Control of Protein Synthesis in Escherichia coli: Strain Differences in ...

Vol. 142, No. 3

JOURNAL OF BACTERIOLOGY, June 1980, p. 888-898 0021-9193/80/06-0888/11 $02.00/0

Control of Protein Synthesis in Escherichia coli: Strain Differences in Control of Translational Initiation After Energy Source Shift-Down LEWIS A. JACOBSON* AND LINDA JEN-JACOBSON Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

We have studied the parameters of protein synthesis in a number of Escherichia coli strains after a shift-down from glucose-minimal to succinate-minimal medium. One group of strains, including K-12(X) (ATCC 10798) and NF162, showed a postshift translational yield of 50 to 65% and a 2- to 2.5-fold increase in the functional lifetime of general messenger ribonucleic acid. There was no change in the lag time for,B-galactosidase induction in these strains after the shift-down. A second group, including Wl and W2, showed no reduction in translational yield, no change in the functional lifetime of messenger ribonucleic acid, and a 50% increase in the lag time for ,8-galactosidase induction. Evidence is presented which indicates that this increased lag time is not the result of a decreased rate of polypeptide chain propagation. A third group of strains, including NF161, CP78, and NF859, showed an intermediate pattern: translational yield was reduced to about 75% of normal, and the messenger ribonucleic acid functional lifetime was increased by about 50%. Calculation of the relative postshift rates of translational initiation gave about 0.2, 1.0, and 0.5, respectively, for the three groups. There was no apparent correlation between the ability to control translation and the genotypes of these strains at the relA, relX, or spoT loci. Measurements of the induction lag for /1-galactosidase during short-term glucose starvation or after a down-shift induced by a-methylglucoside indicated that these regimens elicit responses that are physiologically distinct from those elicited by a glucose-tosuccinate shift-down. Previous reports from this laboratory have described the transient inhibition of protein synthesis that occurs when Escherichia coli undergoes a transition to a lower growth rate (shiftdown) after transfer from glucose-minimal to succinate-minimal medium. Ruscetti and Jacobson (17) showed that the decreased rate of protein synthesis is accompanied by a decrease in the number of polyribosomes and an accumulation of single ribosomes (708 monosomes) which meet empirical criteria for "complexed" rather than free particles. These 70S monosomes are not apparently engaged in polypeptide synthesis but are associated with the majority of pulselabeled RNA (presumably mRNA) in the cell. Ruscetti and Jacobson proposed that the 70S monosomes represent accumulated translational initiation complexes. Further support for this hypothesis was provided by Jacobson and Baldassare (9), who showed by electron microscopy that the 70S monosomes consist of single ribosomes bound at or near the 5' ends of mRNA strands. Westover and Jacobson (21) showed that the translational yield from preformed mRNA is significantly decreased in the period immedi888

ately after the shift-down. The polypeptide chain propagation rate was determined both for ,B-galactosidase and for total protein and was found to be the same (11 to 12 amino acids per s) as during exponential growth. It was also observed that the functional lifetime of general mRNA, but not of f8-galactosidase mRNA, was substantially increased after the shift-down. Westover and Jacobson (21) postulated that the decreased translational yield after shift-down could be quantitatively accounted for by a decrease in the rate of polypeptide chain initiation. The degree of inhibition of initiation was found to be similar for /1-galactosidase and for total protein, suggesting that the mechanism that controls the initiation of translation exerts its effect approximately equally on different mRNA species. Leschine and Jacobson (13) showed that the translation of the Q,B coat protein cistron is similarly affected, suggesting that the control is also exerted on the internal cistrons of polycistronic mRNA's. Johnsen et al. (11), in exploring the effects of a shift-down induced by the addition of a-methylglucoside, found that the rate of translational initiation is not reduced after shift-down, but

