Hence, the radioactivity removed by the ribonuclease action is determined from the difference between the first and second set of samples.
Results are expressed as radioactivity in methyl groups of tRNAs not removed by pancreatic ribonuclease action (see Table I I I ) .
Methylase Preparations. The partially purified methylase source was
treated with streptomycin sulfate, precipitated with ammonium sulfate and
chromatographed on Sephadex G-100 and DEAE-cellulose columns. The crude
extract was prepared by breaking exponentially growing cells by sonication
in 50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM EDTA, 20 mM 2-mercaptoethanol.
After centrifugation, the supernatant fluid was dialyzed overnight in the
cold against the same solution, which also contained ten per cent glycerol.
Hydrolysis of tRNAs. Transfer RNAs were hydrolyzed to the nucleoside
level by two different methods. (1) A mixture of pancreatic ribonuclease,
bacterial alkaline phosphatase (both from Worthington), and phosphodiesterase (Russel's viper's venom, from CalBiochem) was prepared and tRNAs hydrolyzed as described elsewhere . Incubation was carried out in 0.2 M
(MM2CO3 containing 1 mM Mg(OAc)2, pH 8.7 at 37° for 15 h. (2) Transfer
RNAs were f i r s t hydrolyzed with ribonuclease-T2 to nucleotides, and then
dephosphorylated to nucleosides by treatment with alkaline phosphatase .
Separation of Nucleosides.
Nucleosides were separated by anion-ex1251
Nucleic Acids Research
elusion chromatography on a cation-exchange column
.
Results were con-
firmed by cation-exclusion chromatography on an anion-exchange column
.
For the anion-exclusion chromatography, a column of Andnex A-6 (BioRad) (6.35 mm x 40 cm) (bed volume:
12.66 ml; void volume:
3.9 ml) was
eluted with 20 mM (NH1O2CO3 brought t o pH 9.8 by adding concentrated NHi»OH.
The column was maintained at SO". The effluent was monitored at 254 nm
and 280 nm simultaneously with a dual wavelength and dual pen detector,
using a 20 u l , 10 mm l i g h t path flow c e l l (Model 152, Altex S c i e n t i f i c
I n c . , Berkeley, C a l i f o r n i a ) .
Both major and minor nucleosides were quan-
t i t a t e d in the same chromatography by s e t t i n g the two channels at one wavelength (254 nm) but at two different s e n s i t i v i t y ranges.
Peak areas were
calculated from the peak height, H and the peak width at 50% o f the maximum height, W.
(Peak area = H x W x 1.06, where 1.06 i s the correction
factor; see reference 19).
The peak width was expressed in volume u n i t s ,
obtained from the known chart speed and the flow rate.
Each peak area,
corresponding t o the t o t a l absorbance at 254 nm and pH 9 . 8 , was converted
to nanomoles by dividing i t by the appropriate extinction
determined under i d e n t i c a l conditions.
coefficient
Millimolar extinction
coefficients
(at 254 nm) for d i f f e r e n t compounds used in these experiments at pH 9.8
were as follows:
adenosine, 1 3 . 9 ; 6-methyladenosine, 12.5; guanosine,
11.43; 1-methylguanosine, 10.5; c y t i d i n e , 6.88; uridine, 7.36; pseudouridine, 3.05; 5-methyluridine, 5.98.
Radioactivity in the e f f l u e n t was assayed by collecting 0.3 ml fract i o n s in s c i n t i l l a t i o n v i a l s and counting them in a s c i n t i l l a t i o n spectrometer a f t e r the addition of 2 ml s c i n t i l l a t i o n f l u i d (Aquasol, Mew England
Nuclear).
Peaks were characterized by t h e i r elution positions in the anion-exclusion chromatography ( r e f e r t o figure 6 in reference 17).
Their i d e n t i t y
was further confirmed by rechromatography in the cation-exclusion system
a f t e r freeze drying, and by comparison of elution positions with those of
known n u c l e o s i d e s .
RESULTS
Constituents
of B. subtilia
sides from B. stibtilie
in figure l ( a ) .
tRNAs. A typical separation of nucleo-
tRNAs by anion-exclusion chromatography i s shown
Besides the major nucleosides, commonly occuring minor
nucleosides pseudouridine (ijrd) and ribothymidine (SMeUrd) were present.
