sequential transcription of the genes of the lactose operon and … · 2003-02-04 · 4436...

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 241, No. 19, Issue of October 10, pp. 44344443, 1966

Printed in U.S.A.

Sequential Transcription of the Genes of the Lactose Operon and Its Regulation by Protein Synthesis*

(Received for publication, March 24, 1966)

DAVID H. ALPERS

From the Gastrointestinal Laboratory, Massachusetts General Hospital, Department of Medicine, Boston, Massa- chusetts 02114

GORDON M. TOMKINS

From the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, United States Public Health Service, Bethesda, Maryland .$?OOl4

SUMMARY

The initial kinetics of induction and the steady state rates of synthesis of ,&galactosidase and thiogalactoside transacetylase in Escherichia coli were examined under a variety of conditions. Kinetic experiments with 5-fluorouracil and carbon starvation suggest that the z gene is transcribed into messenger ribonucleic acid (mRNA) before the a gene. In general, when protein synthesis was impaired, formation of transacetylase mRNA (defined as enzyme-forming capacity) was delayed, although /3-galactosidase mRNA (similarly denned) occurred at its usual time. When protein synthesis was completely blocked, the z gene could be transcribed but the a gene could not. To explain these findings, a tentative scheme is proposed in which a gene transcription is controlled by the rate at which ribosomes travel along the completed portion of the mRNA. Since ribosomes do not appear to be as critical for the transcription of the z gene, it is suggested that the RNA polymerase maybe able to advance ahead of the leading ribosome by a certain critical distance.

Kinetic experiments also suggested that the transcription of the z gene required a shorter exposure to inducer than did u gene transcription.

Chloramphenicol, which specifically delayed the induction of the transacetylase, also inhibited its steady state production more than the steady state synthesis of /3-galactosidase. At an antibiotic concentration of 2 pg per ml, the synthesis of /3-galactosidase continued, while that of thiogalactoside transacetylase was completely inhibited.

The lac operon in Escherichia coli consists of three lied genes which control the metabolism of lactose. Their order, starting with the gene closest to the operator, is z, which codes for ,8- galactosidase; y, which codes for galactoside permease; and a,

* This work was supported in part by Training Grant TlAM5146 and by Grant AM-01392 from the National Institutes of Health, Bethesda, Maryland 20014.

which codes for thiogalactoside transacetylase. Earlier (l), we found that when an inducer was added to bacterial cultures, /?- galactosidase activity appeared several minutes earlier than thiogalactoside transacetylase. Additional studies suggested that this sequential appearance of enzyme activities actually reflected the order of enzyme synthesis. With the use of a different assay for thiogalactoside transacetylase activity, Kepesl has also found that this enzyme appears in induced cultures later than P-galactosidase, and Goldberger and Berberich (2) have shown that, following derepression by histidine deprivation, the amounts of the enzymes involved in histidine biosynthesis in Salmonella typhi- murium also increased sequentially, with the enzymes controlled by genes nearest the operator region appearing before those controlled by genes located farther away from it.

In our experiments (l), a relatively long interval (2 to 3 min) was required for the appearance of thiogalactoside transacetylase activity after induction of P-galactosidase had started. Kepesl has found a shorter interval than the one we reported, but, in experiments with the histidine system (2), which is several times larger than the Zac operon, about 20 min elapsed between derepression of the earliest and the latest enzymes studied.

In the present communication, we report that various conditions of growth, and chloramphenicol, delay the induction of thiogalactoside transactylase but not that of fi-galactosidase. From these physiological experiments, we infer that the Zuc genes are transcribed sequentially rather than simultaneously, starting at the operator end of the operon. Furthermore, it appears that the rate of transcription of the z gene is relatively insensitive to environmental conditions, and that the rate of transcription of the acetylase gene is regulated in some way by protein synthesis.

In the experiments to be described, messenger ribonucleic acid activity is operationally defined as enzyme-forming capacity, i.e. the inducer-dependent formation of a substance which promotes enzyme synthesis in the absence of the inducer. Num- erous experiments indicate that the synthesis of enzyme-forming capacity requires ribonucleic acid synthesis (3), and that mRNA2

1 Personal communication. 2 The abbreviations used are: mRNA, messenger ribonucleic

acid; IPTG, isopropyl-P-n-thiogalactoside.

4434

by guest on Novem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Issue of October 10, 1966 D. H. Alpers and G. M. Tomkins 4435

formation is directly or indirect,ly stimulated by the inducer (4-6). Therefore, we shall adopt the convention that enzyme- forming capacity represents mRNh, although entirely analogous arguments can be constructed assuming that it reflects, for example, a specific transfer RNA or a group of ribosomes.

010

.o I 6

.o I4

EXPERIMENTAL PROCEDURE 012

Methods

Bacterial Strains-All bacteria used were E. coli, K-12. In most experiments, strain AT2322 (obtained from A. L. Taylor, as described in Reference 1) was used. Its relevant genotype is i+z+y-afmelthi-, which signifies that it carries normally in- ducible P-galactoside and thiogalactoside transacetylase, but is galactoside-permeaseless and auxotrophic for methionine and thiamine. AT2322 is a nonpolar mutation. Several experiments were carried out with AT2345 (i+z-y+a+met-thi-, also obtained from A. L. Taylor), HfrC (i+z+y+a+thi-), and 4680 (i+z- (leaky) y+a+thi-, from Dr. Henry Wu, Massachusetts General Hospital).