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889

that the rate of polypeptide chain elongation is added at 50 ,g/ml and thiamine was added at 100 ,ug/ reduced nearly twofold. They further reported ml. Shifts between media containing different carbon that reUA strains recover a normal peptide sources were performed by rapid filtration (21). aMethylglucoside was prepared as a 2 M solution in chain elongation rate within 10 min after the water and was sterilized by filtration. shift-down, but that chain growth remains slow Induction and assay of ,-galactosidase. The in reA strains for as much as 20 min after the synthesis of f?-galactosidase was induced by the addishift. tion of 0.6 mM isopropyl-,8-D-thiogalactopyranoside To resolve the apparent discrepancies be- (IPTG). Culture samples were treated with toluene tween our earlier results and the findings of (16), and ,B-galactosidase was assayed either spectroJohnsen et al. (11), we set out to examine the photometrically (21), with o-nitrophenyl-f8-D-galactoparameters of protein synthesis in a number of pyranoside as substrate, or fluorometrically (13) using E. coli strains subjected to several kinds of en- 4-methylumbelliferyl-fl-D-galactopyranoside. Measurements of isotope incorporation. Samergy source shift-down. We have found that the of labeled culture (0.05 ml) were placed on 2.4-cm E. coli strain [K-12(X)] used in our earlier studies ples circles of Whatman 3MM filter paper. The papers on glucose-to-succinate shift-down responds were dropped into CC13COOH and processed as desomewhat differently to glucose starvation or to scribed previously (8). Initial treatment was with iceshift-down induced by a-methylglucoside. Fur- cold 5% CC13COOH for measurement of uracil incorthermore, we have observed that different E. poration into RNA, or with 10% CC13COOH at 90°C coli strains respond differently to a glucose-to- for measurement of amino acid incorporation into succinate shift-down. Some strains entirely lack protein. The processed papers were counted as dethe control of translational initiation we had scribed previously (8). techniques. Standard techniques previously observed, and other strains have a (20)Manometric of 02 uptake in a Warburg for measurement control of initiation than our original weaker respirometer were used throughout. The center well strain. of each flask contained 0.2 ml of 10% KOH. The We have designated the pertinent phenotype oxidation substrate, when present, was 10 mM disoas Tic (for translational initiation control). Tic' dium succinate. All measurements of respiration were strains have strong control over translation, Tic- made at 37°C. strains lack this control, and Tic (Int) strains Chemicals and radiochemicals. Rifampin, amethylglucoside, 4-methyl-umbelliferyl-,8-D-galactohave weak control. pyranoside, IPTG, and o-nitrophenyl-,8-D-galactopyranoside were from Sigma Chemical Co., St. Louis, MATERIALS AND METHODS Bacterial strains. The bacterial strains used are Mo. Chloramphenicol was from Calbiochem, Los Anand shown in Table 1. the relA and spoT phenotypes of geles, Calif. ["4C]leucine, ["4C]uracil, [3H]uridine, were from these strains were checked occasionally by treatment 3H-algal profile protein hydrolysate Orangeburg, N.Y. ['4C]lysine and of 32P-labeled cultures with 1 mM ,B-2-thienylalanine Schwarz/Mann,were from New England Nuclear Corp., (1). Thin-layer chromatography of culture extracts ['4C]isoleucine (13) showed accumulation of ppGpp in relAU but not in relA mutants, with pppGpp also appearing in relAU spoT+ strains. Strain K-12(A) was shown by this test to be reUA+ spoT+. Our earlier designation (17) of this strain as "relaxed" was evidently caused by its unsuspected resistance to inhibition by L-valine. Growth conditions. Cultures were grown at 37°C in phosphate-buffered minimal medium as described previously (13). Where required, amino acids were TABLE 1. Bacterial strains used Strain

K-12(X) NF161 NF162 CP78 CP79 wi

W2 NF859FucNF1035

Genotype relA+ spoT+ argA metB relA+ spoTI argA metB relAl spoTI argH thr leu thi his relA+ spoT+ argH thr leu thi his relAll spoT+ argE thr leu thi his pro reUA spoT+ lacY argE thr leu thi his pro relA+ spoTI lacY argA metB fuc relAU spoT+ metB retAUl relX spoT+

Source ATCC 10798 R. A. Lazzarini R. A. Lazzarini J. Gallant J. Gallant J. Gallant J. Gallant J. Gallant

J. Gallant

Boston, Mass.

