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Effects of amino acid starvation on constitutive tryptophan messenger ribonucleic acid synthesis
J. Mol. Bid. (1970) 51, 717-726
Effects of Amino Acid Starvation on Constitutive Tryptophan Messenger Ribonucleic Acid Synthesis stringent (reZ+) and R-&p relaxed (Tel-) strains of EscMh In both R-trp coli, starvation for arginine, leucine, histidine or isoleucine resulted in a marked decrease in the level of tryptophan messenger RNA es aesayed by competitive hybridization experiments with DNA from a non-defective tryptophan transducing phage (+3Opt190). Using the level of tryptophan messenger RNA in a wild-type genetically repressed strain as a measure, this was found to be reduced from roughly ten times the repressed level to 1.5 times this level in the case of starvation for arginine, leucine or isoleucine and three times this amount in the case of histidine starvation. Tryptophsn messenger RNA synthesis, as reflected in the rate of labeling by [3H]uridine, is also greatly decreased by amino acid starvation in both strains. The results show that tryptophan messenger RNA is subject to a type of ammo acid control distinct from the rel+ (RCatr) system described by Stent & Brenner (1961).
pair of Escherichia: coli strains which carried either the stringent (reZ+)t or relaxed (reE-) allele but were otherwise isogenic (arg, his, Zeu, thi, R-.&p), it was previously found that arginine starvation greatly decreased both the level of tryptophan messenger RNA and its rate of synthesis in both the stringent and relaxed strains (Stubbs t Hall, 19683). In view of the possibility of a generalized model for the regulation of RNA synthesis associated with an understanding of the effects of the rel allele (Cashel & Gallant, 1969; Fiil, 1969) and the ambiguity concerning the effects of the rel allele on messenger RNA synthesis (Friesen, 1969; see review by Edlin & Broda, 1968), it was of interest to determine whether the effects we had previously observed on Trp-mRNA were unique to arginine starvation or held for a variety of amino acids. This paper presents the effects on Trp-mRNA resulting from starvation of the trp-constitutive rel+ rel- pair of E. coli strains for histidine, leucine and isoleuoine. The construction and characteristics of the R-trp CP79 (rel-) and R-&p CP78 (rel+) pair of E. coli strains, the isolation of purified [3H]Trp-mRNA, the isolation of non-radioactive competing RNA and pulse-labeled total RNA, and the techniques of the competition experiments and direct DNA-RNA hybridization experiments with $80 DNA and $80 pt190 DNA (480 DNA containing the tryptophan operon) are described in detail in Stubbs & Hall (1968aJ). Some pertinent points for this discussion are as follows. The R-trp rel+ strain requires arginine, histidine, threonine, leucine and thiamin for growth. The requirements of the R-trp rel- are identical with the In an auxotrophic
t Abbreviations used: rel+ (formerly BF’) for the stringent allele in 1. COG strain R-&p CP78 aud reZ- (formerly RCF) for the relaxed allele in 1. c& strain R-trp CP79 (Stubbs & Hall, 196%); arg, hG, Zeu, th%, requirement for arginine, histidiue, leucine and thiamin; R-trp, mutation in the R-trp allele resulting in constitutive synthesis of tryptophan messenger RNA; [3H]Trp.mRNA, [3H]uridine-labeled, pursed tryptophan messenger RNA preparation (Stubbe & Hall, 196&x); +8OpU90 DNA, phage 980 DNA oonteining the oomplete tryptophan operon from a non-defective tryptophan-tranaduoing derivative of phage 480. 717
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exception that it is thr+ . Apparently, in constructing this strain with Plkc, thr+ was introduced from the trp-constitutive donor strain into CP79 by co-transduction with the closely linked R-&p marker. Both strains are judged to be constitutive since the specifio activity of tryptophan synthetase A protein is 60-fold higher than in the repressed wild type (Ymel) and is unaffected by extreme changes (0 to 60 pg/ml.) in the tryptophsn concentration of the growth medium. The purified [3H]Trp-mRNA preparation used in these experiments hybridizes specifically with @Opt190 DNA to
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FIU. 1. Uptake of [sH]uridine in R-trp reE+ and R-trp rd- straim. Bacteria wars grown in minimal medium supplemented with 60 pg arginine/ml., 40 pg histidinel ml., 40 H leuoine/ml., 40 pg isoleuoine/ml. (Alfijldi, Stsnt, Hoogs & Hill, 1903), 200 ccg threonine/ ml., 20 erg t,ryptophan/ml., 2 w thiamin/ml. and 0.25% glucose (complete medium) to a density of 6.4 x 10s baoteria/ml. The cells were oollsoted by fltration onto a Milhpore filter, washed with an equal volume of oompleta medium minus histidine and glucose at 37”C, and resuspended in the same medium. The suspension of eaoh strain was divided into two portions. One portion was (70 m~oles/ml., supplemented with histidine (40 &ml.), gl uoose (0*26%), and [3H]uridine O-4 &nl.). The measured speoifio radioactivity of the [sH]uridine supplement was 3*4x lo6 ots/min/m~ole.) The other portion was supplemented only with gluoose and [sH]uridine. Aeration was begun immediately at 0 min. O-l-ml. samples were removed at intervals, placed in cold 10% triohloroacetio aoid with oarrier bovine serum albumin, and the resulting precipitats was oolleated on glass-fiber f%lt.ers. The radioactivity in the dried samples was determined in a liquid-scintillation speotrometer. -+-+-, R-trp rel+ plus hi&dine; -X-X-, R-trp reE+ minus histidine; ---e---a---, R-trp rel- minus histidine. ---O---O---, R-trp reZ- plus histidine;
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the extent of 66% of the input radioactively labeled RNA at saturating concentrations of DNA, whereas 5% is bound to $80 DNA. This is in good agreement with our previous observations. The R-trp pair were tested for rel+/reE- phenotype by measuring their ability to incorporate [3H]uridine in the presence or absence of various amino acids. A representative result (for histidine starvation) is given in Figure 1. Table 1 gives a complete TABLE
rel-/rel+ uultnre I
str8int
Phnotype of R-Trp
.
medim@
rel+
+
+Wl+ mlrelrel+ fez+ rdretrel+ re1+ rdrelrel+ ret!+ rdTea-
- His + - His + -As + - b + - Leu, -1le + - La, - 110 + t-w + Val (400 /&g/ml.) + t-w + Val(400 pg/ml.)