Media-Most experiments were carried out in a minimal medium with added Casamino acids (Medium A), which was modi- fied from Medium 3XD of Fraser and Jerrel (7) to contain NasHPOd, 0.074 M; KHz,PO~, 0.033 M; NH&I, 0.02 M; MgSOc 7 HzO, 0.0012 M; Casamino acids (Difco), 2.5 mg per ml; glycerol, 0.054 M (0.5% w/v). The pH was adjustedto 7.0 with5 N NaOH and, after diluting the medium to full volume, CaC& was added to a final concentration of 0.0001 M. After the medium was auto- claved, a sterile solution of thiamine hydrochloride was added to a final concentration of 0.5 pg per ml. In certain experiments, a glycerol-salts-thiamine medium (Medium 63) (8), was used.

Growth of Bacteria-Bacteria were grown in a New Brunswick gyrotory shaker model G-76 at 37” under the conditions described below. Growth was monitored by following the optical density of the cultures at 650 rnp in a Coleman Junior spectrophotometer. Before readings were made, aliquots of the cultures were diluted with Tris-HCl buffer, 0.05 M, pH 7.4, to give optical densities less than 0.200. Most experiments were performed with cultures that had been grown to a concentration of about 1 X log cells per ml, which corresponded to an optical density of roughly 0.5.

Materials

Isopropyl-P-n-thiogalactoside, o-nitrophenyl-fi-n-galactoside, and acetyl-CoA were purchased from Mann; 5,5’-dithiobis-2- nitrobenzoic acid was purchased from Aldrich; 5-fluorouracil was a gift of Roche Pharmaceutical Corporation; deoxycholate was purchased from Fisher; thymidine was from Sigma; P-n-galacto- pyranosyl-(l-o-)-n-glycerol was a gift from Dr. G. Ames; and 5-fluorouracil-2-‘4C was purchased from Calbiochem. Chloram- phenicol was a gift from Park, Davis; puromycin HCl was purchased from Nutritional Biochemicals.

Enzyme Assays-Aliquots of the bacterial culture were removed periodically, added to sodium azide at a final concentration of 0.02 M, and immediately chilled. Previous studies had shown that this procedure stops enzyme synthesis within 30 set (1).

Extracts prepared by sonic disintegration and centrifugation were assayed for enzyme activities as described previously (1). Because of the relatively great thermostability of thiogalactoside transacetylase, extracts were heated at 70” for 30 min and coagu- lated protein was removed by centrifugation at 12,000 X g for

2 ,010 - \ s

g .000 - 4

,006 -

I I I 1 0 IO 20 30 40 50 60 70 80 90 100

pl EXTRACT

FIG. 1. Thiogalactoside transacetylase activity as a function of the concentration of bacterial extract. Extracts of E. coli AT2322, grown to 1 X lo9 cells per ml and induced with IPTG, 5 X lo4 M, for 30 min, were prepared as described in “Methods.”

10 min prior to assay. Over this period, no loss of thiogalactoside transacetylase activity was noted, whereas the background reaction (IPTG-independent liberation of thionitrobenzoate) was greatly diminished. For the thiogalactoside transacetylase assays, disodium EDTA was added to the reaction mixtures to give a final concentration of 10e4 M, and the reaction rates were corrected for liberation of thionitrobenzoate not dependent on the addition of IPTG. The concentration of IPTG used as the acetyl acceptor was varied from 0.05 to 0.3 M, according to the levels of thiogalactoside transacetylase in the various extracts. Since over this range the rate of thiogalactoside transacetylase reaction is linearly dependent on the concentration of IPTG (9), the raw data are expressed as values which would have been obtained with the use of 0.05 M IPTG.

One unit of @-galactosidase is delined as the amount of enzyme which catalyzes the hydrolysis of 1 mpmole of o-nitrophenyl-fl- n-galactoside in 1 min at pH 7.0 and at 37” and 1 unit of thiogalactoside transacetylase activity is defined as the amount of enzyme which, in the presence of 0.05 M IPTG, catalyzes the production of 1 mpmole of thionitrobenzoate per min at pH 7.8 at 37”. All enzyme assays were performed at least in duplicate. Protein concentrations were determined by the method of Lowry et al. (10).