RESULTS

Macromolecule accumulation rates. The growth of two E. coli strains, K-12(A) and Wl, after a shift from glucose-minimal to succinateminimal medium, is shown in Fig. 1. In both cases a growth lag of appreciable duration was observed before the onset of growth at the rate characteristic of the succinate medium. It has been reported (3, 17) that E. coli sustains a reduced rate of protein synthesis after such a shift-down. The two relA' spoT+ strains of E. coli did not inhibit protein synthesis to an equal degree after the shift, as shown in Fig. 2. The postshift rate of protein accumulation in strain K-12(X) was about 15 to 20% of the rate in a glucose culture (Fig. 2A), whereas in strain Wl it was about 65% (Fig. 2B). This difference does not reflect a general immunity of Wl to the physiological effects of the shift-down, since RNA accumulation (as measured by ['4C]uracil incorporation) in strain Wl (see also Fig. 8) was

J. BACTERIOL.

JACOBSON AND JEN-JACOBSON

890

.6

--

0.4

0.3 s 0.24

O

,1t -60 -30 0 30

0.1

60 90 120 MINUTES AFTER SHIFT

FIG. 1. Growth of strain K-12(N (0) and strain Wl (U) before and after a shift from glucose-minimal to succinate-minimal medium.

40O0 3000 E 2000 100

0~~~~~. C0

-O

010 O 10 MINUTES AFTER SHIFT

10 10

FIG. 2. Protein labeling after shift-down. Cultures growing in glucose-minimal medium (absorbancy at 600 nm = 0.3) were shifted by filtration and suspended half in fresh glucose medium (E) and half in succinate medium (0). Immediately after shift, cultures were labeled with 1 ,uCi of ["4Clisoleucine per ml (26 juCi/pmol). Samples (0.05 ml) were withdrawn at the indicated times for the determination of acid-insoluble radioactivity. (A) Strain K-12(;V; (B) strain WI.

inhibited to about the same extent as in strain K-12(X). Strain differences in response to various shifts. We initially chose to examine the lag time for fl-galactosidase induction as an index of changes in macromolecular synthesis in response to nutritional stress. The method for deternination of "rise time" is illustrated in Fig. 3. The square root of the increment in enzyme activity is plotted against time after addition of inducer, and the intercept of the resulting straight line on the time axis is the time required for the synthesis of the first ,B-galactosidase polypeptide (18). The rise time represents a composite of several processes, each of which takes a potentially variable time. These processes are: inducer entry

2

2 3 4 3 4 MINUTES AFTER IPTG

5

6

FIG. 3. ,B-Galactosidase rise times in strain K-

12(N). Details are given in the text and by Leschine and Jacobson (13). (Ol) Glucose-to-glucose shift,

IPTG immediately after shift; (0), glucose-to-succinate shift, IPTG immediately after shift; (A) glucoseto-succinate shift, IPTG 10 min after shift; (0) glucose-to-glucose + a-methylglucoside (10:1) shift, IPTG immediately after shift; (A) glucose to glucose + a-methylglucoside, IPTG 10 min after shift; (x) glucose-to-no carbon shift, IPTG immediately after shift. In each case, the initial enzyme level (Eo) was the average of at least four measurements.

into the cell; derepression; initiation of transcription; RNA chain propagation; and translational initiation and propagation. A survey of fl-galactosidase rise times for various E. coli strains is shown in Table 2. Data are shown for glucose-to-succinate and glucose-tono carbon shifts, as well as for cells shifted to medium containing a-methylglucoside and glucose in a molar ratio of 10:1 (7). All strains showed rise times of 96 to 100 s in glucose medium, giving minimum estimates of polypeptide chain growth rate of 10 to 11 amino acids per s for the 1,021 amino acids of f)-galactosidase. (The rise times are the same as those that led to our earlier calculation of 11 to 12 amino acids per s [21]. The number of amino acids in the f8-galactosidase polypeptide has since been revised from 1,170 to 1,021 [2], and the present lower estimate of chain growth rate reflects the use of the newer value.) After a shift from glucose to succinate medium, there was no change in the rise times for strains K-12(X), NF161, NF162, CP78, and CP79. In these cases, we may justifiably conclude that there is no change in the polypeptide chain elongation rate. In strains Wl and W2, there was a substantial increase in the ,B-galactosidase rise time, but as we shall show later, this does not reflect a reduced polypeptide chain propagation rate. By contrast, all strains tested showed substantial increases in /-galactosidase rise time after a shift from glucose medium to medium lacking a