1
R*tio upt8ke 15 mill
[3H]uridine (non-starved) ’ (stied) 30 mill
I 7.3
8.5
1.16
1.32
z 7.50
9.25
> O-96 < 15.0 z
1.07
.
0.97
14.9 l-19
3.56
5.79
1.06
1.03
pair Cell density (beoteria/ml.) ( x 108) 60 min 0 min 6.37 6.37 6.25 6.25 54 5.5 5.67 5.67 6.63 6.63 5.17 5.17 6.25 6.25 6.37 6.37
11.1 6.63 Il.5 7.1 9.34 6.12 9-82 6.37 12.8 7.0 10.34 6.12 10.0 7.2 11.2 8.6
t rd+ indic.stas R-Trp CP78 (stringent); rel- indicstas R-Trp CP79 (relaxed). $ A + in this column indicates thet portion of the washed cells grown in complete medium that was supplemented with the tie aoid of which the other portion was deprived. Experimental conditions for each amino aoid are analogous to those desoribed in Fig. 1, with the exception of the isoleuoinestarvation imposedby exoeaa valine addition. In this oese, emh culture wea simply divided into two portions, and [*H]uridine added to one portion at zero time whereas C3H]uridine plus 400 ccg v8line/ml. wa8 added to the other. 8 Trichloroaoetio mid-pmoipitclble ots/min in 0.1 ml. of oomplete medium divided by trichloroaoetic said-preoipitable ota/min in 0.1 ml. of starvation medium at 15 end 30 min after addition of r3H]uridine.
summary for all the amino acids tested. If it is assumed that the rate of uridine uptake reflects the rate of net RNA synthesis, these results indicate that the pair of strains exhibits the Tel+/tel- phenotype in response to the amino acids used. The least pronounced effect was obtained in the attempt to starve indirectly for isoleucine by addition of valine. In the relaxed strain the amount of uridine uptake at 15 minutes was approximately the same in the starved cultures compared to the non-starved cultures for all amino acids tested, although by 30 minutes the uptake in the starved cultures had dropped off in some cases. Because the tryptophan biosynthetic enzymes are permanently derepressed in
J. D. STUBBS
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I 0
I too
I 200 Unlabeled
AND
I 300 competing
E. A. STUBBS
I 400
I 500
RNA/reaction
I 600 @g)
FIU. 2. Competition between RNA from non-starved and histidine-starved R-&-p ml+ or R-trp rel- strains of E. coli and [sH]Trp-mRNA for binding sites on @Opt190 DNA. For each point, hybridization was carried out using approximately 0.2 pg 14C-labeled 48Opt190 DNA immoblized on a nitrocellulose titer membrane disc (Schleicher & Sohuell, B0, cut to 22.5 mm2 area), a saturating amount (13,600 cts/min) of r3H)Trp-mRNA, and the indicated amount of unlabeled RNA. Total volume of each incubation mixture was 0.25 ml. in a solution of 0.5 MKC1 plus 0.01 M-Tris-chloride, pH 7.3. Incubation was carried out at 67°C for 16 hr. Filters were rinsed in 67°C solutions (0.5 w-KC1 plus 0.01 ad-Tris, pH 7.3) and washed on both faces by suction were determined by liquid-scintillation filtration. After thorough drying, 3H and 14C radioaotivities oounting with appropriate correction for 3H and W overlap. The corrected l*C activity for each filter was used to normalize the corrected 3H activity to a DNA content of 0.2 pg/tilter disc. (The variable content of DNA, 0.2 & 0.03 pg/fdter, is attributable to variation in initial loading. Less than 5% of the DNA was lost from the discs during hybridization.) At zero concentration of competing RNA, the ots/min of the saturating level of [“H]Trp-mRNA bound to 480ptl90 DNA varied from 7 to 8% of the input radioactivity, whereas with +SO DNA less than 0.5% bound. 100% hybridization on the ordinate represents the ots/min bound specifically to 480@190 DNA at zero concentration of oompeting RNA. m--, RNA prepared from R-trp rel- grown in complete medium (Fig. 1) to a cell -o---density of 6 x loo/ml. ; - A-A-, RNA from R-trp Tel+ grown in complete medium to a cell density of 5 x 10B/ml. ; - 0 0 -, RNA prepared from R-h-p rel - grown in complete medium to a oell density of 6 x lO*/ml., filtered and washed as in Fig. 1, and aerated in complete medium minus histidine for 15 min; -A--A-, RNA prepared from R-&v reZ+ grown in complete medium to a cell density of 5 x 10B/ml., filtered and washed as in Fig. 1, and aerated in complete medium minus histidine for 16 min. -X -X -, RNA prepared from Ymel (wild type R+ trp) grown in minimal medium supplemented with 0.1% Casamino acids, 0.26% glucose, and 20 pg tryptophan/ml. to a cell density of 6 x 10e/ml.
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Unlabeled competing RNA/reaction
721
(pg)
Era. 3. Competing ability of RNA from non-starved end hi&line-starved and R-trp CP79 (rel-) for tryptophan operon-specific hybridization. Same date and symbols as in Fig. 2 plotted as described in text.