Initially, certain possibilities were investigated which might artifactually delay the appearance of thiogalactoside transacetylase activity in cultures following the addition of inducer. One such possibility might be the existence of a threshold in the thiogalactoside transacetylase assay, i.e. a critical level of thiogalactoside transacetylase, below which activity could not be detected. Despite the fact that earlier investigations (9) gave no such indication, the question was reinvestigated with the use of very small amounts of bacterial extract containing thiogalactoside transacetylase. The experiments illustrated in Fig. 1 show that no threshold exists, since all levels of enzyme measured felI on a line which passes through the origin when the volume of bacterial extract was plotted with respect to thiogalactoside

by guest on Novem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

4436 Sequential Transcription of lac Genes Vol. 241, No. 19

0

L / / /-

/ /?-Gab~cfosidase~

/ /I Jmnsacefylase

/ /I

O/

6 8 MINUTES

I

-(

FIG. 2. Kinetics of induction of enzymes of the lactose operon in whole cell suspensions. E. coli AT2322 was grown to 1 X lo9 cells per ml and induced with IPTG, 5 X 1O-4 M, at zero time; lo-ml aliquots were removed at various times into 0.02 M NaN3 and chilled. After washing once with Tris, 0.05 M, pH 7.8, the cells were resuspended in 1.0 ml of Tris, 0.05 M, pH 7.8, to which were added 0.05 ml of toluene and 0.05 ml of a 1% (w/v) solution of sodium deoxycholate. The cells were shaken at 37” for 30 min and aliquots were removed for protein and fi-galactosidase assays. For transacetylase activity, the toluene-deoxycholate-treated cell suspension was heated at 70” for 30 min and vigorously shaken to disperse the precipitated material. The IPTG-independent formation of thionitrobenzoate was 2 to 4 times greater than that obtained when the particulate material was not included in the assay. Zero time values for bot,h enzymes were subtracted from each recorded value.

TABLE I Effect of inducer concentration on kinetics of induction of enzymes

of lactose operon

E. coli AT2322 was grown to a concentration of 8 X 108 cells per ml and induced with IPTG concentrations as noted above, and lo-ml samples were removed at 0, 1, 2, 3,4, 5, 6, 8, 10, and 12 min. Aliquots were treated and assayed as in “Methods,” and the results were plotted as in Fig. 2. The lags and steady state enzyme rates were derived from the resulting curves.

Differential rate of synthesis Lag before Lag before

IPTG steady, state steady state Di~erence concentration synthesls,of @- syntehsls of

&Galac- Thiog&cto- galactosldase tIanSaCetylEX (II _ I)

tosidase side trans- (1) (11) acetylase

M

5 x 10-d 1 x 10-d 5 x 10-s

2.5 x 10-h

I

units /mg protein/min

44.2 0.16 40 0.13 24.5 0.09 10 0.032

I I?&

1.9 4.0 2.1 2.1 3.9 1.8 2.1 3.7 1.6 1.7 3.7 2.0

transacetylase activity. The thiogalactoside transacetylase activities rneasured in these experiments are of the same order as those observed in the early minutes of thiogalactoside transacetylase induction reported in an earlier communication (1) and in more detail below. The lowest level of transacetylase activity which could be quantitatively determined with this assay was about 0.015 unit. Values below this limit are recorded as zero.

Earlier experiments (1) also showed that during induction no inhibitors were present which might have artifactually diminished the thiogalactoside transacetylase activity measured. Since enzyme assays were usually performed on centrifuged extracts of sonically extracted bacteria, the delayed appearance of thiogalactoside transacetylase might have resulted from discarding the enzyme molecules synthesized earliest if they were associated with the cell surface or membrane. To test this possibility, kinetic experiments were performed in which both @-galactosidase and thiogalactoside transacetyla,se activities were assayed in whole cells treated with deoxycholate and toluene. The results of such an experiment, illustrated in Fig. 2, show that the steady state rate of P-galactosidase synthesis began 1.6 min, and the steady state rate of thiogalactoside transacetylase synthesis began 4 min, after the addition of IPTG. These intervals are very similar to those seen when sonic extracts were used (l), showing that the delay in synthesis of thiogalactoside transacetylase does not arise from artifacts involving intracellular compart- mentalization of the enzyme.

RESULTS

Conditions A$ecting Kinetics of Induction of P-Galactosidase and Thiogalactoside Transacetylase

Medium-Comparative kinetic experiments were carried out in Medium A, containing Casamino acids, and in a minimal medium (Medium 63). With 5 x 1O-4 M IPTG as the inducer, the times of appearance of P-galactosidase and thiogalactoside transacetylase in both media were similar to those reported earlier (1). For example, in one experiment, with the use of the methionineless strain AT2322 growing in Medium 63, with added methionine, 1.6 min were required for the steady state rate of fl-gaiactosidase synthesis to begin after the addition of inducer, and an additional 1.8 min elapsed before the synthesisof thiogalactoside transacetylase was detected. Experiments done with strain HfrC, which is prototrophic for amino acids, gave practically the same result in both Medium 63 and Medium ,4.

Inducer Concentration-Table I illustrates that a 20.fold de- crease in the inducer concentration caused a 4- to 5-fold reduc- tion in the steady state rates of synthesis of both P-galactosidase and thiogalactoside transacetylase, but did not affect the initial kinetics of induction of either enzyme. Other inducers (5.0 X 10m4 M thiomethylgalactoside, 1.0 X lo+ M /3-n-galactopy- ranosyl-l-O-glycerol (ll), and 1.0 X 1OW M lactose) produced approximately the same initial kinetics of induction as IPTG, even though with lactose the steady state rates of both P-g-a- lactosidase and thiogalactoside transacetylase synthesis were about 10% of those seen with IPTG. Thus, it is clear that slowing the rate of enzyme synthesis by the use of either subopti- ma1 concentrations of inducer or of a less effective inducer does not affect the times of onset of fl-galactosidase and thiogalactoside transacetylase syntheses.