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TABLE 2. f,-Galactosidase rise times Rise time (s)a for strain (genotype): Growth condition

Time IPTG added (min after shift)

CP78 (reUA+ spor)

CP79 (relA

NF161 (relA+

NF162 (relA

WI (reIA+

spoT) 100

150 150

150 180

102 100

213b

210b

231b 216b

186 138

138 105

138 120

150 100

0

100

spOr) 100

Sp07) 100

spoT) 96

SpoT) 99

Glu Succ

0 10

100 100

100 102

99 96

ggb 104b

Glu - No carbon

0 10

152 117

249b

-c -

111 111 133 133 0 111 10 107 123 96 a Rise times are given in seconds from the addition of IPTG. bDetermined by fluorescent assay with 4-methylumbelliferyl-galactoside. c No rise detected within 10 min. d a-MG, a-Methylglucoside.

Glu

a-MGd (10:1)

carbon source. In strains NF161 and NF162 this effect was so pronounced that we could detect no increase in enzyme over basal level even at 10 min after addition of inducer. We have not pursued the mechanism of this effect further and include it only to demonstrate that the physiological effects of very short-term carbon source starvation may be quite different from those of a shift to even a poorly metabolized carbon source. This point is further demonstrated by measurements of respiration. Even though strain K12(X) showed no growth for about an hour after a glucose-to-succinate shift (Fig. 1), its initial respiration rate in succinate medium (Qo2 = 28 PI of 02 per mg of dryin weight per h) was significantly greater than carbon-free medium (Qo2 = 7 ,ul of 02 per mg of dry weight per h). Cells fully adapted to succinate showed Qo2 = 220 p1 Of 02 per mg of dry weight per h. After a shift to medium containing a-methylglucoside, we saw still another pattern. All strains tested showed an increased /?-galactosidase rise time, in accord with the results of Johnsen et al. (11), although the magnitude of the effect was considerably less in our strains than in theirs. As reported by Johnsen et al., reA mutants do not recover to a normal rise time as rapidly as relA+ strains. Parameters of translation after shiftdown. To further explore translational differences between strains, we have measured the total translation of mRNA that was formed before shift-down. The method and experimental logic have been given by Westover and Jacobson (21). A single culture, prelabeled with 3H-amino acids for normalization of recoveries, was filtered, washed, and divided into two cultures, one in glucose medium and one in succinate

-

K-12 (relA+

99

Glu Glu

173b

W2 (reLA+ SpoT)

medium. Rifampin was added to the suspending medium, such that no new transcription was initiated after the shift. At zero time of the shift, each culture was labeled with a large mass (to minimize pool effects) of a '4C-amino acid, and incorporation was followed with time. The total time course of protein synthesis in three strains is shown in Fig. 4. This experiment measures the "translational yield," that is, the total amount of protein synthesized by a given cellular complement of mRNA over the lifetime of that mRNA. As previously reported (21), the translational yield for strain K-12(A) in the succinate culture was about 64% of the yield in the glucose culture. By contrast, translational yield was reduced not at all in strain Wl and was about 77% in strain CP78. The curves of Fig. 4 may be transformed to show the exponential decline in protein-forming capacity as mRNA is inactivated. These transformations are shown in Fig. 5. In all cases, these "functional decay curves" were linear, indicating that the functional inactivation of total cellular mRNA may be represented as a single first-order process. As previously observed by Westover and Jacobson (21), the down-shift led to a substantial decrease in the rate of mRNA inactivation in strain K-12(X). By contrast, strain Wl showed no significant decrease in mRNA inactivation rate. In strain CP78, the rate in the succinate culture was significantly less than in the glucose culture, but the decrease in inactivation rate was not so pronounced as in K-12(X). Based upon the analysis of Jacquet and Kepes (10), these data may be used to calculate the relative rates of polypeptide chain initiation in the succinate and glucose cultures. The appropriate equation is: Y = [(Tp)s + fT8]/[(Tp)g +