R-trp
CP78 (rd+)
R-Trp CP78 and R- Trp CP79 and these strains show the rel- Jrel+ phenotype in response to plus or minus histidine, arginine, leucine or isoleucine, these bacteria provide an ideal system for assessing the effects of amino acid starvation on constitutive Trp-mRNA synthesis. RNA preparations were isolated from each strain, grown either in complete medium or under conditions of amino starvation. Detailed conditions are given in the legend of Table 2. The content of Trp-mRNA in each preparation was assayed by competitive RNA-DNA hybridization. Figure 2 shows the competition curves obtained for the RNA preparations from relaxed and stringent strains grown in complete medium or starved of histidine for 16 minutes. The data for wild-type E. WU (R+Tq) grown in repressing levels of tryptophan is idudeafor comparison. Since the labeled RNA hybridized decreases monotonically, but not linearly, with increasing concentration of unlabeled RNA competitor, it is convenient to represent the data as a plot of (&” - &“)I& versus unlabeled competitor added. &” is the sH radioactivity bound to DNA when no competitor is present, and &” is the radioectivity bound at unlabeled competitor concentration c. This converts the data to a linear form in which the slope provides a direct measure of the Trp-mRNA content of the unlabeled RNA preparation (Stubbs & Hall, 1968a). The data represented in this fashion are shown in Figure 3. Table 2 contains a compilation of all the competition data for the various amino acid-starvation conditions represented as the slopes of plots similar to those in Figure 3. This Table also includes the data from an RNA preparation of E. wli strain T,RY, a derivtdive of Ymel in which the entire trp operon has been deleted (Stubbs & Hall, 1968a). The competition power of this preparation which contains no Trp-mRNA indicates the lower limit of detection associated with our technique and allows the conclusion that competition from outside the trp region does not signifkantly affect the results. When either the stringent or
722
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relaxed strain was grown in complete medium, the Trp-mRNA level was high, competition slopes relative to RNA from repressed wild type being 7.4 and 11.0, respectively. The reason for the decreased level of Trp-mRNA in the stringent compared to the relaxed strain is not clear but is in agreement with our previous results. Starvation for arginine, histidine, leucine or isoleucine decreased the level of Trp-mRNA in both the stringent and relaxed strains. As seen from Table 2, the amount of this decrease varies according to the starvation conditions. If it can be assumed that the competing ability of an unlabeled RNA preparation is directly proportional to its Trp-mRNA content, as is indicated by the competing ability of trp deletion RNA from strain T,RY, then the results indicate that starvation for arginine and leucine decreases the level of Trp-mRNA to approximately l-5 times that present in a repressed strain. This same decrease is observed in our isoleucine starvation conditions for R- Trp CP79. The higher value obtained for R - Trp CP78 with valine addition might well be a reflection of the relative ineffectiveness in provoking stringency of net RNA synthesis in this strain by this technique as is indicated in Table 1. Histidine starvation results in a Trp-mRNA decrease to approximately three times that present in the repressed wild-type strain. These results indicate that the effects on constitutive Trp-mRNA synthesis previously observed for arginine starvation are not unique and support the hypothesis of a definite amino acid control of Trp-mRNA level apparently unrelated to the rel +-reZ- control of net RNAsynthesis. The possibility that the observed decrease in the level of Trp-mRNA was a consequence of an increased rate of messenger RNA degradation was ruled out in the case of arginine starvation by a series of RNA pulse-labeling experiments (Stubbs & Hall, 19683). Analogous experiments were performed, and are reported here, for leucine starvation in order to ascertain whether the decreased level of Trp-mRNA obtained in this case was also due to amino acid starvation causing a decreased rate of constitutive Trp-mRNA synthesis. Short-term [3H]uridine labeling of RNA was carried out in R-Trp CP78 and R-TV CP79 cultures, both in complete medium and in complete medium minus leucine and isoleucine (seelegend to Table 3 for procedure). Total incorporation of [3H]uridine into RNA was measured on a portion of the culture for each of the four cases (Table 3, column 2). These data, together with the specific activities of the purified RNA preparations, affirm that total RNA incorporation was stringent for R-Trp CP78 and relaxed for R-Trp CP79 within these experiments. Specific hybridization of each labeled RNA preparation with $8Optl90 DNA under conditions>f DNA excess provides an estimate of the proportion of [3H]uridinewhich entired Trp-mRNA during the pulses (Table 3, column 4). A pronounced effect of leucine starvation is evident. As previously discussed (Stubbs & Hall, 1968b), a more significant measure of Trp-mRNA synthesis is obtained by multiplying the percentage hybridization by the total incorporation (column 2 x column 4), averaging in each case the 30-second and the 60-second points. Table 4 gives the relative rates of labeling calculated in this fashion. These results are in agreement with the observations on the level of Trp-mRNA. The rate of labeling of Trp-mRNA decreases by more than an order of magnitude in both stringent and relaxed strains. The results reported in this paper indicate that amino acid starvation for a variety of amino acid blocks constitutive Trp-mRNA synthesis by some mechanism which rel+ and rel- bacteria possess. An exception to this appears to occur when the starvation prooedure simultaneously imposes physiological conditions which lead to the transcriptional derepression of a polycistronic operon (starvation for tryptophan of
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723
TABLET
Competing ability of RNA for typtophun operon-sp@c hybri&zatioon from R-Trp rel- &aim of E. coli a~ aflected by uarious amino acid-staruation
rel+ and R-Trp
conditions
streint
rul+ mlreJ+ rulrul+ rulreJ+ rul*cl+ 9x1TiRY (ty, deletion) Ymel (wild-type)
Growth oonditiont
Complete (Fig. 3) Complete (Fig. 3) - His, 16 min (Fig. 3) -HiHis,15min (Fig. 3) - Arg, 16 mm - Arg, 16 min - Leu and Ile, 16 mm - Leu end Ile, 15 min + 400 pgv8l/ml. 30 mill + 400 pg val/ml. 30 mill IXliIlhal+ Cesamino soids + 20 m Trp/ml. IIlhlhd+ Cwemino acids + 20 pg TV/~. (Fig. 3)
Increase in (8” - W/Q0 per H oompeting RNA (x 1O-4)#
Slope relative to repressed wild-type (Ymel, R + Trp)
1.48
7.41
2.20
11.0
0.546
2.73
0.587
2.93
0.260 0.284
1.30 1.42
0.337 0.282
1.68 1.41
0.944
4.74
0.327
1.03
0.066
0.28
0.201
1.00
7 rcl+ indicates R-Trp CP78 (stringent); rd- indicates R-Trp CP79 (relexed). $ Complete medium is minimal medium supplemented with arginine, hi&line, leuoine, isoleuoine, thraonine, tryptophan, thiamin and gluooae w described in the legend to Fig. 1. Starvation for a&nine, histidine and leuoine was accomplished by growing oultures to a cell density of 5 x lO*/ml., filtering end washing as in Fig. 1 and then aerating in complete medium minus the respective amino tutids indioated in the seoond oolumn. Isoleuoine starvation was attempted by growing the cultures in oompleta medium minus isoleuoine to a oell density of 6 x lO’/ml. and then adding 400 H v&e/ml. RNA was isolated in this ease 30 min after valine addition, whereas for the other starvation conditions RNA WES isolated after 15 min inoubstion in the defioient medium. 0 Hybridization oonditiona in eaoh oaae were the same es described in Fig. 2. Slopes were oaloulated from the best fit lines for date plotted 88 in Fig. 3.