Genotype of lac Operon-Comparative kinetic studies were

by guest on Novem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Issue of October 10, 1966 D. H. Alpers and G. M. Tom/&s 4437

performed and described in ‘%ethods,” with the use of E. coli strains 4680 (i+z- (leaky) y+a+) and AT2345 (i+z-~+a+). Al- though 4680 makes only barely detectable levels of P-galactosidase, and AT2345 none at all, the lag between addition of inducer and the onset of steady state synthesis of thiogalactoside transacetylase was found to be 3.8 min in 4680 and 4.2 min in 2345. This result does not differ significantly from the Z+ strains tested. Thus, it appears that active P-galactosidase is not required for the normal induction of thiogalactoside transacetylase.

-Aeration-As illustrated in Table II, the degree of aeration of the cultures proved to be important in determining the length of the delay in thiogalactoside transacetylase synthesis, although it did not significantly influence the initial kinetics of P-galactosidase synthesis. The degree of aeration was adjusted to vary the doubling time of the organisms between 52 and 129 min. Within these limits, there was relatively little change in the differential rate of ,&galactosidase synthesis and only a small, possibly insignificant, effect on the time of onset of steady state synthesis of P-galactosidase. However, the length of the delay in thiogalactoside transacetylase synthesis clearly depended on aeration. With vigorous aeration, the interval between the times of appearance of @-galactosidase and thiogalactoside transacetylase was 1.2 min, while with less aeration this interval increased to 3.2 min. The conditions of aeration used in our previous study (1) produced a doubling time of about 75 to 80 min, correspond- ing roughly to the data shown in Row 2 of Table II, and in the remainder of the experiments described in this communication similar conditions were used.

The initial kinetics of induction was also examined as a function of cell density. When the inducer was added to a density of 4 x lo8 cells per ml, fl-galactosidase was synthesized with its normal kinetics and thiogalactoside transacetylase synthesis began about 1.7 min later. At a density of 1.2 x lOi cells per ml, the interval between the enzymes was about, 2.0 min, which is probably not significantly different from the value observed at the lower density.

Studies on Mechanism of Sequential Appearance of P-Galactosidase and Thiogalactoside

Transacetylase

To analyze the reasons that the synthesis of thiogalactoside transacetylase begins later than that of ,&galactosidase, we have tried to consider separately two of the steps in enzyme induction:gene transcription and messenger translation. It has been established previously (12) that transcription of the z gene occurs almost immediately after addition of the inducer. In the experiments described below, we tried to determine whether the delayed appearance of thiogalactoside transacetylase is attributable to a delay in the transcription of the a gene or to its subse- quent translation. The results suggest that gene transcription is the slower step in thiogalactoside transacetylase formation. In Fig. 39, 5.fluorouracil was added to cultures of growing bacteria at different times after induction had been started. The analogue is thought to be rapidly incorporated in to newly synthesized messenger RNA, causing the synthesis of enzymically inactive p- galactosidase (3, 13, 14) and thiogalactoside transacetylase (see below). As illustrated in Fig. 3B, the incorporation of 14C-labeled 5-fluorouracil into trichloracetic acid-precipitable material in growing bacteria was linear from the time of its addition to the culture.

TABLE II

Effect of aeration on kinetics of induction of enzymes of lactose operon

E. coli AT2322 was grown at 37” to a concentration of 8 X 108 cells per ml to 1 X log per ml. To obtain a doubling time of 52 min, the organisms were grown in a New Brunswick gyrotory shaker G52 with a 2-inch stroke motion at about 250 rpm. The organisms grew with a doubling time of 72 and 129 min in a New Brunswick gyrotory shaker G-76 with a t-inch stroke motion at a setting of 11 and 4, respectively. Samples were removed, treated, and assayed as in Table I, and the results were similarly derived.

If the synthesis of the mRNA and thiogalactoside transacetylase occurs significantly later than that of galactosidase, then 5- fluorouracil added at short enough intervals after the inducer might inhibit thiogalactoside transacetylase but not P-galactosidase synthesis. The experiments illustrated in Fig. 3.1 show that, when the analogue was added 2 min later than the inducer (when the induced synthesis of P-galactosidase had already be- gun), thiogalactoside transacetylase activity could never be detected. However, if the addition of 5-fluorouracil was delayed until 3, 4, or 5 min after the addition of inducer, thiogalactoside transacetylase was synthesized. The curves marked control il- lustrate the synthesis of P-galactosidase and thiogalactoside transacetylase in a culture to which no analogue was added. The experiment illustrated is one of a series of six, in all of which similar results were obtained. These experiments suggest that the messenger for P-galactosidase is transcribed, and even translated, before the transcription of the a gene is completed.

Evidence That Synthesis of mRNds for P-Galactosidase and Thiogalactoside Transacetylase Can Be Dissociated-From the preceding experiments, it seems that after the addition of inducer up to 2 min may elapse before the transcription of the a gene is completed, while functional messenger for P-galactosidase appears almost immediately upon addition of the inducer (12). The length of time required to complete a gene transcription, as determined in the 5-fluourouracil experiments, corresponds closely with the time noted between the onset of steady state rates of synthesis for both enzymes. Thus, it seems likely that the onset of thiogalactoside transacetylase synthesis is limited by transcription of the a gene.