Other modified constituents included 5-hydroxyuridine (SHOUrd), 4-thiouridine (4Srd), 5-methylaminomethyl-2-thiouridine (5MeNHMe2Srd), an uncharac-
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ELUTION VOLUME.ml
(a)
(b)
5
en
o
I
<D
I
ID
-ft—-
Ml
M2
M3
120
ELUTION TIME, mln
Figure 1:
(a) Separation of tRNAs hydrolysate of B. subtilis by anionexclusion chromatography1 . Aminex A-6 column was eluted with
20 mM (NHO2CO3, pH 9.80; 0.20 ml per minute, and 50°.
(b) Elution positions of constituents containing f1 "*C]methyl
groups of tRNAs (S. subtilis) in anion-exclusion chromatography.
Incorporation of label into deoxyribonucleosides and the major
ribonucleosides was suppressed by adding adenosine, guanosine,
and thymidine to the medium.
terized pyrimidine nucleoside (MePyd) , 1-methyl guano sine (lMeGuo) , 2-methylguanosine (2MeGuo), 7-methylguanosine (7MeGuo), and 6-methyladenosine
(6MeAdo). The uridine peak also included the isocytosine derivative derived from the partial degradation of 7-methylguanosine during alkaline and
temperature conditions of the enzymatic digestion . The value of 6-methyladenosine includes 1-methyladenosine, which rearranges into 7-methyladenosine under alkaline conditions . Strongly adsorbed 2-methylthio-6(2-isopentenyl)adenosine was also present in these tRNAs. This constituent was
desorbed from the anion-exclusion column by the normal eluant to which an
equal volume of ethanol was added. It was not quantitated in the present
studies. Though tRNA preparations were treated with deoxyribonuclease,
some samples s t i l l indicated DNA contamination. Apparently the deoxynucleoside peaks in figure 1 (a) were derived from the contaminant DNA
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hydrolyzed with tRNAs.
As a further aid in identification, tRNAs were labeled in vivo
using L- [methy I-1 ^Qmethionine as the methyl donor. The elution position
of these methylated species in anion-exclusion chromatography i s shown in
figure 100 (peak positions shown as horizontal bars). Though a peak
corresponding to 5-methyluridine was characterized in figure l ( a ) , no
radioactivity was associated with this component in figure l(b) (see
Discussion).
Change8 in tRNAs as a Function of Growth Medium. Table I shows the
composition of B. subtilis
tRNAs grown in two different media, rich tryptone-yeast medium and minimal-salt medium supplemented with glucose,
casein hydrolysate and tryptophan (see Methods). Further, the composition
of tRNAs in each medium at three different growth stages is shown. I t is
important to note that after 24 hours, cells in the rich tryptone-yeast
medium were present as spores (about 90%), but cells in the minimal salt
medium were s t i l l in the vegetative stage.
TABLE I
Changes in the composition?
of tRNAs {Bacillus subtilis
168trpC2) , harvesompi
ted at three growth
wth stages ana grown in two different culture media
Culture medium
Cell growth s t a g e s :
Nucleosides
Ado
Exponen-
15.6
6MeAdo?
0.13
13.1
Urd
Stationary*!
15.3
0.11
13.9
Spore/
15.1
0.15
13.8
Minimal Culture Medium"
Exponen- Station- 24h/
tial d
ary*
15.3
0.17
13.7
15.1
0.14
14.1
14.5
0.15
13.2
tired
1.67
1.84
1.08
1.S7
1.76
1.52
SMeUrd
0.92
0.9.1
l.OS
1.06
1.04
0.96
26.2
Guo
lMeGuo
Cyd
6
Normal Culture Medium
0.09
22.3
25.6
0.09
22.3
27.0
0.09
21.7
25.2
0.11
22.9
25.0
0.08
22.8
27.9
0.10
21.7
^Expressed as moles per 80 nucleoside residues.
Tryptone-yeast extract medium .
"spizizen's Minimal Medium , sporulation efficiency in this medium after
72h i s about 0.21%.
'exponential growth phase.
Stationary growth phase, lOh of growth, just before the appearance of heat
resistant spores.
^Spore growth phase, 24h of growth (the stage used to purify spores from
the parental strain grown on sporulation medium).
^6-Methyladenosine i s mainly derived from 1-methyladenosine under the
alkaline conditions of hydrolysis and chromatography.