an R +Trp tqptophan auxotroph: Edliu, Stent, Baker & Yanofsky, 1968 ; Lavalld & De Hauwer, 1968 ; or starvation for histidine of a hi&dine auxotroph of ~abnonella typhimurium: Venetianer, 1969), in which case the operon-specific messenger RNA is produced in the stringent strain. The nature of this generalized amino acid control cannot be deduced from these experiments. Our results support either the conclusion that Trp-mRNA synthesis is independent of stringent oontrol mediated by the rel+ allele in E. co& but is responsive to an unrelated generalized amino acid control or,
J. D.
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3
TABLE
Properties
of pulse-labeled
Total incorporation (acid ppt. cts/min in 1.0 ml. of culture)
RNA preparation?
R - !&p rel + , complete medium 30-set pulse 60-set pulse 46-s.% pulse R-Trp reZ+, -Leu, Ile 30.set pulse 60-set pulse 4%see pulse R - Trp reZ -, complete medium 30-set pulse 60-see pulse 45-se0 pulse R-Trp rd-, -Lou Ile 30-se0 pulse 60-am? pulse 46.se0 pulse
STUBBS
RNA preparations Specific activity of RNAS W/~n/&
% hybridization with @Opt190 DNA5
3580 8140
0.62 0.60
450 950
0.26 0.34
3260 6990
0.70 0.67
2570 6770
0.028 0.061
236,000
20,000
192,500
170,000
t 16 min after resuspension of the bacteria (5 x 10s cells/ml.) in either complete medium or complete medium minus leucine and isoleucine, [3H]uridine (2 c/m-mole) was added to give a uridine concentration of 0.59 M/ml. (4.8 +/ml.). RNA was isolated from portions of the cultures at 30 set and 60 sec. Total incorporation was determined from the 10% trichloroacetic acidpreoipitable radioactivity from a portion of the cultures taken at 45 sec. # Specific activities are given as trichloroacetic acid-precipitable cts/min/pg of each RNA preparation as determined by 260 nm absorption. 8 Hybridizations were accomplished by adding 32 pg of each RNA preparation to either 48Optl90 or 480 DNA immobilized on a 26-mm nitrocellulose filter (approximately 80 pg DNA/ filter). Volume of incubation mixtures was 1.0 ml. in 0.5 M-KC1 + 0.01 M-Tris chloride, pH 7.3. Following 16 hr incubation at 67”C, filters were washed and treated with RNase as previously described (Stubbs & Hall, 196%). The percentage hybridization is the (cts/min on 480pt190 DNA) minus (cts/min on 480 DNA) divided by input cts/min x 100. TABLE
4
Relative rate of [H3]uridine
Incorporation Trp-mRNAt (cts/min)
RNA preparation R - Trp reZ + , complete
R-Trp R-Trp
R-Trp
-Leu,
into
Leucine starvation effect upon Trp-mRNA$
1434
reZ+, -Leu, Ile rel-, complete
rel-,
entry into Trp-mRNA
Ile
complete ~ -Leu, Ile
= 24
complete -Lou, Ile
= 16
60 1229
76
t Obtained by multiplying the average o/0hybridization with 480pt190 DNA (Table 3, column 4) times total incorporation (Table 3, column 2). 1 Obtained by dividing line 2, column 2, into line 1, and line 4 into line 3.
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alternatively, that this generalized ammo acid control dominates an escape from stringency for Trp-mRNA in relaxed strains (rel-) of E. coli. This work wae supported Institutes of Health.
by grant no. lROlCA10942-01
Department of Cell and Molecular S&n Francisco State College San Francisco, Calif. 94132 U.S.A.
Biology
MBC from the U.S. National
J. D. fhTJBBS E. ANN &UEBS
Received 6 March 1970 REFERENCES Alfiildi, L., Stent, G., Hoogs, M. & Hill, R. (1963). 2. Vererbungelehe, 94, 286. Casrhel, M. BEGallant, J. (1969). Nature, 221, 838. Edlin, G. & Broda, P. (1968). Bact. Rew. 32, 206. Edlin, G., Stent, G., Baker, R. & Yanofsky, C. (1968). J. Mol. Biol. 37, 267. Fiil, N. (1969). J. Mol. Biol. 45, 196. Friesen, J. D. (1969). J. Mol. BioZ. 46, 349. Lavalle, R. t De Hauwer, G. (1968). J. Mol. BioZ. 37, 269. Stent, G. t Brenner, S. (1961). Proc. Nat. Acud Sci., W’u&. 47, 2006. Stubbs, J. BEHall, B. (1968a). J. Mol. BioZ. 37, 289. Stubbs, J. & Hall, B. (1968b). J. Mol. BioZ. 37, 303. Venetianer, P. (1969). J. Mol. BioZ. 45, 375.