The experiments described in Table II showed that decreasing aeration prolonged the time required for thiogalactoside transacetylase appearance. In view of the 5-fluorouracil studies just presented, it was decided to investigate whether this prolonga- tion was attributable to inhibition of a gene transcription. For this purpose, the initial kinetic characteristics of induction of both enzymes were compared during carbon starvation, a more convenient means of limiting energy production than partial anaerobiosis.

by guest on Novem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

4438 Sequential Transcriptim of lac Genes Vol. 241, No. 19

r 440

t

-,9GALACTOSIDASE --THIOGALACTOSIDE

TRANSACETYLASE

400-

360-

320- z Y ,0280- LL

5FUADDED AT: 0 Smin 0 4min n 3min A 2min

0 2 4 6 8 IO 12 14 A MINUTES

FIG. 3. A, the effect of 5-fluorouracil on the kinetics of induction of enzymes and the lactose operon. E. coli AT2322 was grown at 37” to a density of 1 X log cells per ml with a doubling time of 75 min, and induced at zero time with 5 X 1O-4 M IPTG. At the times noted on the CUTWS, 5-fluorouracil (final concentration, 20 rg per ml) and thymidine (final concentration, 40 pg per ml) were added to the culture. Aliquots of 10 ml of the culture were removed at the times plotted and extracts were prepared and assayed as in “Methods.” B, the incorporation of 5-fluorouracil-2-W into the trichloracetic acid-insoluble fraction of E. coli. E. coli AT2322 was grown at 37” to a cell density of 8 X IO* per ml with a

Fig 4. illustrates one of a series of four experiments in which cells, grown to the middle of the log phase on glycerol, were centrifuged, washed, and resuspended in a glycerol-free medium. At zero time, inducer was added and the kinetics of appearance of P-galactosidase and thiogalactoside transacetylase was determined. As illustrated, the onset of /?-galactoside synthesis was not delayed by the absence of a carbon source although, of course, its rate of synthesis was greatly decreased. However, 18 min elapsed before thiogalactoside transacetylase appeared.3 Therefore, carbon starvation, like partial anaerobiosis, can delay the synthesis of thiogalactoside transacetylase without affecting the time of appearance of /!?-galactosidase.

To investigate whether the delayed onset of thiogalactoside transacetylase synthesis under these conditions is attributable to delayed transcription of the a gene, an experiment similar to that illustrated in Fig. 4 was performed. At intervals between 0 and 20 min, inducer was removed (to stop the synthesis of additional mRNA) and glycerol was added to allow translation of already completed messenger (Fig. 5). By this means, the presence or absence of thiogalactoside transacetylase messenger was deter-

3 In control experiments, preinduced cells were suspended in a similar glycerol-free medium not containing inducer. The levels of p-galactosidase and thioglactoside transacetylase remained constant for at least 20 min, suggesting that enzyme turnover did not produce the long lag in thiogalactoside transacetylase appearance illustrated in Fig. 4.

-i 600 2 E I

400 l

I/

l 200

l

l

/ 0

OY

B0 2 4 6 8 IO 12 14

MINUTES

doubling time of 75 min. At zero time, 20 pg per ml of 5-fluorouracil-2-W were added, giving a final concentration of radioac- tivity of 0.05 pC per ml. Thymidine (40 pg/ml) was also added at the same time. Aliquots of 1 ml were removed at the times noted and added to 1 ml of chilled 10% trichloracetic acid. The suspensions were filtered through 0.45-r 25.mm Millipore filters, washed three times with 5 ml of 5% trichloracetic acid, mounted on copper planchettes, dried, and counted in a gas flow counter (Nu- clear-Chicago) with an over-all efficiency of 12%. The background counts have been subtracted from the observed counts.

mined 5, 10, 15, and 20 min after the initiation of induction (only the latter three points are illustrated in the figure). When inducer was removed and glycerol was added before 20.min, there was an immediate increase in P-galactosidase activity, but no formation of thiogalactoside transacetylase. However, 20 min after inducer had been added (at times when the transacetylase activity was present even in the absence of a carbon source), the addition of glycerol produced a burst of both P-galactosidase and thiogalactoside transacetylase synthesis. These results confirm those of the 5-fluorouracil experiments in showing that the mRNA for /?-galactosidase is transcribed before that for thiogalactoside transacetylase. In addition, the present experiment shows that, under starvation conditions, the z gene is promptly transcribed, but completion of the transcription of the Zac operon is greatly delayed, as if the transcriptions of the z and a genes were no longer coordinate.

Since a delay in the transcription of the a gene occurs under conditions of carbon starvation and partial anaerobiosis, during which protein synthesis is partially inhibited, we were led to inquire whether complete inhibition of protein synthesis could abolish transcription of the a gene altogether.

