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Modification of leucyl-sRNA after bacteriophage infection
J. MOE. Biol. (1966) 20, 183-209
Modifkation
of Leucyl-sFkNA after Bacteriophage Infection T.
&LNO-SUEOKA
AND
N.
&JEOKA
Department of Biology Princeton University Princeton, New Jersey, U.S.A. (Received 15 February 1966, and in revised form 9 May 1966) Aminoaoyl-sRNA’s of Escherichia coli B with and without bacteriophage T2 infection have been examined using methylated albumin kieselguhr column chromatography. Out of 17 aminoacyl-sRNA’s examined, only one, leucyl-sRNA, was found to be clearly different before and after the infection. This alteration lies in sRNA and not in leucyl-sRNA synthetase, and is one of the earliest events after the infection. Phage T4 and T6 infections show the same modification of leucyl-sRNA, whereas infection with Tl, T3, T6 and Ti’ and A induction do not induce the modification. Leucyl-sRNA was examined in Shigelkz dysenteriue 60 and in various strains of E. coli after T2 infection, and in permissive and nonpermissive hosts after infection with various early amber mutants of T4. In all cases,the same modification of the leucyl-sRNA was observed. It is not excluded that the leucyl-sRNA change may take some role in the arrest of host-protein synthesis.
1. Introduction The role of sRNA as the adaptor between the template and the polypeptide suggests the possibility that structural modification of sRNA may be involved as one of the key steps in the regulation of cell metabolism and cell differentiation, since the translation of messenger RNA should be affected if a motication of sRNA leads to a change of its codon recognition or its amino acid acceptor activity (adaptor modi&&tion hypothesis, Sueoka & Kano-Sueoka, 1964). A test of such a hypothesis was initiated by examining aminoacyl-sRNA from T2-infected Escherichia wli as a model system (Sueoka & Kano-Sueoka, 1964, 1965). Two major metabolic transitions exist in the E. wlGT2 phage system: (1) cessation of the synthesis of host DNA, RNA and protein upon phage infection (Cohen, 1949); (2) transition from the early to the late phase of phage protein synthesis, the mechanisms of which are unknown (see review articles by Luria, 1962; Champe, 1963; Cohen, 1963). The main technique used in this study is the methylated albumin column fractionation of aminoacyl-sRNA (Sueoka & Yamane, 1962). Leucyl-sRNA shows an appreciable difference after T2 infection. Moreover, injection of phage DNA and protein synthesis after infection were found to be necessary for this sRNA alteration (Sueoka & Kano-Sueoka, 1964). The present paper describes the further development of this work. Specificity of the involvement of phage has been firmly established. The time course of the alteration of leucyl-sRNA revealed that this phenomenon is one of the earliest events taking place after phage infection. This and other evidence reported here have eliminated the possibility that the leucyl-sRNA modification is related to transition from the early to the late phase of phage protein synthesis. Also, the alteration of leucyl-sRNA is shown to be speoilic only to the T-even phages and not dependent on the hosts used for the infection. 183
184
T. KANO-SUEOKA
AND
N. SUEOKA
2. Materials and Methods Strains of bacteria and phages used in the present study are as follows: E. coli B (laboratory stock); E. COG C, E. coli K12(X) and E. coli K12X sensitive (from A. B. Pardee); E. co&i CR63 (from R. S. Edgar); E. coli F (from Y. & F. La&); an RNase-less mutant (A19) and RNaee-less and polynucleotidephosphorylase-less double mutant (Q13) of E. coli Hfr strain AB301 (Gesteland, 1965, from J. D. Watson); ShigeZZa dyeenteriue 60 (from S. E. Luria); phages T2 and T4 (from laboratory stock); T6 (from R. S. Edgar); Tl and T3 (from J. Hurwitz); T5 st (from Y. & F. Lanni); T7 (from J. E. Smith); T4 amber mutants and their hosts (from R. S. Edgar). Yeast was baker’s yeast. Radioactive ammo acids were obtained from the New England Nuclear Corp., Volk Radiochemioal Company, and Nuclear Chicago Corp. Purified colicin E2, mitomycin C and anti-T3 serum were gifts from D. Helinski, Y. Takagi and A. H. Doermann, respectively. Kieselgubr was purchased from E. H. Sargent & Company and bovine albumin powder (fraction V from bovine plasma) from the Armour Pharmaceutical Company. (a) Preparation
of p?uzges
T-even phages: E. coli cells were grown in tris C medium, a modification of C medium by Roberts, Abelson, Cowie, Bolton & Britten (1957), at 37°C (for T4 infection 100 pg/ml. tryptophan was added to the medium), infected with T2, T4 or T6 at the multiplicity of 0.6 to 1, and the culture shaken until lysis. After the cell debris was spun down at 6000 rev./min for 15 min, the supernatant fraction was passed through kieselguhr (40 g/l. of the supernatant) and then through a Millipore filter (pore size 0.45 p). Phage was collected by centrifugation at 22,000 g for 1 hr and suspended in a buffered salt solution (2 g NH&I, 5 g NaCl, 0.37 g KCl, 0.01 g MgClz.GHzO, 0.026 g Na,SOI, 0.09 g NasHPOd and 0.046 g KHIPOI in 1 liter of 0.1 M-Tris-Hcl, pH 7.3). Thephage suspension was then treated with DNase and RNase (5 pg/ml. each), in some cases with DNase only, and the phage was spun down again at 22,000 8 for 1 hr. The buffered salt solution was poured onto the phage pellet and kept in the cold for at least 24 hr before suspending the phage and assaying the titer. T-odd phagee: The method of preparing T-odd phages was principally the same ae that of T-even phages, except that the filtration through kieselguhr and the Millipore filter and the treatment with nucleases were omitted. (b) Conditions of phage infection E. coli cells were grown overnight at 37°C in a nutrient broth medium (8 g Difco nutrient broth and 5 g NaCl in 1 liter water, pH 7.0), and diluted 20 times with the same medium. When the cell conoentration reached 6 x lOs/ml., phage were added, yielding a multiplicity of 10, unless otherwise stated. Samples were taken to assay infective centers and non-infected bacteria between 1 and 3 mm after infection, depending on the nature of the experiment. After an appropriate period of infection, the infected bacteria were chilled as fast s.e possible in a beaker containing packed crushed ice, which was immersed in ice water with salt. The infected bacteria were collected by centrifugation and sRNA was extracted. In the case of T4 infection, a Bactotryptone medium (10 g Bactotryptone, 10 g NaCl and 5 g yeast extract in 1 liter water, at pH 7.0) wae used for cell growth and infection. For T5 infection, 10m3 M-Cd& was added to the medium. (c) Preparation of eRNA and aminoacyZ-sRNA eyrdhetuae sRNA was prepared by the phenol procedure described by von Ehrenstein & Lipmann (1961). Essentially the method of Yamane & Sueoka (1963) was used to prepare the enzyme fraction. Bacteria were grown at 37°C in 1 liter of the nutrient brothmediumdescribedabove. The cells were harvested in the logarithmic phase (6 x 10s cells/ml.) and ground with 4 g of alumina (levigated alumina from Norton Abrasives, Worcester, Mass.) in the cold. To the paste, 6.6 ml. of Tris-magnesium buffer (0.01 M-Tris-HCl buffer (pH 7.3) plus 0.001 M-MgClz) was added and the mixture was centrifuged at 8000 rev./min in a Servall centrifuge for 16: min. The supernatant fraction was centrifuged at 106,000 8 for 3 hr at 0°C and the upper two-thirds of the supernatant was dialyzed against 1 liter Tris-magnesium
MODIFICATION
OF LEUCYL-sRNA
185
buffer plus O-006 M-mercaptoethanol at 4°C for 3 hr, changing the buffer every hour. In order to remove sRNA, 105,000 g supernatant was applied to a 1 cm x 6 cm column of DEAE cellulose equilibrated with potassium phosphate buffer (0.02 M, pH 7.7). The charged column was washed with 10 ml. of the phosphate buffer, and the enzyme was eluted with the 0.02 M-potassium phosphate buffer containing 0.35 M-NaCl. Fractions were collected in 1.5-ml. portions, tubes with high absorbancies were combined, divided into small portions, and stored at - 80°C. Yeast was grown in the following medium: 5 g Bactotryptone, 2.5 g yeast extract, 0.25 g CaCl,, 2 g (NH&S04, 1 g KHaP04, 0.25 g MgSOe and 40 g glucose in 1 liter water. For the enzyme preparation, yeast cells were ground with sea sand (Merck & Company) instead of alumina. (d) Preparation of amirwacyl-sl?NA Radioactive aminoacyl-sRNA was prepared basically according to Berg, Bergmann, Ofengand & Dieckmann (1961). The reaction mixture contained the following compounds in a total volume of 1 ml.: 100 pmoles of Tris-HCI buffer (pH 7.3); 5 pmoles of magnesium acetate; 3 pmoles of ATP; 4 wmoles of glutathione (reduced); the enzyme fraction corresponding to about 0.2 mg of protein; 0 to 2 mg of isolated sRNA; and an appropriate amount of W- or sH-labeled ammo acid plus 19 remaining non-radioactive ammo acids (1 pmole of each). The reaction mixture was incubated at 37°C for 15 min and deproteinized by shaking with 1 vol. of water-saturated phenol. Phenol was removed by shaking with ether, which was removed by bubbling air through the mixture; aminoacyl-sRNA was then precipitated with 2.5 vol. of cold alcohol. The precipitate was dissolved in 0.05 ~-sodium phosphate buffer (pH 6.3) and stored frozen. (e) Methylated albumin column The simplified MAKt column described by Sueoka & Cheng (1962) was used to fractionate aminoacyl-sRNA’s. The MAK (30 ml., capacity up to 3 mg of sRNA) was prepared by suspending 6 g of kieselguhr in 30 ml. of 0.1 M-NaCl in 0.05 M-sodium phosphate buffer (pH 6~3), followed by boiling and cooling the suspension. Then 1.5 ml. of 1% methylated albumin solution in water was stirred into the suspension slowly. Paper powder mixed with 0.1 M-NaCl in 0.05 M-sodium phosphate buffer (pH 6.3) was put in the bottom of the column to protect the fritted glass disc. The MAK was poured onto the paper powder layer. A suspension of 2 g kieselguhr in 0.1 M-NaCl-sodium phosphate buffer was layered on top as a protective layer. Paper powder suspension, MAK, or the protective layer was poured into the column without allowing any flow of liquid through the column, by closing the clamp at the bottom portion of the column; this allowed even packing of MAK. After being poured, each layer was packed by air pressure. The column was washed with 100 ml. of the starting buffer. The column was charged with 1 to 2 mg of sRNA suspended in 40 ml. starting buffer and washed again with 40 ml. starting buffer. For routine work, a salt gradient of 0.2 to 1.1 M-NaCl was used for eluting sRNA, and fractions of 2 ml. were collected at the rate of about 1 ml./min. Optical density was measured at 260 v for every other fraction, and crude salmon sperm DNA (200 pg/sample) and cold trichloroacetic acid (final concentration 10%) were added to each sample. The precipitate was collected on a coarse membrane filter (from Carl Schleicher & Schuell Company) and radioactivities were counted in a Packard liquid-scintillation counter. (f) Ultraviolet irrdktion of phage TZ The phage was suspended in Tris C medium (without glucose) at a concentration of 5.2 x lOi particles/ml. 10 ml. of the above suspension in a glass Petri dish was irradiated for 1.5 mm under a germicidal lamp, 15 w, at 50 cm distance with constant &al&g. The phage survival under the above conditions was 8 x 10-S. (g) Phage h induction of E. coli K12(h) Mitomycin C was used as an inducing agent (Otsuji, Sekiguchi, Iijima & Takagi, 1959). E. coli K12(X) was grown in a nutrient broth medium, described previously, with 0.2% t Abbreviation
used: MAE, methyl&ted albumin kieeelguhr.