Effects of Amino Acid Starvation on Constitutive Tryptophan Messenger Ribonucleic Acid Synthesis stringent (reZ+) and R-&p relaxed (Tel-) strains of EscMh In both R-trp coli, starvation for arginine, leucine, histidine or isoleucine resulted in a marked decrease in the level of tryptophan messenger RNA es aesayed by competitive hybridization experiments with DNA from a non-defective tryptophan transducing phage (+3Opt190). Using the level of tryptophan messenger RNA in a wild-type genetically repressed strain as a measure, this was found to be reduced from roughly ten times the repressed level to 1.5 times this level in the case of starvation for arginine, leucine or isoleucine and three times this amount in the case of histidine starvation. Tryptophsn messenger RNA synthesis, as reflected in the rate of labeling by [3H]uridine, is also greatly decreased by amino acid starvation in both strains. The results show that tryptophan messenger RNA is subject to a type of ammo acid control distinct from the rel+ (RCatr) system described by Stent & Brenner (1961).
pair of Escherichia: coli strains which carried either the stringent (reZ+)t or relaxed (reE-) allele but were otherwise isogenic (arg, his, Zeu, thi, R-.&p), it was previously found that arginine starvation greatly decreased both the level of tryptophan messenger RNA and its rate of synthesis in both the stringent and relaxed strains (Stubbs t Hall, 19683). In view of the possibility of a generalized model for the regulation of RNA synthesis associated with an understanding of the effects of the rel allele (Cashel & Gallant, 1969; Fiil, 1969) and the ambiguity concerning the effects of the rel allele on messenger RNA synthesis (Friesen, 1969; see review by Edlin & Broda, 1968), it was of interest to determine whether the effects we had previously observed on Trp-mRNA were unique to arginine starvation or held for a variety of amino acids. This paper presents the effects on Trp-mRNA resulting from starvation of the trp-constitutive rel+ rel- pair of E. coli strains for histidine, leucine and isoleuoine. The construction and characteristics of the R-trp CP79 (rel-) and R-&p CP78 (rel+) pair of E. coli strains, the isolation of purified [3H]Trp-mRNA, the isolation of non-radioactive competing RNA and pulse-labeled total RNA, and the techniques of the competition experiments and direct DNA-RNA hybridization experiments with $80 DNA and $80 pt190 DNA (480 DNA containing the tryptophan operon) are described in detail in Stubbs & Hall (1968aJ). Some pertinent points for this discussion are as follows. The R-trp rel+ strain requires arginine, histidine, threonine, leucine and thiamin for growth. The requirements of the R-trp rel- are identical with the In an auxotrophic
t Abbreviations used: rel+ (formerly BF’) for the stringent allele in 1. COG strain R-&p CP78 aud reZ- (formerly RCF) for the relaxed allele in 1. c& strain R-trp CP79 (Stubbs & Hall, 196%); arg, hG, Zeu, th%, requirement for arginine, histidiue, leucine and thiamin; R-trp, mutation in the R-trp allele resulting in constitutive synthesis of tryptophan messenger RNA; [3H]Trp.mRNA, [3H]uridine-labeled, pursed tryptophan messenger RNA preparation (Stubbe & Hall, 196&x); +8OpU90 DNA, phage 980 DNA oonteining the oomplete tryptophan operon from a non-defective tryptophan-tranaduoing derivative of phage 480. 717
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exception that it is thr+ . Apparently, in constructing this strain with Plkc, thr+ was introduced from the trp-constitutive donor strain into CP79 by co-transduction with the closely linked R-&p marker. Both strains are judged to be constitutive since the specifio activity of tryptophan synthetase A protein is 60-fold higher than in the repressed wild type (Ymel) and is unaffected by extreme changes (0 to 60 pg/ml.) in the tryptophsn concentration of the growth medium. The purified [3H]Trp-mRNA preparation used in these experiments hybridizes specifically with @Opt190 DNA to
YX
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40
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Time (mid
FIU. 1. Uptake of [sH]uridine in R-trp reE+ and R-trp rd- straim. Bacteria wars grown in minimal medium supplemented with 60 pg arginine/ml., 40 pg histidinel ml., 40 H leuoine/ml., 40 pg isoleuoine/ml. (Alfijldi, Stsnt, Hoogs & Hill, 1903), 200 ccg threonine/ ml., 20 erg t,ryptophan/ml., 2 w thiamin/ml. and 0.25% glucose (complete medium) to a density of 6.4 x 10s baoteria/ml. The cells were oollsoted by fltration onto a Milhpore filter, washed with an equal volume of oompleta medium minus histidine and glucose at 37”C, and resuspended in the same medium. The suspension of eaoh strain was divided into two portions. One portion was (70 m~oles/ml., supplemented with histidine (40 &ml.), gl uoose (0*26%), and [3H]uridine O-4 &nl.). The measured speoifio radioactivity of the [sH]uridine supplement was 3*4x lo6 ots/min/m~ole.) The other portion was supplemented only with gluoose and [sH]uridine. Aeration was begun immediately at 0 min. O-l-ml. samples were removed at intervals, placed in cold 10% triohloroacetio aoid with oarrier bovine serum albumin, and the resulting precipitats was oolleated on glass-fiber f%lt.ers. The radioactivity in the dried samples was determined in a liquid-scintillation speotrometer. -+-+-, R-trp rel+ plus hi&dine; -X-X-, R-trp reE+ minus histidine; ---e---a---, R-trp rel- minus histidine. ---O---O---, R-trp reZ- plus histidine;
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EDITOR
the extent of 66% of the input radioactively labeled RNA at saturating concentrations of DNA, whereas 5% is bound to $80 DNA. This is in good agreement with our previous observations. The R-trp pair were tested for rel+/reE- phenotype by measuring their ability to incorporate [3H]uridine in the presence or absence of various amino acids. A representative result (for histidine starvation) is given in Figure 1. Table 1 gives a complete TABLE
rel-/rel+ uultnre I
str8int
Phnotype of R-Trp
.
medim@
rel+
+
+Wl+ mlrelrel+ fez+ rdretrel+ re1+ rdrelrel+ ret!+ rdTea-
- His + - His + -As + - b + - Leu, -1le + - La, - 110 + t-w + Val (400 /&g/ml.) + t-w + Val(400 pg/ml.)