Cells were exposed to inducer for 20 min in the presence either of chloramphenicol or of puromycin (Fig. 6). Under these conditions, as shown by Nakada and Magasanik (14), the z gene can be transcribed, provided no carbon source is present. At

by guest on Novem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Issue of October 10, 1966 D. H. Alpers and G. M. Tomkins 4439

24

Thiogo/octoside 1 Jronsocefylase

I’

I 3 3 6 9 12 15 18 21 6 9 12 15 18 21 MINUTES

FIG. 4. The effect of carbon starvation on the kinetics of induction of enzymes of the lactose operon. A culture of E. coli AT2322, grown at 37” to a density of 1.5 X lo9 cells per ml in Medium A, was filtered on a 0.8-p 142.mm Millipore filter and washed with 50 ml of Medium A not containing glycerol or Casamino acids. The cells were resuspended in the initial volume of glycerol and Casamino acid-free Medium A to which n-methionine (20 pg per ml) had been added. The cells were shaken for 15 min at 37”, and then induced with 5 X 1W M IPTG. Aliquots of 20 to 40 ml were removed at times noted above, washed as described in “Methods,” resuspended in 1 ml of Tris, 0.05 M, pH 7.8, and soni- tally extracted and assayed as described.

the end of the induction period, both the inducer and inhibitor were removed and glycerol was added in order to observe the activity of whatever mRNAs had been synthesized. As expected, there was an immediate synthesis of a small amount of P-galactosidase (14). However, no thiogalactoside transacetylase was formed. Apparently, when protein synthesis is prevented, the a gene is not transcribed into mRNA, but the production of mRXA from the z gene does not seem to be coupled, at least to the same degree, to concomitant translation of its mRNA.

To look further into the relationship between a gene transcription and protein synthesis, the initial kinetics of induction was examined in the presence of submaximal levels of chloramphenicol and puromycin. Fig. 7 and Table III give the results of these experiments. Increasing the chloramphenicol concentration from 0.5 to 1.5 pg per ml (Fig. 7) had several effects: growth of the organisms was progressively inhibited; the steady state differential rates of ,&galactosidase and thiogalactoside transacetylase synthesis were progressively depressed; and, most significantly for the present argument, the time of onset of thiogalactoside transacetylase synthesis was progressively delayed while the time of ,8galactosidase appearance remained unalter- ed. Fig. 7 and Table III show that puromycin, although it slowed the growth of the bacteria and depressed the differential rate of P-galactosidase synthesis, did not affect the time of appearance of either enzyme. Therefore, the actions of chloramphenicol and pyromycin were quite different and the possible im- plications of these differences are discussed below.

Action of Inducer-Genetic evidence clearly indicates a single

site of inducer action for all the enzymes of the Zuc operon. This site appears to be the product of the i gene, the repressor, which in turn, is thought to control the synthesis of all the lac gene products by interaction with the operator locus. Since both P-galactosidase and thiogalactoside transacetylase are synthesized constitutively in 0” mutants, one operator presumably controls all the Zac genes (15).

According to this model, if inducer has been in contact with repressor long enough to allow t,ranscription of the z gene, transcription of the a gene should not require the continued presence of the inducer. In view of the possible dissociation of the transcription of the z gene from that of the a gene, illustrated by the foregoing experiment, we questioned whether the inducer might influence the synthesis of the two enzymes differentially. To examine the question, exponentially growing cells were exposed to short pulses of IPTG, and the kinetics of appearance of p- galactosidase and thiogalactosidase transacetylase was studied. The results are illustrated in Fig. 8. If cells remained in contact with IPTG for 33 min, then, even when the inducer was removed by dilution, /?-galactosidase and thiogalactoside transacetylase syntheses were observed. Suprisingly, however, when exposure to IPTG was limited to 3 min, only P-galactosidase was formed; thiogalactoside transacetylase did not appear. Therefore, a longer exposure to inducer is required to induce thiogalactoside transacetylase synthesis than to induce P-galactosidase. It is

I-

,-

/ b 0

o-9 Thiogolactoside Transacctylose

t d

~~,~-o--P---M!-+-w--+-L-

( GLYCEROL ADDED)

0.40

P 0.35 k

8

P 0.30 $j

5 2 h

0.25 2

a z

2 0.20 p 3

FIG. 5. The induction of enzymes of the lactose operon in the absence of a carbon source. E. coli AT2322 was grown at 37” to density of 1.5 X lo9 cells per ml in Medium A, filtered on a 0.8-p 142.mm Millipore filter, washed with 50 ml of Medium A not containing glycerol or Casamino acids, and resuspended in the initial volume of the same medium free of glycerol and Casamino acids to which was added methionine, 20 pg per ml. The cells were then shaken at 37” for 15 min in this medium to deplete their carbon source further. At zero time, 5 X lo4 M IPTG was added to the culture. Ten minutes later (as indicated by the arrow) one-third of the volume was removed, filtered, and washed and the cells were resuspended in a complete medium containing glycerol and Casamino acids but lacking inducer. The cells were allowed to incubate an additional 5 min, during which samples were removed into NaN3 for enzyme assay as shown. Similar operations were carried out for cells induced in the absence of glycerol for 15 and 20 min as indicated above.

by guest on Novem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

4440 Sequential Transcription of lac Genes Vol. 241, iXo. 19

d II TABLE III $ Effect of puromycin on kinetics of induction and steady state P synthesis of P-galactosidase and galactoside transacetylase z

iti

E. coli, AT2322, was grown at 37” to a concentration of 5 X 108 cells per ml. Samples were removed, heated, and assayed as

-2 c z -

I

-n I

z described under “Methods.” The delay in steady state synthesis -I was derived from curves obtained as in Table I. To obtain the P 5

differential rate of enzyme synthesis, E. coli, AT2322, was grown

z

to a concentration of 1 X lo* cells per ml, and the experiment was carried out over at least one doubling time for each puromycin

2 concentration. At least five samples were removed for enzyme 5 R

assay, and the differential rate of enzyme synthesis was obtained

2 from a plot of enzyme activity with respect to milligrams of bac-

0 terial protein.