-
-
-
-
3
400
‘C 800
no.
Fxa. 1.
Fraction
250
‘C
600
’ @2
0.4
oc
#
Histidine
50
Alanine
I
60 I
70
T2 B-min sRNA (“Cl
1
I
‘C
3
-400
-800
'C
200
400- 400
Jt
g '% 2
52 -5 :: ,v x Y .$
('J!u.'IW)
~l+l~DO!pDt( 0
0
x
0
0
f?
2 ‘E 5:
0 z P
.v
v8
4
8
Leucinc
sRNA
40
(‘4C)
12 8-min sRNA
(“Cl
E. co/i
400-
‘H
c3H)
B .rA
sRNA
I,
(‘4C)
3H
4oo
-1
‘4C
FIG.
Fraction no. 1. Elution profiles of aminnecyl-sRNA’s of E. coZi B before and after T2 infection, E. wli ceils were infected with T2 at the mllltiplicity of 10. At the 8th min, the culture was chilled quickly and the cells mere harvested. Radioactive arninoacyl-&NA’s were prepared according to the method described in Materials and Methods. The TZ-infected sRNA was chromatogrephed on HAK column together with E. coli sRNA charged with zl different isotope from that of TZ-infected sRNA. The oonoentrstion of N&l in the starting buffer was 0.2 &I and in the final buffer was 1.1 M, except for leucyl-sRNA where the N&l gradient was from 0.35 to 0.8 SL
L
T2 8-min
I
T2/8-min
190
T. KANO-SUEOKA
AND
N. SUEOKA
glucose, and the next morning the cells were diluted 20-fold in a fresh medium. When the cell concentration reached 60 Klett u&s (approximately 4 x lo* cells/ml.), 6 pg/ml. of mitomycin C was added, and samples were taken at 0, lo,20 and 30 miu after the addition of mitomycin C for isolating sRNA. 46 min after the addition of mitomycin, phage h began to appear in the culture medium and by 90 to 100 mm the cells were lysed. The yield was about 40 to 50 phage particles/cell. 100 min after induction, the viable cell count was reduced to 3 x 1Oa cells/ml., while the control oulture (without induction) contained viable cells, 1 X 10e cells/ml.
3. Results (a) Comparison of aminoacyl-sRNA’s
before and after TZ infection
The aminoacyl-sRNA’s of normal E. wli B and of E. wli B infected with phage T2 were compared by chromatography on MAK columns. Seventeen aminoacyl-sRNA’s other than asparagyl-, glutamyl-, and cysteinyl-sRNA were examined. In only one case, leuoine, was there found a clear difference in profile between sRNA isolated from infected cells and from non-infected cells (Sueoka & Kane-Sueoka, 1964). Fifteen aminoacyl-sRNA’s were compared directly on the same column by charging sRNA’s prepared from cells before and after T2 infection with each amino acid labeled with a different isotope, l*C or 3H. Figure 1 summarizes the results. Two other aminoacylsRNA’s (threonyl and glutamyl) were also examined by comparing two separate chromatograms before and after T2 infection, but did not show appreciable alteration. Only leucyl-sRNA shows a clear difference in elution proties before and after the infection. Normal leucyl-sRNA of E. wli B shows two major peaks in MAK column chromatography, Leu I and Leu II, Leu I being the majority component. The RNA from cells infected with T2 for eight minutes shows that Leu I decreases to less than half the level in the non-infected case. This change of leucyl-sRNA upon T2 infection lies in aRKA and not in leucyl-sRXA synthetase (Sueoka & Kane-Sueoka, 1964). Some ambiguity remains concerning seryl- and isoleucyl- sRNA. Two preparations of T2 eight-minute infected t-RNA were examined for serine, and both showed a slight but consistent difference between the sRNA’s with and without infection. The profile of isoleucyl-sRNA of E. coli B is somewhat variable in different sRNA preparations for unknown reasons. Accordiugly, even though some discrepancy is found in the profile of isoleucyl-sRNA before and after the infection, its significance is not clear at the moment (Fig. 1). (b) &eci$city
of the alteration of leucyl-sRNA after T2 infection
The necessity of phage DNA and the requirement of protein synthesis for the alteration of leucyl-sRNA were shown previously (Sueoka & Kane-Sueoka, 1964). An RNase-less mutant (A19) and an RNase-less and polynucleotidephosphorylaseless mutant (Q13) were used as hosts for T2 infection and for examination of leucyl&WA. In both cases the normal alteration of leucyl-sRNA was observed (Fig. 2(a) and (b)). This excludes the possibility of the involvement of those two host enzymes in altering leucyl-sRNA. The ghost of phage T2 adsorbed on E. wli B did not induce the alteration of the leucyl-sRNA proGle (Sueoke & Kane-Sueoka, 1964). Killing the cells with colicin E2 also did not alter leuoyl-sRNA of 1. wli B (Fig. 2(c)). Cells were grown in Tris C medium at 37°C. When the density reached 6 x 10s cells/ml., the cells were washed once and resuspended in the same fresh medium. Then pu~%ed colicin E2 was added at a oonoentration of 7 x lo* molecules/cell and 16 minutes later the cells were bar-
MODIFICATION
OF LEUCYL-sRNA
04
191
T2 8-min sRNA (“Cl
I
1
I
3H “C
(b) 2
s;;
0"
-E
c3H)
0.4
rx 'G z -u 5 y
2
E. co/i B sRNA
I I
1 T28-min sRNA C4C)
0.2
’ I
”
I 7
t
: .t8' 60 I
I
70 I
1
3H 14C
(cl
I
A
Colicin-E
co/i 6
I
E. co//' B sRNA t3H)
600
100 ,O.D.
0
60 Fraction
70
00
no.
Fro. 2. Elution profiles of leucyl-sRNA. (a) E. wli A19, RNase-less mutant of E. cdi Hfr strain AB301 was used as the host for T2 infection. sRNA was isolated from the cells infected for 8 min. [i%]Leucyl-sRNA was prepared and compared with that of 1. coli on s MAE column. (b) E. coli 413, an RN&se-less and polynuoleotide phosphorylase-less mutant of E. wli Hfr Al3301 was used as host for T2 infection. Experimental conditions were the same as (a). (c) sRNA was taken from E. COWB cells exposed to colicin E2 for 16 min and its leucyl-sRNA was compsxed to that of E. cd B.
T. KANO-STJEOKA
192
AND
I
I
0.3-
N. SUEOKA
I
#:-E.co/i \
(b)
3”
sRNA-Ecoli-ent
T2 S-min
sRNA-E.co/i-
IO00
I I
0.5 0.4 -
(cl
,/
./-
)H
E.co// sRNA-~.co/~‘-enz.(~H)
~2 B-min
sRNA-Ecd-enr
“C
(“Cl
0 D.
400
/
0.3-
I 14r
('H)
600 300
‘\,
2oo--100 *-_ _ 60
FractKxlno. Fm.
3
- --
I
70
a-c.
0.6 i
Cd)
600
400
200
"0 0 0.6
0.4 T2 l-min
sRNA
0.2
(9) Edi
sRNA
T2 2-min sRNA (“Cly
40
50
60 Fraction no. ma.
70
3 (cont.) see next page
194
T. KANO-SUEOKA
AND
N. SUEOKA
vested to isolate sRNA. The survival of bacteria was 0.1’70~ after 15 minutes exposure to colicin E2. During their preparation, the T-even phage stocks had been treated with pancreatic RNase (5 pg/ml.) as well as with DNase. Although the chance seemed remote that a trace amount of RNase might remain in our phage stock to cause the alteration of leucyl-sRNA after infection, this possibility was also examined by preparing T2 stock without RNase treatment and infecting cells with this T2 stock. Leucyl-sRNA taken from such cells showed the same altered profile as that shown previously with RNase-treated phage stock. All the above results support the idea that this alteration is specific to T2 infection. (c) Time course of the alteration of leucyl-sRNA after T2 injection In the work previously reported (Sueoka & Kano-Sueoka, 1964), the time course of the change of leucyl-sRNA profiles was followed by preparing sRNA from the cells three, five and eight minutes after the infection. The elution profile of leucyl-sRNA from three-minute infected cells showed a new component in front of the Leu I peak not seen in the eight-minute sRNA (Fig. 3(a)). Five minutes after infection, the new component has increased, while the Leu I peak has decreased considerably (Fig. 3(b)). In the eight-minute infected sRNA the new front component has disappeared (Fig. 3(c)). The timing of the change in leucyl-sRNA profile, however, does not seem to be similar with different phage preparations and conditions of infection. Previously, the phage was prepared by acid precipitation from the lysate and differential centrifugation (Sueoka & Kano-Sueoka, 1964). The survival of bacteria three minutes after infecting E. coli B with this phage at the multiplicity of ten was 10m3to 3 x 10w2.A different method of T2 preparation, which avoided the acid-precipitation step (see Materials & Methods), gave a much higher e%ciency of infection (in the order of 10w4); and the synchrony of infection was good. With this new preparation of phage T2 the leucyl-sRNA alteration was found to be completed at a very early period of infection. Examples are shown in Fig. 3(d), (e), (f) and (g). 0 ne minute after infection, cells infected with one such stock of T2 gave an almost completely altered profile, i.e., like T2 eight-minute infected leucyl-sRNA (Fig. 3(e)). With another stock of T2, it gave an intermediary profile one minute after infection (Fig. 3(f)) and a completed profile two minutes after infection (Fig. 3(g)). The addition of glucose (0*2%) to the nutrient broth medium also seemed to accelerate the alteration of leucyl-sRNA after infection. To terminate the process of infection, the infected cells were routinely poured into a beaker containing plastic bags packed with crushed ice, which was immersed in ice water with salt, as described in Materials & Methods. It took about 30 seconds to cool one liter of the culture down to 10°C and about one minute down to 4 to 5°C. Accordingly, the timing we have described above is not exact. FIQ. 3. Leucyl-sRNA at different times after T2 infection. sRNA was prepared from the cells infected for various lengths of time, and also with different batches of T2 preparation. Charging was made by E. ooli enzyme. The profiles of the leucyl-sRNA were compared with that of E. wli B by eluting together on a MAK column. (a), (b) and (c) sRNA was taken from the cells infected with T2 for 3, 5 and 8 min. Phage T2 was prepared by aoid precipitation method. (d) and (e) Cells were infected with T2 prepared without acid-precipitation for 50 set and 1 min. (f) and (g) A different batch of the phage was prepared as in the case of(d) and (e). sRNA was taken from 1 min- and 2 min-infected cells.
MODIFICATION
195
OF LEUCYL-sRNA
1n order to stop the cell metabolism instantaneously at a desired moment, the infected cells were mixed rapidly with two volumes of cold lOOo/oethanol (- WC). The cells were centrifuged down and sRNA was isolated in the ordinary manner. Comparison of T2 one-minute and three-minute infected leucyl-sRNA isolated from cells treated with ice and with alcohol shows that there was little difference between the two sRNA preparations in the extent of leucyl-sRNA alteration. This implies that the process of leucyl-sRNA alteration is indeed one of the earliest steps in the infection process.
(d) Infection with ultraviolet light-irradiated
T2
According to Dirksen, Wiberg, Koerner & Buchanan (1960), the early phage specific protein synthesis upon the infection of E. coli by ultraviolet-irradiated
U.V. irradiated
-
.60
70 Fraction
T2
/ 80
I
no,
FIG 4. Leucyl-sRNA from E. coli B infected with ultraviolet light irradiated T2. The phage suspension was irradiated withultraviolet light up to the survival of the phage (8 x 10-a). E. co& cells were infected with the irradiated phage at the multiplicity of five. sRNA was taken from cells infected for 8 min and 15 min and charged with normal E. coli enzyme. (a) sRNA from 8 min infected cells with ultraviolet light-irradiated phage. (b) sRNA from 15 min infected cells with ultraviolet light-irradiated phage.