Note:
1254
The results indicated above were derived from at least triplicate
analyses. (For typical deviations in ion-exclusion chronatography,
see Tables I to VI in reference 23.)
Nucleic Acids Research
The results indicate that about the same modified nucleosides are
present in both tRNAs whether isolated from rich culture medium or from
the minimal culture medium. The data in Table I also indicate that the
chemical composition of the tRNAs remains unchanged in exponential and
stationary phases. Hence, limitations in cell nutrients have no effect in
the "maturation" of tRNAs.
Changes in tRNAs as a Function of Cell Development.
The nucleoside
composition of tRNAs from cells harvested at three developmental stages
are shown in Table I .
Transfer RNAs appear to be modified as much in the
early developmental stage (exponentially growing cells) as in the stationary growth phase.
The chemical nature of tRNAs appears to remain unaltered
in general, even in spore tRNAs.
However, the loss in pseudouridine and
the gain in guanosine of tRNAs from spores exceed simple experimental variations.
A similar increase in guanosine, but no change in pseudouridine of
tRNAs from the minimal s a l t medium after 24 hours of growth, i s noticeable.
Changes in 4-thiouridine.
fied tRNAs from Escherichia
The work of Lipsett and Doctor
aoli,
, for puri-
shows that an absorbance at 335 nm equal
to 4.03 per cent of the t o t a l absorbance at 260 nm indicates two 4-thiouridines for each molecule of tRNA.
From this relationship, the tRNAs from
exponentially growing cells and tRNAs from spores were found to contain
0.12 and 0.11 residues per tRNA, respectively.
ninth tRNA contains one 4-thiouridine.
Thus, every eighth or
The small difference between the
two values suggest that the degree of thiolation in tRNAs do not vary
substantially when cells transform from vegetative to spore phase.
Methylation of Preformed tRNAs in vitro.
To determine i f the tRNA
i s undeT-methylated (lacking the full complement of methylated constituents
) , methylation of tRNAs in vitro was attempted with the methyl donor,
S-adenosyl-L-[met/ij/Z-1'*C]methionine and a homologous methylase.
As a con-
t r o l for homologous methyl acceptance, tRNA was prepared from a relaxed,
methionine requiring s t r a i n , B. subtilis
reference 24).
168 trpC2 metBS rel- (#27) (see
The tRNA thus prepared was 57-67% less methylated than
that from the parent strain of B. subtilis.
A p a r t i a l l y purified methylase
was used for experiments described in Table I I .
Transfer of the methyl
group into tRNAs was confirmed by the ribonuclease sensitivity of the product (see Methods).
To establish the absence of any methyl-transfer inhi-
bitor in tRNA preparations, mixing experiments were performed.
Transfer
RNAs from exponentially growing cells or from spores were combined with
tRNAs from the mutant requiring methionine (strain #27).
This showed no
loss in the methyl acceptance by tRNAs from the mutant s t r a i n .
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TABLE II
Methylation of transfer RNAs in
Origin of tRNA
Concentration
of tRNA
(A-,, units/reaction)
vitro1
Incorporation
of
[1!*C] methyl group
(cpm/A260 unit)
Methylation
capacity*
91
109
(a) Mutant
s t r a i n #27 e
3.0
2.0
1,880
2,260
(b) Exponential d
growth stage
7.0
54
23
2.6
1.1
(c) Spores^
1.4
7.0
24
12
1.2
0.6
using S-adenosyl-L- [met% J- 1 uC]methionine as methyl donor.
unit of tRNA i s taken
The incorporation of 2070 cpm of methyl-^^C per A,,
z b n0
as the full methylation (100%).
Undermethylated tRNA obtained from a relaxed, methionine-requiring mutant:
Bacillus 8ubtilis 168trpC2metBSr*el- (#27).
Transfer RNAs from Bacillus svbtilis
168irpC2; tRNAs were isolated from
exponentially growing cells and from the purified spores.
The results (Table II) indicate some lack of methylation in tRNAs both
from exponentially growing cells (up to 2.6%) and from spores (up to 1.2%).
Since only about 50% of tRNAs from the mutant strain weTe under-methylated,
lack of the methyl group in tRNAs was in fact twice as much.