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JACOBSON AND JEN-JACOBSON

o.o0 NO8 0.6~~~~~~~~~

tO4 02 00

0

10

20

30

0

30 10 20 MINUTES AFTER SHIFT

0

10

20

30

FIG. 4. Protein synthesis after rifampin treatment. Cells growing in glucose medium were labeled for 80 to 90 min with 1.2 IACi of either [3H]leucine (A) or 3H-algal profile protein hydrolysate (B and C) per ml, then shifted to minimal medium containing 200 ug of rifampin per ml, 1 ,uCi of "4C-amino acid per ml, and either glucose (0) or succinate (a). Duplicate samples (0.05 ml) were taken at the indicated times for measurement of acid-insoluble radioactivity. In each case, the average plateau 14C/3H ratio in the glucose culture has been set as unity, and all data are normalized accordingly to facilitate comparison. (A) Strain K-12(V), postshift labeling with ['4C]leucine (10 uCis/pmol). (B) Strain Wl, postshift labeling with ['4Cl1ysine (10 ,ACi/lmol). (C) Strain CP78, postshift labeling with [14C]lysine (10 lsCi/pmol).

MINUTES AFTER SHIFT

FIG. 5. Decay ofprotein-forming capacity after rifampin treatment. Data taken from Fig. 4. Symbols are the same as those used in Fig. 4. Percent proteinforming capacity remaining = [(Rf - RdI/Rd x 100, where Rf = final 14C/3H ratio and Rt = 14C/3H ratio at time t. (A) Strain K-12(AJ; (B) strain WI; (C) strain CP78. T], where Y is the relative translational yield in the succinate and glucose cultures (the ratio of the plateau values in Fig. 4); T. and Tg are the mean functional lifetimes (T = t1/2/ln2) ofmRNA in the succinate and glucose cultures, respectively; f is the relative frequency of translational initiation in the succinate culture (setting the initiation frequency in the glucose culture as unity); and (Tp)s and (TP)R are the polypeptide chain completion times in the succinate and glucose cultures, respectively. (This equation is slightly different from the incorrect equation of Westover and Jacobson [21], who multiplied the

right-hand side of this equation by T,/5T. Recalculation of the earlier results [21] using the present equation shows no qualitative effect on the earlier conclusions.)

The values of Tp are obtained from the data of Fig. 5 by determining the time at which the curves intersect the 100% level of protein-forming capacity (10). The data for all three strains in Fig. 5 give values of Tp of about 15 s. Since in all cases XT>> Tp, the calculations of f are quite insensitive to errors in the estimate of Tp. The data and collected values of f for the strains shown in Fig. 4 and 5, as well as for several other strains we have tested, are collected in Table 3. The strains may be divided into three groups, differing in their abilities to reduce the frequency of translational initiations after a glucose-to-succinate shift-down. We designate this phenotype as Tic (for translational initiation control). This is intended as purely a phenotypic designation, and implies nothing about the genetic basis for the phenotypes. Tic+ strains show strong control, with postshift translational initiation frequencies of 0.2 to 0.24. Ticstrains show little or no reduction in translational initiation frequency after shift-down. Tic(Int) (intermediate) strains show postshift initiation frequencies of 0.45 to 0.5. We note that the Tic phenotypes of these strains do not correspond with the genotypes at the relA or spoT loci. For example, relA+ spoT+ strains may be Tic' [K-12(A)], Tic- (Wl), or Tic(Int) (CP78). Tic+ strains may be reUA spoT+ [K-12(A)] or relA spoT (NF162). Tic(Int) strains carrying a reUA allele may be spoT (NF161) or spoT+ (CP78). The Tic- phenotype is likewise independent of spoT (compare Wl and W2). A relX reA derivative of NF859, strain NF1035 (15), remains, like its ancestor, Tic(Int). Thus the Tic phenotype does not correlate with alleles of reLX.