196
T. KANO-SUEOKA
AND
N. SUEOKA
phage does not stop, but continues. Moreover, contrary to the normal phage infection case, the bter protein synthesis is inhibited. If leucyl-sRNA alteration is responsible for the cessation of early protein synthesis, we may expect the absence of the alteration by ultraviolet-irradiated phage infection. The results of such experiments are given in Fig. 4. T2 were inactivated to the range where Dirksen et al. (1960) found the continuation of deoxycytidylate hydroxymethylase synthesis, and cells were infected with the irradiated phage at a multiplicity of five. The presence of normally altered leucylsRNA from cells infected with irradiated phage for eight minutes suggests that this is not responsible for the switch-off of early protein synthesis. This notion has been supported also by the fast timing of the change in leucyl-sRNA after infection. (e) Nature oj’ the alteration of leucyl-sRNA T2-infected sRNA was charged with radioactive leucine by yeast enzyme which was known to charge leucine only to the Leu I peak of E. coli sRNA. As shown
T2 5-min
sRNA-yeast
T2 5-min
\I XI \
X k
‘X
sRNA-
600400 sjt;
200 200 100
X..
Fraction
no.
FIQ. 6. Leuoyl-sRNA of TB-infected cells charged by yeast enzyme. Enzyme extract of yeast W&B freed from RNA by a DEAE-oellulose columu and used to charge radioactive leuciue to T2infected sRNA. The elution profile of the leucyl-sRNA was compared with that of T2-infected sRNA charged by 1. co& enzyme. (a) T2 3-min sRNA. (b) T2 6-min sRNA (Sueoka & Kane-Sueoka, 1964).
MODIFICATION
19;
OF LEUCYL-sRNA TABLE
1
Ratio of leucine to other an&w acids attached to sRNA before and after T2 infection E. coli aRNA ( l$$)
Ratio
T2 S-min sRNA ( $y;ti)
Ratio
0
Leu Ser
0.04 0.003
[“H]Leucine
5
Leu SEW
1.43 0.13
[l4C]Serine
10
Leu Ser
1.76 0.25
7.04
1.47 0.20
7.50
15
Leu SW
1.65 0.34
4.85
1.38 0.28
4*93
0
Leu Gly
0.03 0.004
[ 3H]Leuoine
5
Leu GUY
1.36 0.19
7.17
1.36 0.19
7.32
[r4]Glycine
10
Leu Q~Y
1.55 0.35
4.43
1.47 0.35
4.30
15
Leu GUY
1.54 0.47
3.28
1.54 0.47
3.28
0
Leu Val
0.07 0.28
[eH]Leucine
5
Leu Val
1.79 1.40
1.28
1.66 1.07
1.55
[14C]Valine
10
Leu Val
1.97 1.24
1.59
2.03 1.23
1.65
15
Leu Val
1.87 1.31
1.43
1.81 1.13
1.60
0
Leu Phe
0.06 0.008
[eH]Leucine
5
Leu Phe
1.87 0.04
44.5
1.84 0.04
46.7
[14~~jlhe~yl-
10
Phe Leu
0.08 2.16
27.7
0.07 1.93
20.5
15
Leu Phe
2.25 0.100
22.5
2.04 0.08
25.2
0
LYS Leu
0.03 0.007
5
Lys Leu
1.53 0.63
2.43
1.56 O-68
2.30
10
LYS Leu
3.00 0.95
3.16
3.36 1.09
3.08
15
LYS Leu
3.79 l-04
3.64
3.60 1.03
3.50
[sH]Lysine
[14C]Leucine
0.03 0.003 11.0
1.26 0.10
12.6
0.03 0.003
0.07 0.32
0.06 0.008
0.01 0.002
The amount of ammo acids attached to sRNA is expressed as pmoles/lOO ratios of lsucine to the other 5 amino acids was calculated for every sample.
pg sRNA
and the
198
T. KANO-SUEOKA
AND
N. SUEOKA
in Fig. 5, in addition to the Leu I peak, the new front component and a part of the Leu II region were also charged with leucine. This indicates that two kinds of new leucyl-sRNA components (Leu F and Leu R, Fig. 11) are closely related to Leu I (Sueoka & Kano-Sueoka, 1964). In order to find out whether there is a sizable amount of new synthesis or degradation of leucyl-sRNA after the infection, the ratios between leucine and other amino acids attached to sRNA before and after the infection were compared. E. coli sRNA and T2 eight-minute infected sRNA were charged with radioactive valine, phenylalanine, lysine, serine and glycine; each reaction mixture also contained radioactive leucine labeled with a different radioisotope (14C or 3H) as a direct reference. As shown in Table 1, there is little difference in the ratio of leucine to other ammo acids attached before and after the infection. Two possibilities exist as to the cause of the alteration of leucyl-sRNA profile after T2 infection: one, the synthesis of one or more new sRNA’s; the other, the partial degradation or modification of one or more pre-existing leucyl-sRNA’s eluted at the Leu I peak. The fact that both new components of leucyl-sRNA (front and rear) after T2 infection can be charged by yeast enzyme (Fig. 5) and that the relative amount of leucyl-sRNA to other aminoacyl-sRNA’s does not appreciably change after T2 infection (Table 1) renders the modification of pre-existing leucyl-sRNA (some of Leu I) more likely. Another possibility, although unlikely, is that equivalent amounts of degradation of pre-existent host sRNA and synthesis of new sRNA occur simultaneously. A unique characteristic of the front component (Leu F) is its loss of amino acidaccepting capacity once the leucine is discharged by high pH. TZ-infected sRNA which contained the front component was treated in 0.5 M-Tris-HCl (pH 8.8) for 45 I
I
I
I
v 1%
;i
sc
400
+ z
+J z. 06 “E Iv
T2 3-min
sRNA (‘4C)
300
:z 4.J :: 2
0
; .+
0.4
200
0”
@2
100
Fraction
no.
FIG. 6. Stability of the front component of T2 leucyl-sRNA. An sRNA preparation which contained the front leucyl-sRNA component was prepared by infecting E. co&i B with T2 at 37% under slow shaking of the culture. The sRNA was treated in 0.5 M-Tris-HC1 buffer (pH 8.8) for 45 min at 37”C, reprecipitated with alcohol twice to remove any free amino acid and recharged with [Wlleucine. This was compared with the same sRNA but non-treated.
MODIFICATION
OF LEUCYL-sRNd
19!J
minutes at 37”C, reprecipitated and recharged with radioactive leucine. This RNA was compared directly on a MAK column with the same sRNA without the high pH treatment. The front component was not detected after the alkaline treatment whereas the other component (peaks I and II) remained the same (Fig. 6). This loss of rechargeability by high pH was also confirmed by isolating the Leu F first, and then treating with high pH and trying to recharge with radioactive leucine. It may be that either the structure of the sRNA of the front component is unstable or the intact primary structure of the sRNA is lost, so that by raising the pH it may have disintegrated. The possibility of recharging with other amino acids has not been examined. (f) Leucyl-sRNA
of various hosts infected with T2
Leucyl-sRNA for the following strains of E. coli were compared for the MAK column profile with E. coli B, E. coli C, E. coli K12(/\), and E. coli CR63. Within the resolving power of the column, the profiles were very similar to that of E. coli I3 (Fig. 7). Leucyl-sRNA was also compared after T2 infection on those different hosts, which showed profiles of leucyl-sRNA very similar to those of infected E. coli B (Fig. 7). The case of Shigella dysenteriae 60 is of special interest. The elution pattern of leucyl-sRNA from Shigella 60 is different from the profile of E. coli sRNA. At first glance, Shigella leucyl-sRNA does not seem to have components corresponding to Lcu I of E. coli (Fig. 8). However, when Shigella 60 was infected with T2, and its leucyl-sRNA compared with the non-infected case, the difference between the two was clear. On a MAK column profile the front portion of leucyl-sRNA disappeared, while the rear portion seemed to increase eight minutes after T2 infection. Moreover, the leucyl-sRNA profile of Shigellu charged with yeast enzyme occupied the front part of the normal profile charged by E. coli enzyme (Fig. 8). This implies that although the elution characteristic is distinct, Shigella has species of leucyl-sRNA corresponding to Leu I of E. coli which was modified after T2 infection. Therefore, in principle, T2 exerts the same effect on Shigella leucyl-sRNA as on E. coli. (8) Leucyl-sRNA
of E. coli B infected with various $qes
Leucyl-sRNA’s from E. coli B after infection with various T phages and from E. coli K12(/\) during the induction of h were examined. Alteration similar to the case of T2 infection was observed with T4 and T6 infection, which may be expected by the closeness of T4 and T6 to T2 (Fig. 9). On the other hand, infection with T-odd phages, Tl, T3, T5 and T7 did not show the alteration (Fig. 9). Samples of sRNA from T-odd phage-infected cells taken at periods of infection different from the ones shown in Fig. 9 (five-minute infected sRNA for T3, lo-minute infected sRNA for T5 and seven-minute infected sRNA for T7) have also been analyzed. No alteration of leucyl-sRNA was detected. Phage X was induced by an addition Pm. 7. Leucyl-sRNA of various hosts before and after infection with T2. Leucyl-sRNA’s of E. COGC, E. coli K12(X) and E. COG CR63 were compared with that of E. coli B. (a) E. coli C; (b) E. coli K12(A); (c) E. coli CR63. The three strains of E. coli were infected with T2 and sRNA was isolated from cells 8 min after infection. Each leucyl-sRNA preparation was compared with that of its own host cells. (d) T2 S-mm E. wli; (e) T2 S-min E. coli K12(A); (f) T2 S-min E. coli CR63. see next page
R
sRNA t
(14C) C3H)
600
3t 1000
(bf
K12 (A)
sRNA
c3H)
800 E. co/i
B sRNA
400
4C
600
(‘4C) -
500 600 400 400
300 200
200 100
3H
(cl
14C
600 E. co/i
B sRNA c3H)
- 200
200
50
60
70 Fraction
no.
Fra. 5
80
90
- 100
3ki
Cd)
"C
E co/i C sRNA (3H) Ob-
403 T2
8-min
E.co/~
C
203 02-
5n
60
70
80
3H
(4
i
.f
i
600
0. T2
k
400
8-min
-300 0 200 0.1
100
80
70
60
90 ‘C
CR 63 sRNA (3H) r*, ’ ? T2
CR 63 sRNA
8-min
600.
1
/ 300
150
40
50
60 Fraction
Frc.
i
70 no.
(cont.)
F ;
,x :z z :: 0 cz
E,co/i 6
sRNA
t3H)
T2 8-min
Shigella
E. co/i,enzyme
..
_. . . . 50
60
70
Shlgelia 60 sRNA (14C)
yeast
Fraction
FIG. 8
no.
enzyme
ci4C)
80
sRNA ,nn
MODIFICATION
OF LEUCYL-sRNA
203
of mitomycin C (5 pg/ml.) to E. coli K12(A) culture and sRNA was isolated from the cells 10 minutes, 20 minutes and 30 minutes after the addition of mitomycin C. Here again, the alteration was not observed. (h) Leucyl-sRNA
after infection
with various amber mutants
-4mber mutants of T4 infecting non-permissive host (E. coli B) are of particular interest because of their known phenotypic effects and arrangements of the genes along the chromosome. If the alteration of leucyl-sRNA were due to the action of a phage-specific enzyme and essential for the phage development, it should be possible to locate the genes for this protein, which could be an amber-type mutant. Several amber mutants for early functions were obtained from Dr R. S. Edgar, and the MAK column profiles of leucyl-sRNA were compared before and after the infection on non-permissive host. All mutants so far examined have given normal modified profiles like that of the T4 wild type, except that some mutants, gene no. 39, 52 and 57, still had a small amount of the front component eight minutes after infection. However, there seems to be no correlation between characteristics of the mutants and the presence of the front component. A summary of the characteristics of the mutants examined is shown in Fig. 10.