1
R*tio upt8ke 15 mill
[3H]uridine (non-starved) ’ (stied) 30 mill
I 7.3
8.5
1.16
1.32
z 7.50
9.25
> O-96 < 15.0 z
1.07
.
0.97
14.9 l-19
3.56
5.79
1.06
1.03
pair Cell density (beoteria/ml.) ( x 108) 60 min 0 min 6.37 6.37 6.25 6.25 54 5.5 5.67 5.67 6.63 6.63 5.17 5.17 6.25 6.25 6.37 6.37
11.1 6.63 Il.5 7.1 9.34 6.12 9-82 6.37 12.8 7.0 10.34 6.12 10.0 7.2 11.2 8.6
t rd+ indic.stas R-Trp CP78 (stringent); rel- indicstas R-Trp CP79 (relaxed). $ A + in this column indicates thet portion of the washed cells grown in complete medium that was supplemented with the tie aoid of which the other portion was deprived. Experimental conditions for each amino aoid are analogous to those desoribed in Fig. 1, with the exception of the isoleuoinestarvation imposedby exoeaa valine addition. In this oese, emh culture wea simply divided into two portions, and [*H]uridine added to one portion at zero time whereas C3H]uridine plus 400 ccg v8line/ml. wa8 added to the other. 8 Trichloroaoetio mid-pmoipitclble ots/min in 0.1 ml. of oomplete medium divided by trichloroaoetic said-preoipitable ota/min in 0.1 ml. of starvation medium at 15 end 30 min after addition of r3H]uridine.
summary for all the amino acids tested. If it is assumed that the rate of uridine uptake reflects the rate of net RNA synthesis, these results indicate that the pair of strains exhibits the Tel+/tel- phenotype in response to the amino acids used. The least pronounced effect was obtained in the attempt to starve indirectly for isoleucine by addition of valine. In the relaxed strain the amount of uridine uptake at 15 minutes was approximately the same in the starved cultures compared to the non-starved cultures for all amino acids tested, although by 30 minutes the uptake in the starved cultures had dropped off in some cases. Because the tryptophan biosynthetic enzymes are permanently derepressed in
J. D. STUBBS
720
I 0
I too
I 200 Unlabeled
AND
I 300 competing
E. A. STUBBS
I 400
I 500
RNA/reaction
I 600 @g)
FIU. 2. Competition between RNA from non-starved and histidine-starved R-&-p ml+ or R-trp rel- strains of E. coli and [sH]Trp-mRNA for binding sites on @Opt190 DNA. For each point, hybridization was carried out using approximately 0.2 pg 14C-labeled 48Opt190 DNA immoblized on a nitrocellulose titer membrane disc (Schleicher & Sohuell, B0, cut to 22.5 mm2 area), a saturating amount (13,600 cts/min) of r3H)Trp-mRNA, and the indicated amount of unlabeled RNA. Total volume of each incubation mixture was 0.25 ml. in a solution of 0.5 MKC1 plus 0.01 M-Tris-chloride, pH 7.3. Incubation was carried out at 67°C for 16 hr. Filters were rinsed in 67°C solutions (0.5 w-KC1 plus 0.01 ad-Tris, pH 7.3) and washed on both faces by suction were determined by liquid-scintillation filtration. After thorough drying, 3H and 14C radioaotivities oounting with appropriate correction for 3H and W overlap. The corrected l*C activity for each filter was used to normalize the corrected 3H activity to a DNA content of 0.2 pg/tilter disc. (The variable content of DNA, 0.2 & 0.03 pg/fdter, is attributable to variation in initial loading. Less than 5% of the DNA was lost from the discs during hybridization.) At zero concentration of competing RNA, the ots/min of the saturating level of [“H]Trp-mRNA bound to 480ptl90 DNA varied from 7 to 8% of the input radioactivity, whereas with +SO DNA less than 0.5% bound. 100% hybridization on the ordinate represents the ots/min bound specifically to 480@190 DNA at zero concentration of oompeting RNA. m--, RNA prepared from R-trp rel- grown in complete medium (Fig. 1) to a cell -o---density of 6 x loo/ml. ; - A-A-, RNA from R-trp Tel+ grown in complete medium to a cell density of 5 x 10B/ml. ; - 0 0 -, RNA prepared from R-h-p rel - grown in complete medium to a oell density of 6 x lO*/ml., filtered and washed as in Fig. 1, and aerated in complete medium minus histidine for 15 min; -A--A-, RNA prepared from R-&v reZ+ grown in complete medium to a cell density of 5 x 10B/ml., filtered and washed as in Fig. 1, and aerated in complete medium minus histidine for 16 min. -X -X -, RNA prepared from Ymel (wild type R+ trp) grown in minimal medium supplemented with 0.1% Casamino acids, 0.26% glucose, and 20 pg tryptophan/ml. to a cell density of 6 x 10e/ml.
LETTERS
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EDITOR
Unlabeled competing RNA/reaction
721
(pg)
Era. 3. Competing ability of RNA from non-starved end hi&line-starved and R-trp CP79 (rel-) for tryptophan operon-specific hybridization. Same date and symbols as in Fig. 2 plotted as described in text.