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893

TABLE 3. Parameters of protein synthesis Strain

mRNA lifetime (8)a Translation yielda, Tbg Ts T/T,,

Relative initiation frequency'

(f)

Tic' 0.24 212 550 2.6 0.64 K-12(A) 0.20 316 606 1.9 0.42 NF162 Tic0.93 213 213 1.0 0.97 WI 1.02 286 286 1.0 1.02 W2 Tic(Int) 0.49 234 359 1.5 0.76 CP78 0.49 333 463 1.4 0.70 NF161 0.45 286 403 1.4 0.65 NF859 0.47 394 593 1.5 0.71 NF1035d a From experiments similar to those shown in Fig. 4 and 5. b = Y Succ/Glu. 'Calculated as described in the text. d All experiments with NF1035 were performed at

300C.

We also note that the degree of functional stabilization of mRNA (Ts/T,, Table 3) falls into groups corresponding to the Tic phenotypes of these strains. This correlation is currently being investigated further in our laboratory. To further demonstrate that Tic' and Ticstrains differ in their ability to control translational initiation, we used strains K-12(X) and Wl in experiments similar to those of Fig. 4, but examined protein synthesis at very short times after shift. The presence of rifampin in these experiments insures that differences in the rate of protein synthesis between the glucose and the succinate cultures cannot arise from differences in transcription, since we are measuring only the translation of preformed mRNA. A calculation from the formulas of Jacquet and Kepes (10) indicates that about 7% of global mRNA should be incompletely transcribed at steady state. Therefore, even a complete failure of transcriptional completion would have a negligible effect on these experiments. The data for strain K-12(X) are shown in Fig. 6. It may be seen that for the first 15 s after shift the rate of protein labeling in the two cultures is the same, as would be expected if ribosomes that had initiated before shift-down continued polypeptide chain propagation at an undiminished rate after the shift. The break in the rate of accumulation at about 15 s is consistent with the polypeptide chain completion time (T,) of 15 s which we obtained from Fig. 5. The inference that the rate of polypeptide chain propagation is unaffected by the shift is also consistent with the unaltered ,B-galactosidase rise time (Table 2). After 15 s, the rate of protein labeling

003 , 0.2

0.1I 0.0

120 60 0 SECONDS AFTER SHIFT + RIF

FIG. 6. Early time course of protein labeling in strain K-12(A) treated with rifampin. Cells growing -in glucose medium were prelabeled with 3H-algalprofile protein hydrolysate as described in the legend to Fig. 4. Cultures were shifted by filtration either to fresh glucose medium (l) or to succinate medium (0), both containing rifampin (200 pg/ml) and ['4CJleucine (1 ,uCi/ml; 312 uCi/,inol). Samples (0.05 ml) were withdrawn at the indicated times and immediately precipitated with CCl3COOH. The two data points at t = 15 s were exactly coincident.

dropped abruptly to 13% of the rate in the glucose culture. This would indicate a relative translational initiation frequency of 0.13, about half the value obtained in Table 3 from Fig. 4 and 5. We do not regard this degree of discrepancy as too surprising, since the experiment of Fig. 6 requires that we use [14C]leucine at much lower mass concentration (higher specific radioactivity) than in the experiment of Fig. 4. We believe that the intracellular leucine pool would be more likely to depress the rate of incorporation in Fig. 6, where we presumably have not flooded the pool, thus giving a lower estimate of f. We also note that the calculation of f from total translational yield (Table 3) weights each protein according to the functional lifetime (T) of its particular mRNA, whereas short-term labeling (Fig. 6) does not involve mRNA-lifetime weighting. The resulting estimates of f thus should not agree precisely. This is further confirmed by the data of Table 4, in which we have measured the translational yield of 8l-galactosidase after shift-down. In combination with the unaltered value of 100 s for Tp (Table 2) and the unaltered value of 82 s for the functional lifetime of 81-galactosidase mRNA (21), we calculate a value of f of 0.27 for ,Bgalactosidase, in good agreement with the value for total protein (Table 3). Inasmuch as the