4. Discussion The degeneracy of the code has been established now beyond a reasonable doubt (Nirenberg et al., 1963; Speyer et al., 1963; So11 et al., 1965). The corresponding degeneracy (heterogeneity) in sRNA has been reported in leucyl-sRNA (Weisblum, Benzer & Holley, 1962; Bennett, Goldstein & Lipmann, 1963; Yamane, Cheng & Sueoka, 1963; Leder & Nirenberg, 1964; S6ll et al., 1965), although its applicability for other amino acids is not yet clear. When there is degeneracy in sRNA of an amino acid, the modification of one of the synonymous sRNA’s may eliminate the adaptor of the corresponding codon and prevent the translation of mRNA of genes which contain the codon, but not of those which do not contain the codon. If the secondary structure of sRNA is modified so that a different part of the molecule now becomes the new codon-recognizing site, i.e.; the anticodon is changed, then the translation of various mRNA’s should be disturbed. Another possible outcome of the adaptor modification is its change in the site of the aminoacyl-sRNA synthetase recognition, which may lead to loss of amino acid acceptor activity of sRNA or to a change of the kind of amino acid accepted. Also, the possibility of activation of cryptic (non-functional) sRNA should not be ignored. Our work on E. coli sRNA after phage infection is directed to exploring the possibility of adaptor modification being involved in cell differentiation through a change of the code translation system.
FIG. 8. Leucyl-sRNA of Shigella o!yse?ttertie 60. (a) sRNA from Shigella dyeenteriae 60 was charged with [W]leucine by E. coli enzyme and compared on the MAK column with leucyl-sRNA of E. coli. (b) ShigeZZa leucyl-sRNA was compared with that taken from ShigeZZa cells infected with T2 for 8 min. (c) ShigeZZa leucyl-sRNA charged with E. coli B enzyme was compared with that charged with yeast enzyme.
.5
50 T4
I
50
60
I x-x
70
1 : ,/ :yE.
60
I
80
co/i sRNA c3H)
I
90
80
T4 8-min sRNA (‘4C)
70
,200
500
3 'C
I ooo-
soo250
0
0
FIR. 9
Fraction no.
Tl
I
I
E. co//’ B sRNA (‘HI
50
I
I
70
3H “C
300-
;; 0 2
600 Lz
‘H “C
400
60
I
60
:4
I
I
70
200
40 I
60
1.2
T3
50
a
206
T. KANO-SUEOKA
AND
N. SUEOKA
The experimental data presented in this paper establish the following facts, (1) Infection of E. wli B with T-even phages leads to a structural modification of some synonymous component of leucyl-sRNA belonging to Leu I of the MAK column proiiles. T-odd phages and X phage do not bring about the modification. Leucyl-sRNA synthetase is normal after phage infection and has nothing to do with the leucyl-sRNA modification. (2) E. wli strains B, C, K12(h), CR63, A19, Q13, and Shigellu dysenteriae 60 show essentially the same modification of leucyl-sRNA upon T2 infection. (3) The modification of leucyl-sRNA has two steps, a summary of which is shown diagrammatically in Fig. 11. (4) The modification is completed shortly after the infection (one to three minutes). Ultraviolet irradiation of phage T2, which prevents the cessation of early enzyme synthesis, does not affect the modification.
rI1 I 00152
FIG. 10. Leucyl-sRNA of amber mutants of phage T4. The map positions of the amber mutants and the nature of mutants which have been analyzed for leucyl-sRNA are indicated on the genetic map of phage T4 according to Epstein et al. (1963). Abbreviations: DA, DNA synthesis is initiated but ceases after a short interval; DO, no detectable DNA synthesis occurs; DD, there is a delay in the onset of DNA synthesis after which DNA synthesis proceeds normally; MD, DNA synthesis appears to be normal, but there seems to be a general defect in maturation. E. coli B (non-permissive host) was infected with amber mutants and sRNA’s were taken 8 min after infection. The elution profiles of leucyl-sRNA were compared with that of E. coli B.
FIG. 9. Leucyl-sRNA of E. coli infected with various phages. Phages T2, T4 and T6 were added to E. coli B cultures at a multiplicity of 10 and sRNA’s were isolated from 8-min infected cells. E.. coli K12(X) was grown in e, nutrient broth medium (plus 0.2% glucose) and mitomycin C (5 pg/ml.) was added to the exponential-phase cells. The sRNA taken from zero-min and 20-min induced cells was analyzed. Phage Tl, T3 and T7 were added to E. coli B cultures at a multiplicity of 10 and sRNA was isolated 8 min later for Tl and T3 infections and 5 min later for T7 infection. E. coli F and phage T5 st were used to obtain fast infection by the phage. The sRNA was taken from cells infected for 30 min and charged with E. co.% enzyme. The profile of leucyl-sRNA of T&infected 1. COGF was compared with that of E. co& B and not with that of E. coli F. However, the leucyl-sRNA profiles of E. coli B and F have been shown to be identical.
MODIFICATION
OF LEUCYL-sRNA
207
(5) T2 ghost and colicin E2 do not induce the modification. Inhibition of protein synthesis by chloramphenicol abolishes the modification by T2 infection. (6) Alanyl-, arginyl-, aspartyl-, glycyl-, histidyl-, lysyl-, methionyl-, phenylalanyl-, prolyl-, tryptophanyl-, tyrosyl-, and valyl-sRNA’s of E. coli B are not altered upon T2 infection, judging from the direct comparison on the column using 14C-3H doublelabeling technique. Isoleucyl- and seryl-sRNA may show a small change, which cannot be definitely established at the moment. Glutamyl- and threonyl-sRNA are not altered by comparison of separate profiles before and after infection. Cysteinyl-, asparaginyl- and glutaminyl-sRNA await further analysis. The present results indicate that the involvement of the modification of leucylsRNA in the transition of the early phase to the late phase of phage protein synthesis is unlikely. This was raised as one of the possibilities (Sueoka & Kane-Sueoka, 1964). Early timing of the completion of the modification has now been established by quickly stopping the metabolism of phage-infected cells with alcohol. Ultraviolet irradiated T2 and amber mutants of T4 which lack early function, both of which
Time 2
Time 3
pi&D&
FIG. 11. Schematic presentation of the time course of leucyl-sRNA modification after T2 infection. Solid line represents leuoyl-sRNA charged with E. coli B enzyme and broken line represents leucyl-sRNA charged with yeast enzyme.
208
T. KANO-SUEOKA
AND
N. SUEOKA
allow the continuation of early enzyme synthesis and inhibit the late protein synthesis, showed a normal alteration upon their infection on E. wli B. The possibility that the leucyl-sRNA change may take 8ome role in the arrest of host-protein synthesis is not excluded. As for the chemical nature of the modification, we have no answer at the moment. There are several possibilities: (1) rearrangement of secondary structure; (2) methylation or demethylation; (3) partial enzymic digestion (Niahimura & Novelli, 1964); (4) oxidation and reduction of thiopyrimidine (Carbon, Hung & Jones, 1965; Lipsett, 1965). It has been reported that the distribution of methylated bases of 8RNA is different before and after T2 infection (Wainfan, Srinivasan & Borek, 1965). In vitro methylation of E. coli sRNA with T2 infected cell extract was carried out in collaboration with Dr Borek’s laboratory. The result so far obtained is negative. The direct test on the triplet response of the modified leucyl-8RNA and the effect on in citro protein synthesis in E. coli are being investigated. Other example8 of 8RNA modification have been observed in valyl-sRNA during spore formation (Kaneko & Doi, 1966), and in tyrosyl-sRNA during spore germination (Sueoka, unpublished results) of Bacillus subtilis. There is also a report by Subak-Sharpe & Hay (1965) claiming that in the presence of herpes virus, the synthesis of virus-specific sRNA is initiated in BHK 21 cells.
We thank Dr A. D. Hershey for his valuable suggestion on the purification of T-even phages. We also acknowledge the excellent technical assistance of Mrs J. V. Taub. This work was supported by grants from the National Institutes of Health, GM10923-04 and the National Science Foundation, GB3445.
REFERENCES Bennett, T. P., Goldstein, J. & Lipmann, F. (1963). Proc. Nat. Acad. Sci., Wash. 49, 850. Berg, P., Bergmann, F. H., Ofengand, E. J. & Dieckmann, M. (1961). J. Biol. Chem. 236, 1726. Carbon, J. A., Hung. L. C Jones, D. S. (1965). Proc. Nut. Acad. Sci., Wash. 53,979. Champe, S. P. (1963). Ann. Rev. Microbial. p. 205. Cohen, S. S. (1949). Bad. Rev. 13, 1. Cohen, S. S. (1963). Ann. Rev. Biochem. 32, 83. Dirksen, M., Wiberg, J. S., Koerner, J. F. & Buchanan, J. M. (1960). I-‘roc. Nut. Acad. Sci., Wash. 46, 1425. Epstein, R. H., Bolle, A., Steinberg, C. M., Kellenberger, E., Boy de la Tour, E., Chevalley, R., Edgar, R. S., Susman, M., Denhardt, G. H. & Lielausis, A. (1963). Cold Spr. Harb. Symp. Quant. Biol. 28, 375. Ehrenstein, G. von & Lipmann, F. (1961). Proc. Nat. Acad. Sci., Wash. 47, 941. Gesteland, R. F. (1965). Fed. Proc. 24, 293. Kaneko, I. & Doi, R. (1966). Proc. Nut. Acud. Sci., Wa-sh. 55, 564. Leder, P. & Nirenberg, M. W. (1964). Proc. Nat. Acad. Sk., Wash. 52, 1521. Lipsett, M. N. (1965). Fed. Proc. 24, 216. Luria, S. E. (1962). Ann. Rev. Microbial. p. 87. Nirenberg, M. W., Jones, 0. W., Leder, P., Clark, B. F. C., Sly, W. S. & Pestka, S. (1963). Cold Spr. Harb. Symp. Quant. Bill. 28, 549. Nishimura, S. & Novelli, G. D. (1964). Biochim. biophys. Act+ 80, 574. Otsuji, N., Sekiguohi, M., Iijima, T. & Takagi, Y. (1959). Nature, 184, 1079. Roberts, R. B., Abelson, P. H., Cowie, D. B., Bolton, B. T. & Britten, R. J. (1957). Pub.?. Cameg. In&n. Washington, no. 607, p. 5. S811,D., Ohtsuka, E., Jones, D. S., Lohrmann, R., Hayatsu, H., Nishimura, S. & Khorana, H. G. (1965). Proc. Nat. Acud. Sk., Wash. 54, 1378.
MODIFICATION Speyer, J. F., Lengyel, Cold Spr. Had.
OF LEUCYL-sRNA
“09
P., Basilio, C., Wahba, A. J., Gardner, R. S. & Ochoa, S. (1963).
Symp.
Quant.
Biol.
28, 559.
Subak-Sharpe I%Hay, J. (1966). J. Mol. Biol. 12, 924. Sueoka, N. & Cheng, T. Y. (1962). J. Mol. Biol. 4, 161. Sueoka, N. 85 Kano-Sueoka, T. (1964). Proc. Nat. Acad. Sci., lFa.&. 52, 1535. Sueoka, N. & Kano-Sueoka, T. (1965). In Developmental and Metabolic Control Mechanisms und Neopkuia, p. 114. Baltimore: Williams & Wilkins Company. Suooka, N. & Yamane, T. (1962). Proc. Nat. Acad. Sci., Wash. 48, 1454. \Vainfan, E., Srinivasan, P. R. & Borek, E. (1965). Biochemistry, 4, 2845. \Veisblum, B., Benzer, S. & Holley, R. W. (1962). Proc. Nat. Acad. Sci., Wash. 48, 1449. A-amane, T., Cheng, T. Y. & Suoeka, N. (1963). Cold Spr. Harb. Symp. Quant. Biol. 28, 569. Yamane, T. & Sueoka, N. (1963). Proc. Nat. Acad. Sci., Wash. 50, 1093.