R-trp
CP78 (rd+)
R-Trp CP78 and R- Trp CP79 and these strains show the rel- Jrel+ phenotype in response to plus or minus histidine, arginine, leucine or isoleucine, these bacteria provide an ideal system for assessing the effects of amino acid starvation on constitutive Trp-mRNA synthesis. RNA preparations were isolated from each strain, grown either in complete medium or under conditions of amino starvation. Detailed conditions are given in the legend of Table 2. The content of Trp-mRNA in each preparation was assayed by competitive RNA-DNA hybridization. Figure 2 shows the competition curves obtained for the RNA preparations from relaxed and stringent strains grown in complete medium or starved of histidine for 16 minutes. The data for wild-type E. WU (R+Tq) grown in repressing levels of tryptophan is idudeafor comparison. Since the labeled RNA hybridized decreases monotonically, but not linearly, with increasing concentration of unlabeled RNA competitor, it is convenient to represent the data as a plot of (&” - &“)I& versus unlabeled competitor added. &” is the sH radioactivity bound to DNA when no competitor is present, and &” is the radioectivity bound at unlabeled competitor concentration c. This converts the data to a linear form in which the slope provides a direct measure of the Trp-mRNA content of the unlabeled RNA preparation (Stubbs & Hall, 1968a). The data represented in this fashion are shown in Figure 3. Table 2 contains a compilation of all the competition data for the various amino acid-starvation conditions represented as the slopes of plots similar to those in Figure 3. This Table also includes the data from an RNA preparation of E. wli strain T,RY, a derivtdive of Ymel in which the entire trp operon has been deleted (Stubbs & Hall, 1968a). The competition power of this preparation which contains no Trp-mRNA indicates the lower limit of detection associated with our technique and allows the conclusion that competition from outside the trp region does not signifkantly affect the results. When either the stringent or
722
J. D.
STUBBS
AND
E. A.
STUBBS
relaxed strain was grown in complete medium, the Trp-mRNA level was high, competition slopes relative to RNA from repressed wild type being 7.4 and 11.0, respectively. The reason for the decreased level of Trp-mRNA in the stringent compared to the relaxed strain is not clear but is in agreement with our previous results. Starvation for arginine, histidine, leucine or isoleucine decreased the level of Trp-mRNA in both the stringent and relaxed strains. As seen from Table 2, the amount of this decrease varies according to the starvation conditions. If it can be assumed that the competing ability of an unlabeled RNA preparation is directly proportional to its Trp-mRNA content, as is indicated by the competing ability of trp deletion RNA from strain T,RY, then the results indicate that starvation for arginine and leucine decreases the level of Trp-mRNA to approximately l-5 times that present in a repressed strain. This same decrease is observed in our isoleucine starvation conditions for R- Trp CP79. The higher value obtained for R - Trp CP78 with valine addition might well be a reflection of the relative ineffectiveness in provoking stringency of net RNA synthesis in this strain by this technique as is indicated in Table 1. Histidine starvation results in a Trp-mRNA decrease to approximately three times that present in the repressed wild-type strain. These results indicate that the effects on constitutive Trp-mRNA synthesis previously observed for arginine starvation are not unique and support the hypothesis of a definite amino acid control of Trp-mRNA level apparently unrelated to the rel +-reZ- control of net RNAsynthesis. The possibility that the observed decrease in the level of Trp-mRNA was a consequence of an increased rate of messenger RNA degradation was ruled out in the case of arginine starvation by a series of RNA pulse-labeling experiments (Stubbs & Hall, 19683). Analogous experiments were performed, and are reported here, for leucine starvation in order to ascertain whether the decreased level of Trp-mRNA obtained in this case was also due to amino acid starvation causing a decreased rate of constitutive Trp-mRNA synthesis. Short-term [3H]uridine labeling of RNA was carried out in R-Trp CP78 and R-TV CP79 cultures, both in complete medium and in complete medium minus leucine and isoleucine (seelegend to Table 3 for procedure). Total incorporation of [3H]uridine into RNA was measured on a portion of the culture for each of the four cases (Table 3, column 2). These data, together with the specific activities of the purified RNA preparations, affirm that total RNA incorporation was stringent for R-Trp CP78 and relaxed for R-Trp CP79 within these experiments. Specific hybridization of each labeled RNA preparation with $8Optl90 DNA under conditions>f DNA excess provides an estimate of the proportion of [3H]uridinewhich entired Trp-mRNA during the pulses (Table 3, column 4). A pronounced effect of leucine starvation is evident. As previously discussed (Stubbs & Hall, 1968b), a more significant measure of Trp-mRNA synthesis is obtained by multiplying the percentage hybridization by the total incorporation (column 2 x column 4), averaging in each case the 30-second and the 60-second points. Table 4 gives the relative rates of labeling calculated in this fashion. These results are in agreement with the observations on the level of Trp-mRNA. The rate of labeling of Trp-mRNA decreases by more than an order of magnitude in both stringent and relaxed strains. The results reported in this paper indicate that amino acid starvation for a variety of amino acid blocks constitutive Trp-mRNA synthesis by some mechanism which rel+ and rel- bacteria possess. An exception to this appears to occur when the starvation prooedure simultaneously imposes physiological conditions which lead to the transcriptional derepression of a polycistronic operon (starvation for tryptophan of
LETTERS
TO
THE
EDITOR
723
TABLET
Competing ability of RNA for typtophun operon-sp@c hybri&zatioon from R-Trp rel- &aim of E. coli a~ aflected by uarious amino acid-staruation
rel+ and R-Trp
conditions
streint
rul+ mlreJ+ rulrul+ rulreJ+ rul*cl+ 9x1TiRY (ty, deletion) Ymel (wild-type)
Growth oonditiont
Complete (Fig. 3) Complete (Fig. 3) - His, 16 min (Fig. 3) -HiHis,15min (Fig. 3) - Arg, 16 mm - Arg, 16 min - Leu and Ile, 16 mm - Leu end Ile, 15 min + 400 pgv8l/ml. 30 mill + 400 pg val/ml. 30 mill IXliIlhal+ Cesamino soids + 20 m Trp/ml. IIlhlhd+ Cwemino acids + 20 pg TV/~. (Fig. 3)
Increase in (8” - W/Q0 per H oompeting RNA (x 1O-4)#
Slope relative to repressed wild-type (Ymel, R + Trp)
1.48
7.41
2.20
11.0
0.546
2.73
0.587
2.93
0.260 0.284
1.30 1.42
0.337 0.282
1.68 1.41
0.944
4.74
0.327
1.03
0.066
0.28
0.201
1.00
7 rcl+ indicates R-Trp CP78 (stringent); rd- indicates R-Trp CP79 (relexed). $ Complete medium is minimal medium supplemented with arginine, hi&line, leuoine, isoleuoine, thraonine, tryptophan, thiamin and gluooae w described in the legend to Fig. 1. Starvation for a&nine, histidine and leuoine was accomplished by growing oultures to a cell density of 5 x lO*/ml., filtering end washing as in Fig. 1 and then aerating in complete medium minus the respective amino tutids indioated in the seoond oolumn. Isoleuoine starvation was attempted by growing the cultures in oompleta medium minus isoleuoine to a oell density of 6 x lO’/ml. and then adding 400 H v&e/ml. RNA was isolated in this ease 30 min after valine addition, whereas for the other starvation conditions RNA WES isolated after 15 min inoubstion in the defioient medium. 0 Hybridization oonditiona in eaoh oaae were the same es described in Fig. 2. Slopes were oaloulated from the best fit lines for date plotted 88 in Fig. 3.