8 A- Od

z Puromycin

concentration

- ,,9-Ga/actosidose _--- Thiogolactoside Transocetyfase

l Chloramphenicol2O~g/ml * Puromycin lOO~g/ml

INDUCER PL us

INHBITOR /No Glycerol,

NO INDUCER OR INHBITOR NO INDUCER OR INHBITOR /With G/ycerolJ /With G/ycerolJ

Delay in steady state synthesis of

~-g&C-

tosidase

Delay in Differential teady state rate of thio- iynthesis of galactoside thiogalac- transace- oside trans- ty1ase acetylase synthesis

Differential rate of @-

rlactosidast synthesis

units/mg protein

32 30 10.2

(3.0 2.G

*---+---*--+ - - - - - - - 4 Doubling time I I I I I t

-- -20 0 I 2 3 4 6

MINUTES

FIG. 6. The induction of enzyme-forming capacity in the presence of puromycin and chloramphenicol. E. coli AT2322 was grown to a density of 7 X 108 cells per ml at 37” in Medium A, filtered, washed, and resuspended, as in Fig. 5, in Medium A free of amino acids and glycerol. The cells were shaken at 37” for 15 min. To one flask was added puromycin (100 rg per ml) and to another, chloramphenicol (20 pg per ml), and IPTG was added to both at a final concentration of 5 X 10m4 M. At the end of 15 min, the cells were transferred as in Fig. 5 to complete Medium A, free of inducer, and inhibitor and samples were removed at times noted above and treated as in Fig. 5.

Lx/ml

0 5

12.5 25 50

-I-

m in

76 75 80 84

122

min

1.7

1.8 2.0

min unit/mg grotein

3.7 0.16 0.17 0.06

3.4 0.04 3.8 0.03 I -L

CLAM 0.5pg/ml / *O c z

1.5 ;1

3.0 500

7.5 400

2.0 3oo 200

I .5 100

I .o 300

3.5 200

0

2.0 100

I .5

0

150 I .o

IOC

o.5 5c

0 C

600

500

z 400 6 & 300 E r 200

G CJ) 100 s G=J P

0

::

2

200

& 150

? 2 100

50

I

CLAM l.Opg /ml *

/ I,

, L

I -

1 -

I* 0

4 0.50 D

5 025 6

CLAM 1.5pg/ml

.

: I--/---

. P

/ 0, /

/, A/ag=fO.5mmin. .,’ a&--&- K’ ’ ’ 10

2 4 6 8 IO I2 I4 I6

4c

Puromycin 5pg /m I

1’ H ,&Ga/actosidase *- * Thhiogoloctoside

Transacetylase

0' 0 2 4 6 8 IO 12 14

MINUTES

FIG. 7. The effect of chloramphenicol and puromycin on the kinetics of induction of enzymes of the lactose operon. E. coli AT2322 was grown to 1 X lo9 cells per ml and induced with 5 X 1W M IPTG at zero time. Puromycin and chloramphenicol, at designated concentrations, were added simultaneously. Aliquots were removed and assayed as described under “Methods.” Levels of enzyme activity at zero time were subtracted from each point on the curve.

by guest on Novem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Issue of October 10, 1966 D. H. Alpers and G. M. Tom/&as 4441

difficult to reconcile these results with the model of inducer-repressor-operator interaction outlined above, and further experiments will be required to clarify their significance.

Control of Steady State Rate of Synthesis of P-Galactosidase and Thiogalactoside Transacetylase-The preceding experiments show that in the first minutes of induction the z gene is transcribed before the a gene and that the z messenger can be translated even before a gene transcription is completed. Although these conclusions were drawn by observing the initial kinetics of induction, they should also apply when the rate of synthesis of the Zac enzymes has become constant. If both the z and a portions of the Zac messenger were inactivated shortly after a gene transcription is completed, then conditions which permit z gene transcription but not a gene transcription should therefore diminish the steady state rate of synthesis of thiogalactoside transacetylase with respect to P-galactosidase. Since the procedures described above appear to have a differential effect on the transcription of the two genes, their influence on the steady state rates of synthesis of the enzymes was examined.