Modifkation
of Leucyl-sFkNA after Bacteriophage Infection T.
&LNO-SUEOKA
AND
N.
&JEOKA
Department of Biology Princeton University Princeton, New Jersey, U.S.A. (Received 15 February 1966, and in revised form 9 May 1966) Aminoaoyl-sRNA’s of Escherichia coli B with and without bacteriophage T2 infection have been examined using methylated albumin kieselguhr column chromatography. Out of 17 aminoacyl-sRNA’s examined, only one, leucyl-sRNA, was found to be clearly different before and after the infection. This alteration lies in sRNA and not in leucyl-sRNA synthetase, and is one of the earliest events after the infection. Phage T4 and T6 infections show the same modification of leucyl-sRNA, whereas infection with Tl, T3, T6 and Ti’ and A induction do not induce the modification. Leucyl-sRNA was examined in Shigelkz dysenteriue 60 and in various strains of E. coli after T2 infection, and in permissive and nonpermissive hosts after infection with various early amber mutants of T4. In all cases,the same modification of the leucyl-sRNA was observed. It is not excluded that the leucyl-sRNA change may take some role in the arrest of host-protein synthesis.
1. Introduction The role of sRNA as the adaptor between the template and the polypeptide suggests the possibility that structural modification of sRNA may be involved as one of the key steps in the regulation of cell metabolism and cell differentiation, since the translation of messenger RNA should be affected if a motication of sRNA leads to a change of its codon recognition or its amino acid acceptor activity (adaptor modi&&tion hypothesis, Sueoka & Kano-Sueoka, 1964). A test of such a hypothesis was initiated by examining aminoacyl-sRNA from T2-infected Escherichia wli as a model system (Sueoka & Kano-Sueoka, 1964, 1965). Two major metabolic transitions exist in the E. wlGT2 phage system: (1) cessation of the synthesis of host DNA, RNA and protein upon phage infection (Cohen, 1949); (2) transition from the early to the late phase of phage protein synthesis, the mechanisms of which are unknown (see review articles by Luria, 1962; Champe, 1963; Cohen, 1963). The main technique used in this study is the methylated albumin column fractionation of aminoacyl-sRNA (Sueoka & Yamane, 1962). Leucyl-sRNA shows an appreciable difference after T2 infection. Moreover, injection of phage DNA and protein synthesis after infection were found to be necessary for this sRNA alteration (Sueoka & Kano-Sueoka, 1964). The present paper describes the further development of this work. Specificity of the involvement of phage has been firmly established. The time course of the alteration of leucyl-sRNA revealed that this phenomenon is one of the earliest events taking place after phage infection. This and other evidence reported here have eliminated the possibility that the leucyl-sRNA modification is related to transition from the early to the late phase of phage protein synthesis. Also, the alteration of leucyl-sRNA is shown to be speoilic only to the T-even phages and not dependent on the hosts used for the infection. 183
184
T. KANO-SUEOKA
AND
N. SUEOKA
2. Materials and Methods Strains of bacteria and phages used in the present study are as follows: E. coli B (laboratory stock); E. COG C, E. coli K12(X) and E. coli K12X sensitive (from A. B. Pardee); E. co&i CR63 (from R. S. Edgar); E. coli F (from Y. & F. La&); an RNase-less mutant (A19) and RNaee-less and polynucleotidephosphorylase-less double mutant (Q13) of E. coli Hfr strain AB301 (Gesteland, 1965, from J. D. Watson); ShigeZZa dyeenteriue 60 (from S. E. Luria); phages T2 and T4 (from laboratory stock); T6 (from R. S. Edgar); Tl and T3 (from J. Hurwitz); T5 st (from Y. & F. Lanni); T7 (from J. E. Smith); T4 amber mutants and their hosts (from R. S. Edgar). Yeast was baker’s yeast. Radioactive ammo acids were obtained from the New England Nuclear Corp., Volk Radiochemioal Company, and Nuclear Chicago Corp. Purified colicin E2, mitomycin C and anti-T3 serum were gifts from D. Helinski, Y. Takagi and A. H. Doermann, respectively. Kieselgubr was purchased from E. H. Sargent & Company and bovine albumin powder (fraction V from bovine plasma) from the Armour Pharmaceutical Company. (a) Preparation
of p?uzges
T-even phages: E. coli cells were grown in tris C medium, a modification of C medium by Roberts, Abelson, Cowie, Bolton & Britten (1957), at 37°C (for T4 infection 100 pg/ml. tryptophan was added to the medium), infected with T2, T4 or T6 at the multiplicity of 0.6 to 1, and the culture shaken until lysis. After the cell debris was spun down at 6000 rev./min for 15 min, the supernatant fraction was passed through kieselguhr (40 g/l. of the supernatant) and then through a Millipore filter (pore size 0.45 p). Phage was collected by centrifugation at 22,000 g for 1 hr and suspended in a buffered salt solution (2 g NH&I, 5 g NaCl, 0.37 g KCl, 0.01 g MgClz.GHzO, 0.026 g Na,SOI, 0.09 g NasHPOd and 0.046 g KHIPOI in 1 liter of 0.1 M-Tris-Hcl, pH 7.3). Thephage suspension was then treated with DNase and RNase (5 pg/ml. each), in some cases with DNase only, and the phage was spun down again at 22,000 8 for 1 hr. The buffered salt solution was poured onto the phage pellet and kept in the cold for at least 24 hr before suspending the phage and assaying the titer. T-odd phagee: The method of preparing T-odd phages was principally the same ae that of T-even phages, except that the filtration through kieselguhr and the Millipore filter and the treatment with nucleases were omitted. (b) Conditions of phage infection E. coli cells were grown overnight at 37°C in a nutrient broth medium (8 g Difco nutrient broth and 5 g NaCl in 1 liter water, pH 7.0), and diluted 20 times with the same medium. When the cell conoentration reached 6 x lOs/ml., phage were added, yielding a multiplicity of 10, unless otherwise stated. Samples were taken to assay infective centers and non-infected bacteria between 1 and 3 mm after infection, depending on the nature of the experiment. After an appropriate period of infection, the infected bacteria were chilled as fast s.e possible in a beaker containing packed crushed ice, which was immersed in ice water with salt. The infected bacteria were collected by centrifugation and sRNA was extracted. In the case of T4 infection, a Bactotryptone medium (10 g Bactotryptone, 10 g NaCl and 5 g yeast extract in 1 liter water, at pH 7.0) wae used for cell growth and infection. For T5 infection, 10m3 M-Cd& was added to the medium. (c) Preparation of eRNA and aminoacyZ-sRNA eyrdhetuae sRNA was prepared by the phenol procedure described by von Ehrenstein & Lipmann (1961). Essentially the method of Yamane & Sueoka (1963) was used to prepare the enzyme fraction. Bacteria were grown at 37°C in 1 liter of the nutrient brothmediumdescribedabove. The cells were harvested in the logarithmic phase (6 x 10s cells/ml.) and ground with 4 g of alumina (levigated alumina from Norton Abrasives, Worcester, Mass.) in the cold. To the paste, 6.6 ml. of Tris-magnesium buffer (0.01 M-Tris-HCl buffer (pH 7.3) plus 0.001 M-MgClz) was added and the mixture was centrifuged at 8000 rev./min in a Servall centrifuge for 16: min. The supernatant fraction was centrifuged at 106,000 8 for 3 hr at 0°C and the upper two-thirds of the supernatant was dialyzed against 1 liter Tris-magnesium
MODIFICATION
OF LEUCYL-sRNA
185
buffer plus O-006 M-mercaptoethanol at 4°C for 3 hr, changing the buffer every hour. In order to remove sRNA, 105,000 g supernatant was applied to a 1 cm x 6 cm column of DEAE cellulose equilibrated with potassium phosphate buffer (0.02 M, pH 7.7). The charged column was washed with 10 ml. of the phosphate buffer, and the enzyme was eluted with the 0.02 M-potassium phosphate buffer containing 0.35 M-NaCl. Fractions were collected in 1.5-ml. portions, tubes with high absorbancies were combined, divided into small portions, and stored at - 80°C. Yeast was grown in the following medium: 5 g Bactotryptone, 2.5 g yeast extract, 0.25 g CaCl,, 2 g (NH&S04, 1 g KHaP04, 0.25 g MgSOe and 40 g glucose in 1 liter water. For the enzyme preparation, yeast cells were ground with sea sand (Merck & Company) instead of alumina. (d) Preparation of amirwacyl-sl?NA Radioactive aminoacyl-sRNA was prepared basically according to Berg, Bergmann, Ofengand & Dieckmann (1961). The reaction mixture contained the following compounds in a total volume of 1 ml.: 100 pmoles of Tris-HCI buffer (pH 7.3); 5 pmoles of magnesium acetate; 3 pmoles of ATP; 4 wmoles of glutathione (reduced); the enzyme fraction corresponding to about 0.2 mg of protein; 0 to 2 mg of isolated sRNA; and an appropriate amount of W- or sH-labeled ammo acid plus 19 remaining non-radioactive ammo acids (1 pmole of each). The reaction mixture was incubated at 37°C for 15 min and deproteinized by shaking with 1 vol. of water-saturated phenol. Phenol was removed by shaking with ether, which was removed by bubbling air through the mixture; aminoacyl-sRNA was then precipitated with 2.5 vol. of cold alcohol. The precipitate was dissolved in 0.05 ~-sodium phosphate buffer (pH 6.3) and stored frozen. (e) Methylated albumin column The simplified MAKt column described by Sueoka & Cheng (1962) was used to fractionate aminoacyl-sRNA’s. The MAK (30 ml., capacity up to 3 mg of sRNA) was prepared by suspending 6 g of kieselguhr in 30 ml. of 0.1 M-NaCl in 0.05 M-sodium phosphate buffer (pH 6~3), followed by boiling and cooling the suspension. Then 1.5 ml. of 1% methylated albumin solution in water was stirred into the suspension slowly. Paper powder mixed with 0.1 M-NaCl in 0.05 M-sodium phosphate buffer (pH 6.3) was put in the bottom of the column to protect the fritted glass disc. The MAK was poured onto the paper powder layer. A suspension of 2 g kieselguhr in 0.1 M-NaCl-sodium phosphate buffer was layered on top as a protective layer. Paper powder suspension, MAK, or the protective layer was poured into the column without allowing any flow of liquid through the column, by closing the clamp at the bottom portion of the column; this allowed even packing of MAK. After being poured, each layer was packed by air pressure. The column was washed with 100 ml. of the starting buffer. The column was charged with 1 to 2 mg of sRNA suspended in 40 ml. starting buffer and washed again with 40 ml. starting buffer. For routine work, a salt gradient of 0.2 to 1.1 M-NaCl was used for eluting sRNA, and fractions of 2 ml. were collected at the rate of about 1 ml./min. Optical density was measured at 260 v for every other fraction, and crude salmon sperm DNA (200 pg/sample) and cold trichloroacetic acid (final concentration 10%) were added to each sample. The precipitate was collected on a coarse membrane filter (from Carl Schleicher & Schuell Company) and radioactivities were counted in a Packard liquid-scintillation counter. (f) Ultraviolet irrdktion of phage TZ The phage was suspended in Tris C medium (without glucose) at a concentration of 5.2 x lOi particles/ml. 10 ml. of the above suspension in a glass Petri dish was irradiated for 1.5 mm under a germicidal lamp, 15 w, at 50 cm distance with constant &al&g. The phage survival under the above conditions was 8 x 10-S. (g) Phage h induction of E. coli K12(h) Mitomycin C was used as an inducing agent (Otsuji, Sekiguchi, Iijima & Takagi, 1959). E. coli K12(X) was grown in a nutrient broth medium, described previously, with 0.2% t Abbreviation
used: MAE, methyl&ted albumin kieeelguhr.
-
-
-
-
3
400
‘C 800
no.