Methylation of tRNAa in vivo. To study the modification of tRNAs at
different growth stages, three cultures were grown and labeled for short
A growth curve showing the labeling
periods with L- [methyl-11*C]methionine.
periods i s shown in figure 2. About 0.5 mg corresponding to 50 uCi of L[metfe/Z-ll4C]methionine was added to each culture at the beginning of the
labeling period and harvested at the end of the labeling period. Cultures
of 200 ml were grown and tRNAs were extracted as described in Methods. The
tRNA from each growth stage i s shown in Table I I I . The cessation of net
tRNA synthesis occurs during or immediately after the exponential stage.
This i s shown by the constant recovery of tRNAs in growth phases 2 and 3.
However, the methyl group from L- [metfe/Z-^Clmethionine continues to be
incorporated into tRNAs although at a reduced rate (see Discussion).
The lower portion of Table III shows the separation of methylated cons t i t u e n t s of tRNAs, methylated in vivo and isolated from three different
development phases (see figure 2). There i s a constant turnover of methyl nu-
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•
i
CELL GROWTH (houra)
Growth curve for B. subtilis
168irpC2 in minimal s a l t medium 13
showing the length of time in which different cultures were
labeled with L-[met?ij/l-1£fC]methionine (see Table I I I ) .
Figure 2:
cleosides as a r e s u l t of tRNA turnover (7-methylguanosine and 5-methylaminomethyl-2-thiouridine) in both actively-dividing cells and stationary c e l l s .
However, as the c e l l passes into the resting stage, the methyl group in.corporation increases in some constituents (guanosine methylated at 1 or
2 p o s i t i o n ) , and decreases in others (uncharacterized methylated pyrimidine). Differences in the 6-methyladenosine value are probably due to
differences in tnethylation of adenosine at the 1 or 6 position.
DISCUSSION
Constituents
of B. subtilis
tRNAs.
from t h i s spore forming bacterium.
composition of Bacillus
exists '
'
'
subtilis
Very l i t t l e i s known about tRNAs
Although some data on the nucleoside
and related Bacillus
species
, only one report has been concerned with differences of
major nucleosides between vegetative c e l l s and spores .
In that r e p o r t ,
the four major nucleosides were compared and the authors concluded that
there were no differences in the major nucleosides of tRNAs from the two
phases .
The composition of the tRNAs (Table I) from B. subtilis
indicates
about equal amounts of adenosine and uridine, and of guanosine and cytidine.
About 3-4 residues are present as modified bases in the average tRNA
molecule.
Modified adenosine i s present as the 1-methyl- or N6-methyl
adenosine and as methylthio(isopentenyl)adenosine.
We were unable t o de-
t e c t 2-methyladenosine, which i s commonly found in E. aoli tRNAs
.
Among
the modified guanosines, 7-methyl derivative i s present in twice the
amount of the 1-methyl derivative.
A very small quantity of 2-methyl-
guanosine (about 2% of a l l methylated species) i s present.
No modified
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cytidines were detected. A modified pyrimidine containing a methyl group
and an anionic charge at alkaline pH i s yet to be characterized. Contrary
to the case in E. coli, we find that the methyl group of 5-methyluridine
tRNAs i s not derived from methionine. Other workers report
in B. subtilis
that i t i s derived from a tetrahydrofolate derivative . The ratio of
pseudouridine to 5-methyluridine in B. subtilis is similar to that found
in E. ooli tRNAs . Among sulfur-containing uridines (see figure 1),
we find 5-methylaminomethyl-2-thiouridine and a very small quantity of
4-thiouridine in B. subtilis
tRNAs, the l a t t e r in about 15% of the amount
28
reported for E. ooli tRNAs . Thus, there appear to be basic differences
subtilis.
between the tRNAs from E. ooli and from B.
Influence
of nutrients
on tRNA Population.
t i o n of tRNAs remains unchanged when B. subtilis
n u t r i t i o n a l l y rich medium or in the poor medium.
The net chemical composii s grown e i t h e r in the
Minor bases appear to
be equally modified in the two different media, and limitations in the
c e l l n u t r i e n t s do not appear t o cause any adverse effect in the "maturat i o n " of tRNAs.
However, differences in the number of serine isoacceptor
tRNAs occur when t h i s bacterium i s grown in two different media 29
Changes in tRNAs during Development of b. subtilis.