an R +Trp tqptophan auxotroph: Edliu, Stent, Baker & Yanofsky, 1968 ; Lavalld & De Hauwer, 1968 ; or starvation for histidine of a hi&dine auxotroph of ~abnonella typhimurium: Venetianer, 1969), in which case the operon-specific messenger RNA is produced in the stringent strain. The nature of this generalized amino acid control cannot be deduced from these experiments. Our results support either the conclusion that Trp-mRNA synthesis is independent of stringent oontrol mediated by the rel+ allele in E. co& but is responsive to an unrelated generalized amino acid control or,
J. D.
724
STUBBS
AND
E. A.
3
TABLE
Properties
of pulse-labeled
Total incorporation (acid ppt. cts/min in 1.0 ml. of culture)
RNA preparation?
R - !&p rel + , complete medium 30-set pulse 60-set pulse 46-s.% pulse R-Trp reZ+, -Leu, Ile 30.set pulse 60-set pulse 4%see pulse R - Trp reZ -, complete medium 30-set pulse 60-see pulse 45-se0 pulse R-Trp rd-, -Lou Ile 30-se0 pulse 60-am? pulse 46.se0 pulse
STUBBS
RNA preparations Specific activity of RNAS W/~n/&
% hybridization with @Opt190 DNA5
3580 8140
0.62 0.60
450 950
0.26 0.34
3260 6990
0.70 0.67
2570 6770
0.028 0.061
236,000
20,000
192,500
170,000
t 16 min after resuspension of the bacteria (5 x 10s cells/ml.) in either complete medium or complete medium minus leucine and isoleucine, [3H]uridine (2 c/m-mole) was added to give a uridine concentration of 0.59 M/ml. (4.8 +/ml.). RNA was isolated from portions of the cultures at 30 set and 60 sec. Total incorporation was determined from the 10% trichloroacetic acidpreoipitable radioactivity from a portion of the cultures taken at 45 sec. # Specific activities are given as trichloroacetic acid-precipitable cts/min/pg of each RNA preparation as determined by 260 nm absorption. 8 Hybridizations were accomplished by adding 32 pg of each RNA preparation to either 48Optl90 or 480 DNA immobilized on a 26-mm nitrocellulose filter (approximately 80 pg DNA/ filter). Volume of incubation mixtures was 1.0 ml. in 0.5 M-KC1 + 0.01 M-Tris chloride, pH 7.3. Following 16 hr incubation at 67”C, filters were washed and treated with RNase as previously described (Stubbs & Hall, 196%). The percentage hybridization is the (cts/min on 480pt190 DNA) minus (cts/min on 480 DNA) divided by input cts/min x 100. TABLE
4
Relative rate of [H3]uridine
Incorporation Trp-mRNAt (cts/min)
RNA preparation R - Trp reZ + , complete
R-Trp R-Trp
R-Trp
-Leu,
into
Leucine starvation effect upon Trp-mRNA$
1434
reZ+, -Leu, Ile rel-, complete
rel-,
entry into Trp-mRNA
Ile
complete ~ -Leu, Ile
= 24
complete -Lou, Ile
= 16
60 1229
76
t Obtained by multiplying the average o/0hybridization with 480pt190 DNA (Table 3, column 4) times total incorporation (Table 3, column 2). 1 Obtained by dividing line 2, column 2, into line 1, and line 4 into line 3.
LETTERS
TO THE
725
EDITOR
alternatively, that this generalized ammo acid control dominates an escape from stringency for Trp-mRNA in relaxed strains (rel-) of E. coli. This work wae supported Institutes of Health.
by grant no. lROlCA10942-01
Department of Cell and Molecular S&n Francisco State College San Francisco, Calif. 94132 U.S.A.
Biology
MBC from the U.S. National
J. D. fhTJBBS E. ANN &UEBS
Received 6 March 1970 REFERENCES Alfiildi, L., Stent, G., Hoogs, M. & Hill, R. (1963). 2. Vererbungelehe, 94, 286. Casrhel, M. BEGallant, J. (1969). Nature, 221, 838. Edlin, G. & Broda, P. (1968). Bact. Rew. 32, 206. Edlin, G., Stent, G., Baker, R. & Yanofsky, C. (1968). J. Mol. Biol. 37, 267. Fiil, N. (1969). J. Mol. Biol. 45, 196. Friesen, J. D. (1969). J. Mol. BioZ. 46, 349. Lavalle, R. t De Hauwer, G. (1968). J. Mol. BioZ. 37, 269. Stent, G. t Brenner, S. (1961). Proc. Nat. Acud Sci., W’u&. 47, 2006. Stubbs, J. BEHall, B. (1968a). J. Mol. BioZ. 37, 289. Stubbs, J. & Hall, B. (1968b). J. Mol. BioZ. 37, 303. Venetianer, P. (1969). J. Mol. BioZ. 45, 375.