Fxa. 1.
Fraction
250
‘C
600
’ @2
0.4
oc
#
Histidine
50
Alanine
I
60 I
70
T2 B-min sRNA (“Cl
1
I
‘C
3
-400
-800
'C
200
400- 400
Jt
g '% 2
52 -5 :: ,v x Y .$
('J!u.'IW)
~l+l~DO!pDt( 0
0
x
0
0
f?
2 ‘E 5:
0 z P
.v
v8
4
8
Leucinc
sRNA
40
(‘4C)
12 8-min sRNA
(“Cl
E. co/i
400-
‘H
c3H)
B .rA
sRNA
I,
(‘4C)
3H
4oo
-1
‘4C
FIG.
Fraction no. 1. Elution profiles of aminnecyl-sRNA’s of E. coZi B before and after T2 infection, E. wli ceils were infected with T2 at the mllltiplicity of 10. At the 8th min, the culture was chilled quickly and the cells mere harvested. Radioactive arninoacyl-&NA’s were prepared according to the method described in Materials and Methods. The TZ-infected sRNA was chromatogrephed on HAK column together with E. coli sRNA charged with zl different isotope from that of TZ-infected sRNA. The oonoentrstion of N&l in the starting buffer was 0.2 &I and in the final buffer was 1.1 M, except for leucyl-sRNA where the N&l gradient was from 0.35 to 0.8 SL
L
T2 8-min
I
T2/8-min
190
T. KANO-SUEOKA
AND
N. SUEOKA
glucose, and the next morning the cells were diluted 20-fold in a fresh medium. When the cell concentration reached 60 Klett u&s (approximately 4 x lo* cells/ml.), 6 pg/ml. of mitomycin C was added, and samples were taken at 0, lo,20 and 30 miu after the addition of mitomycin C for isolating sRNA. 46 min after the addition of mitomycin, phage h began to appear in the culture medium and by 90 to 100 mm the cells were lysed. The yield was about 40 to 50 phage particles/cell. 100 min after induction, the viable cell count was reduced to 3 x 1Oa cells/ml., while the control oulture (without induction) contained viable cells, 1 X 10e cells/ml.
3. Results (a) Comparison of aminoacyl-sRNA’s
before and after TZ infection
The aminoacyl-sRNA’s of normal E. wli B and of E. wli B infected with phage T2 were compared by chromatography on MAK columns. Seventeen aminoacyl-sRNA’s other than asparagyl-, glutamyl-, and cysteinyl-sRNA were examined. In only one case, leuoine, was there found a clear difference in profile between sRNA isolated from infected cells and from non-infected cells (Sueoka & Kane-Sueoka, 1964). Fifteen aminoacyl-sRNA’s were compared directly on the same column by charging sRNA’s prepared from cells before and after T2 infection with each amino acid labeled with a different isotope, l*C or 3H. Figure 1 summarizes the results. Two other aminoacylsRNA’s (threonyl and glutamyl) were also examined by comparing two separate chromatograms before and after T2 infection, but did not show appreciable alteration. Only leucyl-sRNA shows a clear difference in elution proties before and after the infection. Normal leucyl-sRNA of E. wli B shows two major peaks in MAK column chromatography, Leu I and Leu II, Leu I being the majority component. The RNA from cells infected with T2 for eight minutes shows that Leu I decreases to less than half the level in the non-infected case. This change of leucyl-sRNA upon T2 infection lies in aRKA and not in leucyl-sRXA synthetase (Sueoka & Kane-Sueoka, 1964). Some ambiguity remains concerning seryl- and isoleucyl- sRNA. Two preparations of T2 eight-minute infected t-RNA were examined for serine, and both showed a slight but consistent difference between the sRNA’s with and without infection. The profile of isoleucyl-sRNA of E. coli B is somewhat variable in different sRNA preparations for unknown reasons. Accordiugly, even though some discrepancy is found in the profile of isoleucyl-sRNA before and after the infection, its significance is not clear at the moment (Fig. 1). (b) &eci$city
of the alteration of leucyl-sRNA after T2 infection
The necessity of phage DNA and the requirement of protein synthesis for the alteration of leucyl-sRNA were shown previously (Sueoka & Kane-Sueoka, 1964). An RNase-less mutant (A19) and an RNase-less and polynucleotidephosphorylaseless mutant (Q13) were used as hosts for T2 infection and for examination of leucyl&WA. In both cases the normal alteration of leucyl-sRNA was observed (Fig. 2(a) and (b)). This excludes the possibility of the involvement of those two host enzymes in altering leucyl-sRNA. The ghost of phage T2 adsorbed on E. wli B did not induce the alteration of the leucyl-sRNA proGle (Sueoke & Kane-Sueoka, 1964). Killing the cells with colicin E2 also did not alter leuoyl-sRNA of 1. wli B (Fig. 2(c)). Cells were grown in Tris C medium at 37°C. When the density reached 6 x 10s cells/ml., the cells were washed once and resuspended in the same fresh medium. Then pu~%ed colicin E2 was added at a oonoentration of 7 x lo* molecules/cell and 16 minutes later the cells were bar-
MODIFICATION
OF LEUCYL-sRNA
04
191
T2 8-min sRNA (“Cl
I
1
I
3H “C
(b) 2
s;;
0"
-E
c3H)
0.4
rx 'G z -u 5 y
2
E. co/i B sRNA
I I
1 T28-min sRNA C4C)
0.2
’ I
”
I 7
t
: .t8' 60 I
I
70 I
1
3H 14C
(cl
I
A
Colicin-E
co/i 6
I
E. co//' B sRNA t3H)
600
100 ,O.D.
0
60 Fraction
70
00
no.
Fro. 2. Elution profiles of leucyl-sRNA. (a) E. wli A19, RNase-less mutant of E. cdi Hfr strain AB301 was used as the host for T2 infection. sRNA was isolated from the cells infected for 8 min. [i%]Leucyl-sRNA was prepared and compared with that of 1. coli on s MAE column. (b) E. coli 413, an RN&se-less and polynuoleotide phosphorylase-less mutant of E. wli Hfr Al3301 was used as host for T2 infection. Experimental conditions were the same as (a). (c) sRNA was taken from E. COWB cells exposed to colicin E2 for 16 min and its leucyl-sRNA was compsxed to that of E. cd B.
T. KANO-STJEOKA
192
AND
I
I
0.3-
N. SUEOKA
I
#:-E.co/i \
(b)
3”
sRNA-Ecoli-ent
T2 S-min
sRNA-E.co/i-
IO00
I I
0.5 0.4 -
(cl
,/
./-
)H
E.co// sRNA-~.co/~‘-enz.(~H)
~2 B-min
sRNA-Ecd-enr
“C
(“Cl
0 D.
400
/
0.3-
I 14r
('H)
600 300
‘\,
2oo--100 *-_ _ 60
FractKxlno. Fm.
3
- --
I
70
a-c.
0.6 i
Cd)
600
400
200
"0 0 0.6
0.4 T2 l-min
sRNA
0.2
(9) Edi
sRNA
T2 2-min sRNA (“Cly
40
50
60 Fraction no. ma.
70
3 (cont.) see next page
194
T. KANO-SUEOKA
AND
N. SUEOKA
vested to isolate sRNA. The survival of bacteria was 0.1’70~ after 15 minutes exposure to colicin E2. During their preparation, the T-even phage stocks had been treated with pancreatic RNase (5 pg/ml.) as well as with DNase. Although the chance seemed remote that a trace amount of RNase might remain in our phage stock to cause the alteration of leucyl-sRNA after infection, this possibility was also examined by preparing T2 stock without RNase treatment and infecting cells with this T2 stock. Leucyl-sRNA taken from such cells showed the same altered profile as that shown previously with RNase-treated phage stock. All the above results support the idea that this alteration is specific to T2 infection. (c) Time course of the alteration of leucyl-sRNA after T2 injection In the work previously reported (Sueoka & Kano-Sueoka, 1964), the time course of the change of leucyl-sRNA profiles was followed by preparing sRNA from the cells three, five and eight minutes after the infection. The elution profile of leucyl-sRNA from three-minute infected cells showed a new component in front of the Leu I peak not seen in the eight-minute sRNA (Fig. 3(a)). Five minutes after infection, the new component has increased, while the Leu I peak has decreased considerably (Fig. 3(b)). In the eight-minute infected sRNA the new front component has disappeared (Fig. 3(c)). The timing of the change in leucyl-sRNA profile, however, does not seem to be similar with different phage preparations and conditions of infection. Previously, the phage was prepared by acid precipitation from the lysate and differential centrifugation (Sueoka & Kano-Sueoka, 1964). The survival of bacteria three minutes after infecting E. coli B with this phage at the multiplicity of ten was 10m3to 3 x 10w2.A different method of T2 preparation, which avoided the acid-precipitation step (see Materials & Methods), gave a much higher e%ciency of infection (in the order of 10w4); and the synchrony of infection was good. With this new preparation of phage T2 the leucyl-sRNA alteration was found to be completed at a very early period of infection. Examples are shown in Fig. 3(d), (e), (f) and (g). 0 ne minute after infection, cells infected with one such stock of T2 gave an almost completely altered profile, i.e., like T2 eight-minute infected leucyl-sRNA (Fig. 3(e)). With another stock of T2, it gave an intermediary profile one minute after infection (Fig. 3(f)) and a completed profile two minutes after infection (Fig. 3(g)). The addition of glucose (0*2%) to the nutrient broth medium also seemed to accelerate the alteration of leucyl-sRNA after infection. To terminate the process of infection, the infected cells were routinely poured into a beaker containing plastic bags packed with crushed ice, which was immersed in ice water with salt, as described in Materials & Methods. It took about 30 seconds to cool one liter of the culture down to 10°C and about one minute down to 4 to 5°C. Accordingly, the timing we have described above is not exact. FIQ. 3. Leucyl-sRNA at different times after T2 infection. sRNA was prepared from the cells infected for various lengths of time, and also with different batches of T2 preparation. Charging was made by E. ooli enzyme. The profiles of the leucyl-sRNA were compared with that of E. wli B by eluting together on a MAK column. (a), (b) and (c) sRNA was taken from the cells infected with T2 for 3, 5 and 8 min. Phage T2 was prepared by aoid precipitation method. (d) and (e) Cells were infected with T2 prepared without acid-precipitation for 50 set and 1 min. (f) and (g) A different batch of the phage was prepared as in the case of(d) and (e). sRNA was taken from 1 min- and 2 min-infected cells.
MODIFICATION
195
OF LEUCYL-sRNA
1n order to stop the cell metabolism instantaneously at a desired moment, the infected cells were mixed rapidly with two volumes of cold lOOo/oethanol (- WC). The cells were centrifuged down and sRNA was isolated in the ordinary manner. Comparison of T2 one-minute and three-minute infected leucyl-sRNA isolated from cells treated with ice and with alcohol shows that there was little difference between the two sRNA preparations in the extent of leucyl-sRNA alteration. This implies that the process of leucyl-sRNA alteration is indeed one of the earliest steps in the infection process.
(d) Infection with ultraviolet light-irradiated
T2
According to Dirksen, Wiberg, Koerner & Buchanan (1960), the early phage specific protein synthesis upon the infection of E. coli by ultraviolet-irradiated
U.V. irradiated
-
.60
70 Fraction
T2
/ 80
I
no,
FIG 4. Leucyl-sRNA from E. coli B infected with ultraviolet light irradiated T2. The phage suspension was irradiated withultraviolet light up to the survival of the phage (8 x 10-a). E. co& cells were infected with the irradiated phage at the multiplicity of five. sRNA was taken from cells infected for 8 min and 15 min and charged with normal E. coli enzyme. (a) sRNA from 8 min infected cells with ultraviolet light-irradiated phage. (b) sRNA from 15 min infected cells with ultraviolet light-irradiated phage.