The analysis
of the tRNA patterns of sporulating B. subtilis shows that during the
process of morphogenesis a shift occurs in the ratios of two isoacceptor
species of valine tRNA ' . Our results indicate minor differences in
the net composition of tRNAs among the three different phases (Table I ) .
Studies on the methylation of tRNAs in vitro indicate l i t t l e under-methylation of tRNAs (1.3% in exponentially growing cells and 0.6% in spores,
see Table I I ) . (In Table I I , an incorporation of 2070 cpm per A^.. unit of
tRNA i s taken as full methylation. Since the substrate i s already about
50% methylated, a t o t a l l y unmethylated substrate would be expected to accept twice as many methyl groups. On this basis, the methylation capacities
l i s t e d in Table II are divided by two.) The results indicate an uptake of
roughly less than 3 mmoles of [' ^C\methyl groups per mole of tRNA in the
in vitro studies. Though tRNAs from different growth phases (figure 2)
appear to be methylated {in vivo') to different degrees (64% from exponent i a l l y growing c e l l s , 24% from stationary cells and 12% from spores, see
Table I I I ) , they are synthesized at different rates in different growth
phases.
If new isoacceptor species are required for the synthesis of new proteins during cell differentiation , our results indicate that they are
perhaps derived from subtle modifications of certain residues in the
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TABLE III
Methylatlon of B. subtitle
Growth stage, when
cells harvested"
Time of incorporation
of methyl-^C donor
tRNAs in vivo at three growth stages"
(1)
Early exponential
0.5 h
(2)
Late exponentialearly stationary
1.5 h
(3)
Late stationary
2 h
tRNA y i e l d
260
unlts
)
Incorporation o f
['••CJmethyl per 1
A
unit o f tRNA
5.8
26.7
25.1
48,340
18,360
9,330
(cpm)
Relative Proportions o f Methylated Nucleosides
[ ll *C]methyl incorporated to tRNAs
nucleosides"
7MeGuo (M2)
31
30
29
6MeAdo (M6)
28
20
28
MePyd (Ml)
22
25
IS
lMeCuo (M5)
9
13
14
SMeNHMe2Srd (M3)
8
10
8
2MeGuo (M4)
2
2
6
a
Cells were grown in the Minimal Medium following labelling procedure
as described in the Methods section. A growth curve with labelling times
from (1) to (3) is shown in figure 2.
Methylated nucleosides correspond to their elution positions on the anionexclusion column, shown as bars in figure 1 (b), and numbered from Ml to
M6 as eluted. The labelled material 2-methylthic-6-(2-isopentenyl]adenosine which was strongly adsorbed to the column and was eluted with 10%
ethanol was not considered in these calculations. The 6-methyladenosine
peak also contains this compound derived froa 1-methyladenosine during
alkali conditions. The first peak, Ml appears to be a methylated pyrimidine (MePyd).
existing tRNA molecule.
In conclusion, tRNAs of B. subtilis differ from tRNAs of E. coli in
their overall composition and in their modified constituents. Changes in
the nutrients of the growth medium does not appear to cause compositional
changes in the tRNAs. In different growth stages, minor compositional differences in tRNAs exist. However, both in vivo and in vitro methylation
studies indicate that the tRNAs in exponentially growing cells do not contain their full complement of modified bases. More complete modification
is noted in tRNAs from stationary cells or spores. The results also indicate methylation of tRNAs in the stationary growth phase after the
cessation of net tRNA synthesis.
ACKNOWLEDGEMENTS
We thank Arlene Carbone and Nicole Keisel for their excellent technical
help and Dr. Waldo E. Cohn for reviewing the manuscript. The work was
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Nucleic Acids Research
supported by PHS Research Grant (GM 17421) and Research Career Development
Award (GM 23726) from the NIGMS and by the Wichita State University.
Department of Microbiology, Scripps Clinic and Research Foundation, La Jolla,
CA 92037, USA.
Note added in the revision.
Keith et al. report tRNAT^1" and tRNAp^ from
B. subtilis
differ due t o post-transcriptional modification (FEBS Letters,
61, 1976, p. 120). They find tRNA]^1", typical of the exponentially growing
cells contains only isopentenyladenosine, but tRNA^V1", typical of the stationary c e l l s contains i t s full modification, 2-methylthioisopentenyladenosine. The r e s u l t s confirm our findings that after cessation of tRNA trans c r i p t i o n , tRNA structure changes by minor modifications and not be overall
composition.
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