894

JACOBSON AND JEN-JACOBSON

J. BACTERIOL.

value for B-galactosidase is independent of pool effects on labeling rates, the close agreement of the two values indicates that f = 0.24 to 0.27, rather than the lower value of Fig. 6, is correct. Strain Wl, however, showed very different early kinetics of protein labeling. As shown in Fig. 7, there was no diminution in protein labeling rate during the first 2 min after shift-down, indicating that both translational initiation and translational elongation are proceeding at the same rate after the shift. The experiment of Fig. 7 used [14C]lysine for the postshift labeling, but experiments with [14C]isoleucine gave similar results. To show that the down-shift was truly effective in strain Wl at these early times, we measured the rate of [14C]uracil incorporation into RNA. As shown in Fig. 8, the rate of RNA labeling was reduced almost immediately in the down-shifted culture. TABLE 4. Translation of f3-galactosidase Postshift increase in ,-galactosidasea Strain Succinate

Glucose

Y

=

Succ/

Glu

K-12(A)

4.25 ± 0.30 6.34 ± 0.19 0.67 ± 0.08 0.27 2.38 ± 0.21 2.23 ± 0.17 1.07 ± 0.12 1.1 a Mean enzyme units (N = 10) assayed fluorometrically standard error of the mean. Data are normalized to culture absorbancy at 660 nm = 1.0. 'For a discussion of error sources in this calculation, see

0.15 -0 0

0.10

-~~~~

Q05 0005

0

60 120 SECONDS AFTER SHIFT

FIG. 8. Early time course of RNA labeling in strain Wl. Cells growing in glucose medium were prelabeled for 90 min with [3H]uridine (1 ,iCi/ml; 8 Ci/mmol), then filtered and shifted to either fresh glucose medium (U) or to succinate medium (0), both containing/[4C]uracil (1 iCi/ml; 60 t&Ci/mmol). Samples (0.05 ml) were withdrawn at the indicated times and immediately precipitated with cold 5%

CCl3COOH.

WI

To further confirm that polypeptide chain elongation in strain Wl is unaffected by the shift-down, we performed the experiment shown in Fig. 9. Cells growing in glucose medium were (21). using Tp 93 T= Tg= = Tg = 83 for strain K-12(A) (21), and Tp induced with IPTG for 3 min and then filtered 112 for strain Wl (see Fig. 9). and washed, and the culture was divided into glucose medium or succinate medium, each containing rifampin. The kinetics of f8-galactosidase synthesis were measured until enzyme formation i.6 ceased due to the decay of the mRNA. The resulting data were then transformed to show the exponential inactivation of ,B-galactosidaseforming capacity (Fig. 9). It was evident that the rate of inactivation of f8-galactosidase mRNA was the same in both cultures (T = 112 s). More importantly, the extrapolation of the curves to the 100% level of enzyme-forming capacity gave a value for the polypeptide chain completion time of 93 s in both cultures. This agrees reasonably well with the fB-galactosidase rise time of 100 s measured in glucose medium (Table 2) and indicates that the increased rise time (150 s) J measured for strain Wl after the glucose-to-suc0.0 90 60 0 30 cinate shift (Table 2) does not reflect a decrease SECONDS AFTER SHIFT RIF in the polypeptide chain propagation rate. FIG. 7. Early time course of protein labeling in Finally, measurements of the translational strain WI treated with rifampin. Procedure and sym- yield and calculation of the value of f for (Ibols are the same as for Fig. 6, except postshift labeling was with [14C]lysine (I ILCi/ml; 345 ,tCi/ galactosidase in strain Wl (Table 4) confirmed ,umol). '4C/3H ratios are higher than those in Fig. 6 that neither translational yield nor translational because the 3H-amino acid mixture used for prela- initiation frequency was reduced in strain Wl beling is diluted by the unlabeled amino acids in the after the glucose-to-succinate shift-down. culture medium for strain Wi. Effect of shift-down on transcription. If

Westover and Jacobson T

Calculated

100

s

,

1.8

1.4

1.2

I.0