196
T. KANO-SUEOKA
AND
N. SUEOKA
phage does not stop, but continues. Moreover, contrary to the normal phage infection case, the bter protein synthesis is inhibited. If leucyl-sRNA alteration is responsible for the cessation of early protein synthesis, we may expect the absence of the alteration by ultraviolet-irradiated phage infection. The results of such experiments are given in Fig. 4. T2 were inactivated to the range where Dirksen et al. (1960) found the continuation of deoxycytidylate hydroxymethylase synthesis, and cells were infected with the irradiated phage at a multiplicity of five. The presence of normally altered leucylsRNA from cells infected with irradiated phage for eight minutes suggests that this is not responsible for the switch-off of early protein synthesis. This notion has been supported also by the fast timing of the change in leucyl-sRNA after infection. (e) Nature oj’ the alteration of leucyl-sRNA T2-infected sRNA was charged with radioactive leucine by yeast enzyme which was known to charge leucine only to the Leu I peak of E. coli sRNA. As shown
T2 5-min
sRNA-yeast
T2 5-min
\I XI \
X k
‘X
sRNA-
600400 sjt;
200 200 100
X..
Fraction
no.
FIQ. 6. Leuoyl-sRNA of TB-infected cells charged by yeast enzyme. Enzyme extract of yeast W&B freed from RNA by a DEAE-oellulose columu and used to charge radioactive leuciue to T2infected sRNA. The elution profile of the leucyl-sRNA was compared with that of T2-infected sRNA charged by 1. co& enzyme. (a) T2 3-min sRNA. (b) T2 6-min sRNA (Sueoka & Kane-Sueoka, 1964).
MODIFICATION
19;
OF LEUCYL-sRNA TABLE
1
Ratio of leucine to other an&w acids attached to sRNA before and after T2 infection E. coli aRNA ( l$$)
Ratio
T2 S-min sRNA ( $y;ti)
Ratio
0
Leu Ser
0.04 0.003
[“H]Leucine
5
Leu SEW
1.43 0.13
[l4C]Serine
10
Leu Ser
1.76 0.25
7.04
1.47 0.20
7.50
15
Leu SW
1.65 0.34
4.85
1.38 0.28
4*93
0
Leu Gly
0.03 0.004
[ 3H]Leuoine
5
Leu GUY
1.36 0.19
7.17
1.36 0.19
7.32
[r4]Glycine
10
Leu Q~Y
1.55 0.35
4.43
1.47 0.35
4.30
15
Leu GUY
1.54 0.47
3.28
1.54 0.47
3.28
0
Leu Val
0.07 0.28
[eH]Leucine
5
Leu Val
1.79 1.40
1.28
1.66 1.07
1.55
[14C]Valine
10
Leu Val
1.97 1.24
1.59
2.03 1.23
1.65
15
Leu Val
1.87 1.31
1.43
1.81 1.13
1.60
0
Leu Phe
0.06 0.008
[eH]Leucine
5
Leu Phe
1.87 0.04
44.5
1.84 0.04
46.7
[14~~jlhe~yl-
10
Phe Leu
0.08 2.16
27.7
0.07 1.93
20.5
15
Leu Phe
2.25 0.100
22.5
2.04 0.08
25.2
0
LYS Leu
0.03 0.007
5
Lys Leu
1.53 0.63
2.43
1.56 O-68
2.30
10
LYS Leu
3.00 0.95
3.16
3.36 1.09
3.08
15
LYS Leu
3.79 l-04
3.64
3.60 1.03
3.50
[sH]Lysine
[14C]Leucine
0.03 0.003 11.0
1.26 0.10
12.6
0.03 0.003
0.07 0.32
0.06 0.008
0.01 0.002
The amount of ammo acids attached to sRNA is expressed as pmoles/lOO ratios of lsucine to the other 5 amino acids was calculated for every sample.
pg sRNA
and the
198
T. KANO-SUEOKA
AND
N. SUEOKA
in Fig. 5, in addition to the Leu I peak, the new front component and a part of the Leu II region were also charged with leucine. This indicates that two kinds of new leucyl-sRNA components (Leu F and Leu R, Fig. 11) are closely related to Leu I (Sueoka & Kano-Sueoka, 1964). In order to find out whether there is a sizable amount of new synthesis or degradation of leucyl-sRNA after the infection, the ratios between leucine and other amino acids attached to sRNA before and after the infection were compared. E. coli sRNA and T2 eight-minute infected sRNA were charged with radioactive valine, phenylalanine, lysine, serine and glycine; each reaction mixture also contained radioactive leucine labeled with a different radioisotope (14C or 3H) as a direct reference. As shown in Table 1, there is little difference in the ratio of leucine to other ammo acids attached before and after the infection. Two possibilities exist as to the cause of the alteration of leucyl-sRNA profile after T2 infection: one, the synthesis of one or more new sRNA’s; the other, the partial degradation or modification of one or more pre-existing leucyl-sRNA’s eluted at the Leu I peak. The fact that both new components of leucyl-sRNA (front and rear) after T2 infection can be charged by yeast enzyme (Fig. 5) and that the relative amount of leucyl-sRNA to other aminoacyl-sRNA’s does not appreciably change after T2 infection (Table 1) renders the modification of pre-existing leucyl-sRNA (some of Leu I) more likely. Another possibility, although unlikely, is that equivalent amounts of degradation of pre-existent host sRNA and synthesis of new sRNA occur simultaneously. A unique characteristic of the front component (Leu F) is its loss of amino acidaccepting capacity once the leucine is discharged by high pH. TZ-infected sRNA which contained the front component was treated in 0.5 M-Tris-HCl (pH 8.8) for 45 I
I
I
I
v 1%
;i
sc
400
+ z
+J z. 06 “E Iv
T2 3-min
sRNA (‘4C)
300
:z 4.J :: 2
0
; .+
0.4
200
0”
@2
100
Fraction
no.
FIG. 6. Stability of the front component of T2 leucyl-sRNA. An sRNA preparation which contained the front leucyl-sRNA component was prepared by infecting E. co&i B with T2 at 37% under slow shaking of the culture. The sRNA was treated in 0.5 M-Tris-HC1 buffer (pH 8.8) for 45 min at 37”C, reprecipitated with alcohol twice to remove any free amino acid and recharged with [Wlleucine. This was compared with the same sRNA but non-treated.
MODIFICATION
OF LEUCYL-sRNd
19!J
minutes at 37”C, reprecipitated and recharged with radioactive leucine. This RNA was compared directly on a MAK column with the same sRNA without the high pH treatment. The front component was not detected after the alkaline treatment whereas the other component (peaks I and II) remained the same (Fig. 6). This loss of rechargeability by high pH was also confirmed by isolating the Leu F first, and then treating with high pH and trying to recharge with radioactive leucine. It may be that either the structure of the sRNA of the front component is unstable or the intact primary structure of the sRNA is lost, so that by raising the pH it may have disintegrated. The possibility of recharging with other amino acids has not been examined. (f) Leucyl-sRNA
of various hosts infected with T2
Leucyl-sRNA for the following strains of E. coli were compared for the MAK column profile with E. coli B, E. coli C, E. coli K12(/\), and E. coli CR63. Within the resolving power of the column, the profiles were very similar to that of E. coli I3 (Fig. 7). Leucyl-sRNA was also compared after T2 infection on those different hosts, which showed profiles of leucyl-sRNA very similar to those of infected E. coli B (Fig. 7). The case of Shigella dysenteriae 60 is of special interest. The elution pattern of leucyl-sRNA from Shigella 60 is different from the profile of E. coli sRNA. At first glance, Shigella leucyl-sRNA does not seem to have components corresponding to Lcu I of E. coli (Fig. 8). However, when Shigella 60 was infected with T2, and its leucyl-sRNA compared with the non-infected case, the difference between the two was clear. On a MAK column profile the front portion of leucyl-sRNA disappeared, while the rear portion seemed to increase eight minutes after T2 infection. Moreover, the leucyl-sRNA profile of Shigellu charged with yeast enzyme occupied the front part of the normal profile charged by E. coli enzyme (Fig. 8). This implies that although the elution characteristic is distinct, Shigella has species of leucyl-sRNA corresponding to Leu I of E. coli which was modified after T2 infection. Therefore, in principle, T2 exerts the same effect on Shigella leucyl-sRNA as on E. coli. (8) Leucyl-sRNA
of E. coli B infected with various $qes
Leucyl-sRNA’s from E. coli B after infection with various T phages and from E. coli K12(/\) during the induction of h were examined. Alteration similar to the case of T2 infection was observed with T4 and T6 infection, which may be expected by the closeness of T4 and T6 to T2 (Fig. 9). On the other hand, infection with T-odd phages, Tl, T3, T5 and T7 did not show the alteration (Fig. 9). Samples of sRNA from T-odd phage-infected cells taken at periods of infection different from the ones shown in Fig. 9 (five-minute infected sRNA for T3, lo-minute infected sRNA for T5 and seven-minute infected sRNA for T7) have also been analyzed. No alteration of leucyl-sRNA was detected. Phage X was induced by an addition Pm. 7. Leucyl-sRNA of various hosts before and after infection with T2. Leucyl-sRNA’s of E. COGC, E. coli K12(X) and E. COG CR63 were compared with that of E. coli B. (a) E. coli C; (b) E. coli K12(A); (c) E. coli CR63. The three strains of E. coli were infected with T2 and sRNA was isolated from cells 8 min after infection. Each leucyl-sRNA preparation was compared with that of its own host cells. (d) T2 S-mm E. wli; (e) T2 S-min E. coli K12(A); (f) T2 S-min E. coli CR63. see next page
R
sRNA t
(14C) C3H)
600
3t 1000
(bf
K12 (A)
sRNA
c3H)
800 E. co/i
B sRNA
400
4C
600
(‘4C) -
500 600 400 400
300 200
200 100
3H
(cl
14C
600 E. co/i
B sRNA c3H)
- 200
200
50
60
70 Fraction
no.
Fra. 5
80
90
- 100
3ki
Cd)
"C
E co/i C sRNA (3H) Ob-
403 T2
8-min
E.co/~
C
203 02-
5n
60
70
80
3H
(4
i
.f
i
600
0. T2
k
400
8-min
-300 0 200 0.1
100
80
70
60
90 ‘C
CR 63 sRNA (3H) r*, ’ ? T2
CR 63 sRNA
8-min
600.
1
/ 300
150
40
50
60 Fraction
Frc.
i
70 no.
(cont.)
F ;
,x :z z :: 0 cz
E,co/i 6
sRNA
t3H)
T2 8-min
Shigella
E. co/i,enzyme
..
_. . . . 50
60
70
Shlgelia 60 sRNA (14C)
yeast
Fraction
FIG. 8
no.
enzyme
ci4C)
80
sRNA ,nn
MODIFICATION
OF LEUCYL-sRNA
203
of mitomycin C (5 pg/ml.) to E. coli K12(A) culture and sRNA was isolated from the cells 10 minutes, 20 minutes and 30 minutes after the addition of mitomycin C. Here again, the alteration was not observed. (h) Leucyl-sRNA
after infection
with various amber mutants
-4mber mutants of T4 infecting non-permissive host (E. coli B) are of particular interest because of their known phenotypic effects and arrangements of the genes along the chromosome. If the alteration of leucyl-sRNA were due to the action of a phage-specific enzyme and essential for the phage development, it should be possible to locate the genes for this protein, which could be an amber-type mutant. Several amber mutants for early functions were obtained from Dr R. S. Edgar, and the MAK column profiles of leucyl-sRNA were compared before and after the infection on non-permissive host. All mutants so far examined have given normal modified profiles like that of the T4 wild type, except that some mutants, gene no. 39, 52 and 57, still had a small amount of the front component eight minutes after infection. However, there seems to be no correlation between characteristics of the mutants and the presence of the front component. A summary of the characteristics of the mutants examined is shown in Fig. 10.
4. Discussion The degeneracy of the code has been established now beyond a reasonable doubt (Nirenberg et al., 1963; Speyer et al., 1963; So11 et al., 1965). The corresponding degeneracy (heterogeneity) in sRNA has been reported in leucyl-sRNA (Weisblum, Benzer & Holley, 1962; Bennett, Goldstein & Lipmann, 1963; Yamane, Cheng & Sueoka, 1963; Leder & Nirenberg, 1964; S6ll et al., 1965), although its applicability for other amino acids is not yet clear. When there is degeneracy in sRNA of an amino acid, the modification of one of the synonymous sRNA’s may eliminate the adaptor of the corresponding codon and prevent the translation of mRNA of genes which contain the codon, but not of those which do not contain the codon. If the secondary structure of sRNA is modified so that a different part of the molecule now becomes the new codon-recognizing site, i.e.; the anticodon is changed, then the translation of various mRNA’s should be disturbed. Another possible outcome of the adaptor modification is its change in the site of the aminoacyl-sRNA synthetase recognition, which may lead to loss of amino acid acceptor activity of sRNA or to a change of the kind of amino acid accepted. Also, the possibility of activation of cryptic (non-functional) sRNA should not be ignored. Our work on E. coli sRNA after phage infection is directed to exploring the possibility of adaptor modification being involved in cell differentiation through a change of the code translation system.
FIG. 8. Leucyl-sRNA of Shigella o!yse?ttertie 60. (a) sRNA from Shigella dyeenteriae 60 was charged with [W]leucine by E. coli enzyme and compared on the MAK column with leucyl-sRNA of E. coli. (b) ShigeZZa leucyl-sRNA was compared with that taken from ShigeZZa cells infected with T2 for 8 min. (c) ShigeZZa leucyl-sRNA charged with E. coli B enzyme was compared with that charged with yeast enzyme.
.5
50 T4
I
50
60
I x-x
70
1 : ,/ :yE.
60
I
80
co/i sRNA c3H)
I
90
80
T4 8-min sRNA (‘4C)
70
,200
500
3 'C
I ooo-
soo250
0
0
FIR. 9
Fraction no.
Tl
I
I
E. co//’ B sRNA (‘HI
50
I
I
70
3H “C
300-
;; 0 2
600 Lz
‘H “C
400
60
I
60
:4
I
I
70
200
40 I
60
1.2
T3
50
a
206
T. KANO-SUEOKA
AND
N. SUEOKA
The experimental data presented in this paper establish the following facts, (1) Infection of E. wli B with T-even phages leads to a structural modification of some synonymous component of leucyl-sRNA belonging to Leu I of the MAK column proiiles. T-odd phages and X phage do not bring about the modification. Leucyl-sRNA synthetase is normal after phage infection and has nothing to do with the leucyl-sRNA modification. (2) E. wli strains B, C, K12(h), CR63, A19, Q13, and Shigellu dysenteriae 60 show essentially the same modification of leucyl-sRNA upon T2 infection. (3) The modification of leucyl-sRNA has two steps, a summary of which is shown diagrammatically in Fig. 11. (4) The modification is completed shortly after the infection (one to three minutes). Ultraviolet irradiation of phage T2, which prevents the cessation of early enzyme synthesis, does not affect the modification.
rI1 I 00152
FIG. 10. Leucyl-sRNA of amber mutants of phage T4. The map positions of the amber mutants and the nature of mutants which have been analyzed for leucyl-sRNA are indicated on the genetic map of phage T4 according to Epstein et al. (1963). Abbreviations: DA, DNA synthesis is initiated but ceases after a short interval; DO, no detectable DNA synthesis occurs; DD, there is a delay in the onset of DNA synthesis after which DNA synthesis proceeds normally; MD, DNA synthesis appears to be normal, but there seems to be a general defect in maturation. E. coli B (non-permissive host) was infected with amber mutants and sRNA’s were taken 8 min after infection. The elution profiles of leucyl-sRNA were compared with that of E. coli B.
FIG. 9. Leucyl-sRNA of E. coli infected with various phages. Phages T2, T4 and T6 were added to E. coli B cultures at a multiplicity of 10 and sRNA’s were isolated from 8-min infected cells. E.. coli K12(X) was grown in e, nutrient broth medium (plus 0.2% glucose) and mitomycin C (5 pg/ml.) was added to the exponential-phase cells. The sRNA taken from zero-min and 20-min induced cells was analyzed. Phage Tl, T3 and T7 were added to E. coli B cultures at a multiplicity of 10 and sRNA was isolated 8 min later for Tl and T3 infections and 5 min later for T7 infection. E. coli F and phage T5 st were used to obtain fast infection by the phage. The sRNA was taken from cells infected for 30 min and charged with E. co.% enzyme. The profile of leucyl-sRNA of T&infected 1. COGF was compared with that of E. co& B and not with that of E. coli F. However, the leucyl-sRNA profiles of E. coli B and F have been shown to be identical.
MODIFICATION
OF LEUCYL-sRNA
207
(5) T2 ghost and colicin E2 do not induce the modification. Inhibition of protein synthesis by chloramphenicol abolishes the modification by T2 infection. (6) Alanyl-, arginyl-, aspartyl-, glycyl-, histidyl-, lysyl-, methionyl-, phenylalanyl-, prolyl-, tryptophanyl-, tyrosyl-, and valyl-sRNA’s of E. coli B are not altered upon T2 infection, judging from the direct comparison on the column using 14C-3H doublelabeling technique. Isoleucyl- and seryl-sRNA may show a small change, which cannot be definitely established at the moment. Glutamyl- and threonyl-sRNA are not altered by comparison of separate profiles before and after infection. Cysteinyl-, asparaginyl- and glutaminyl-sRNA await further analysis. The present results indicate that the involvement of the modification of leucylsRNA in the transition of the early phase to the late phase of phage protein synthesis is unlikely. This was raised as one of the possibilities (Sueoka & Kane-Sueoka, 1964). Early timing of the completion of the modification has now been established by quickly stopping the metabolism of phage-infected cells with alcohol. Ultraviolet irradiated T2 and amber mutants of T4 which lack early function, both of which
Time 2
Time 3
pi&D&
FIG. 11. Schematic presentation of the time course of leucyl-sRNA modification after T2 infection. Solid line represents leuoyl-sRNA charged with E. coli B enzyme and broken line represents leucyl-sRNA charged with yeast enzyme.
208
T. KANO-SUEOKA
AND
N. SUEOKA
allow the continuation of early enzyme synthesis and inhibit the late protein synthesis, showed a normal alteration upon their infection on E. wli B. The possibility that the leucyl-sRNA change may take 8ome role in the arrest of host-protein synthesis is not excluded. As for the chemical nature of the modification, we have no answer at the moment. There are several possibilities: (1) rearrangement of secondary structure; (2) methylation or demethylation; (3) partial enzymic digestion (Niahimura & Novelli, 1964); (4) oxidation and reduction of thiopyrimidine (Carbon, Hung & Jones, 1965; Lipsett, 1965). It has been reported that the distribution of methylated bases of 8RNA is different before and after T2 infection (Wainfan, Srinivasan & Borek, 1965). In vitro methylation of E. coli sRNA with T2 infected cell extract was carried out in collaboration with Dr Borek’s laboratory. The result so far obtained is negative. The direct test on the triplet response of the modified leucyl-8RNA and the effect on in citro protein synthesis in E. coli are being investigated. Other example8 of 8RNA modification have been observed in valyl-sRNA during spore formation (Kaneko & Doi, 1966), and in tyrosyl-sRNA during spore germination (Sueoka, unpublished results) of Bacillus subtilis. There is also a report by Subak-Sharpe & Hay (1965) claiming that in the presence of herpes virus, the synthesis of virus-specific sRNA is initiated in BHK 21 cells.
We thank Dr A. D. Hershey for his valuable suggestion on the purification of T-even phages. We also acknowledge the excellent technical assistance of Mrs J. V. Taub. This work was supported by grants from the National Institutes of Health, GM10923-04 and the National Science Foundation, GB3445.
REFERENCES Bennett, T. P., Goldstein, J. & Lipmann, F. (1963). Proc. Nat. Acad. Sci., Wash. 49, 850. Berg, P., Bergmann, F. H., Ofengand, E. J. & Dieckmann, M. (1961). J. Biol. Chem. 236, 1726. Carbon, J. A., Hung. L. C Jones, D. S. (1965). Proc. Nut. Acad. Sci., Wash. 53,979. Champe, S. P. (1963). Ann. Rev. Microbial. p. 205. Cohen, S. S. (1949). Bad. Rev. 13, 1. Cohen, S. S. (1963). Ann. Rev. Biochem. 32, 83. Dirksen, M., Wiberg, J. S., Koerner, J. F. & Buchanan, J. M. (1960). I-‘roc. Nut. Acad. Sci., Wash. 46, 1425. Epstein, R. H., Bolle, A., Steinberg, C. M., Kellenberger, E., Boy de la Tour, E., Chevalley, R., Edgar, R. S., Susman, M., Denhardt, G. H. & Lielausis, A. (1963). Cold Spr. Harb. Symp. Quant. Biol. 28, 375. Ehrenstein, G. von & Lipmann, F. (1961). Proc. Nat. Acad. Sci., Wash. 47, 941. Gesteland, R. F. (1965). Fed. Proc. 24, 293. Kaneko, I. & Doi, R. (1966). Proc. Nut. Acud. Sci., Wa-sh. 55, 564. Leder, P. & Nirenberg, M. W. (1964). Proc. Nat. Acad. Sk., Wash. 52, 1521. Lipsett, M. N. (1965). Fed. Proc. 24, 216. Luria, S. E. (1962). Ann. Rev. Microbial. p. 87. Nirenberg, M. W., Jones, 0. W., Leder, P., Clark, B. F. C., Sly, W. S. & Pestka, S. (1963). Cold Spr. Harb. Symp. Quant. Bill. 28, 549. Nishimura, S. & Novelli, G. D. (1964). Biochim. biophys. Act+ 80, 574. Otsuji, N., Sekiguohi, M., Iijima, T. & Takagi, Y. (1959). Nature, 184, 1079. Roberts, R. B., Abelson, P. H., Cowie, D. B., Bolton, B. T. & Britten, R. J. (1957). Pub.?. Cameg. In&n. Washington, no. 607, p. 5. S811,D., Ohtsuka, E., Jones, D. S., Lohrmann, R., Hayatsu, H., Nishimura, S. & Khorana, H. G. (1965). Proc. Nat. Acud. Sk., Wash. 54, 1378.
MODIFICATION Speyer, J. F., Lengyel, Cold Spr. Had.
OF LEUCYL-sRNA
“09
P., Basilio, C., Wahba, A. J., Gardner, R. S. & Ochoa, S. (1963).
Symp.
Quant.
Biol.
28, 559.
Subak-Sharpe I%Hay, J. (1966). J. Mol. Biol. 12, 924. Sueoka, N. & Cheng, T. Y. (1962). J. Mol. Biol. 4, 161. Sueoka, N. 85 Kano-Sueoka, T. (1964). Proc. Nat. Acad. Sci., lFa.&. 52, 1535. Sueoka, N. & Kano-Sueoka, T. (1965). In Developmental and Metabolic Control Mechanisms und Neopkuia, p. 114. Baltimore: Williams & Wilkins Company. Suooka, N. & Yamane, T. (1962). Proc. Nat. Acad. Sci., Wash. 48, 1454. \Vainfan, E., Srinivasan, P. R. & Borek, E. (1965). Biochemistry, 4, 2845. \Veisblum, B., Benzer, S. & Holley, R. W. (1962). Proc. Nat. Acad. Sci., Wash. 48, 1449. A-amane, T., Cheng, T. Y. & Suoeka, N. (1963). Cold Spr. Harb. Symp. Quant. Biol. 28, 569. Yamane, T. & Sueoka, N. (1963). Proc. Nat. Acad. Sci., Wash. 50, 1093.