As shown in Table II, partial anaerobiosis delays the induction of thiogalactoside transacetylase but not that of fl-galactosidase. According to the considerations just presented, lowering the oxygen tension should therefore diminish the steady state rate of thiogalactoside transacetylase synthesis compared to that of P-galactosidase. Under conditions during which the doubling time of the organisms was 52 min (see Table II), the differential rate of P-galactosidase formation was 40 units per mg, while that

IIO-

DO-

go-

560- !s % m 70- + : c%60- - g

4 50- 4’ z

40- r 5 = 30-

MINUTES

FIG. 8. The effect of a pulse of inducer on the kinetics of induction of enzymes of the lactose operon. E. coli AT2322 was grown at 37” to a density of 8 X lo* cells per ml and induced with 8 X 1P M IPTG. At designated times, 25 ml of the culture were added to 225 ml of warm inducer-free Medium A. Thereafter aliquots of 50 ml of the diluted culture were removed at the times noted, concentrated by centrifugation, and resuspended in 1.0 ml Tris, 0.05 M pH 7.8. Extracts were prepared and assayed as under “Methods.”

TABLE IV Effect of chloramph,enic‘ol on steady state rate of synthesis of enzymes

of lactose operon

E. coli AT2322 was grown at 37” to a concentration of 1 X lo* cells per ml. The steady state rate of synthesis was determined as in Table III.

Chloramphenicol concentration

-

- Steady state rate jteady state rate of syn-

of synthesis of thesis of thiogalactoside ,%galactosidase transacety1ase

units/??@ protein

36.3 31.3 17.6 13.2

9.9 8.8 3.3

i

units/mg protein min 0.17 76 0.13 76 0.062 76 0.042 86 0.021 134

Barely detectable 164 0 195

-

_- Doubling time

0 25 .5 75 I .o 1.25 1.5 I .75 2.0 CHLORAMPHENICOL (pg/ml)

FIG. 9. The effect of chloramphenicol on the steady state rate of synthesis of p-galactosidase and transacetylase. The ratio of steady state synthesis of p-galactosidase and transacetylase was obtained from Columns 2 and 3 in Table IV.

of the thiogalactoside transacetylase was 0.29 unit per mg. When the doubling time was 129 min, the rates were 45.6 and 0.22, respectively. While not striking, this change in ratio was observed consistently, and we were stimulated to examine the effect of chloramphenicol on the steady state rates of synthesis of the two enzymes. Chloramphenicol, which delayed induction more than anaerobiosis, did not retard the appearance of fi-galactosidase. The effects of this antibiotic were tested on the steady state rates of enzyme formation. The results in Table IV and Fig. 9 show that, as expected, chloramphenicol inhibited the steady state synthesis of thiogalactoside transacetylase more than

by guest on Novem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

4442 Sequential Transcription of lac Genes Vol. 241, No. 19

d i= 6- 6 El :: k ::. 0 .:.: '/ :/ :. ,, i "

:, ; I I I

0 IO 20 30 40 50 PUROMYCIN (pg/ml)

FIG. 10. The effect of puromycin on the steady state rates of synthesis of &galactosidase and transacetylase. The ratio of steady state synthesis of @-galactosidase and transacetylase was obtained from Columns 4 and 6 of Table III.

I 0 i! Y A

DNA

mRNA /

INDUCED PROTEIN SYNTHESIS INHIBITED

PROTEIN CHAINS

INDUCED PROTEIN SYNTHESIS PERMITTED

FIG. 11. Diagrammatic representation of the effect of protein svnthesis on the transcription of the Zac genes. In I, no ribosomes are shown attached to the mRNA since protein synthesis is completely inhibited by chloramphenicol or puromycin. In this case, the RNA polymerase does not advance into the a gene. The point of inhibition is arbitrarily shown as the end of the z gene, but might equally well be anywhere before the end of the a gene. In II, protein synthesis is permitted and transcription and translation of all three Zac genes occurs.

that of ,8-galactosidase. In fact, at an antibiotic concentration of 2 I.cg per ml, the organisms were still growing (with a doubling time of 195 min) and elaborating /3-galactosidase, but thiogalactoside transacetylase synthesis was completely inhibited. The bar graph in Fig. 9 also illustrates the ratio of the rates of synthesis of thiogalactoside transacetylase to /3-galactosidase as a function of chloramphenicol concentration.

Puromycin inhibits the differential rate of synthesis of /3-galactosidase and thiogalactoside transacetylate but does not delay the induction of either enzyme (Fig. 7; Table III). We, therefore, expect that, unlike chloramphenicol, this antibiotic should not selectively inhibit the formation of thiogalactoside transacetylase. The steady state data in Table III show that this is the case. In fact, the differential rate of P-galactosidase formation was inhibited more by puromycin than the rate of thiogalactoside transacetylase synthesis. This is also illustrated in Fig. 10.

DISCUSSION

The present experiments confirm our earlier report that fl- galactosidase appears earlier than thiogalactoside transacetylase when inducers of the lac operon are added to E. coli. They also show that various conditions under which protein synthesis was inhibited (relative anaerobiosis, carbon starvation, exposure to chloramphenicol) delayed the time of appearance of thiogalactoside transacetylase, but did not influence the onset of P-galactosidase formation. When inhibition of protein synthesis was complete, enzyme-forming capacity of thiogalactoside transacetylase could not be induced at all.

A slower differential rate of enzyme synthesis was not sufficient in itself to delay the appearance of thiogalactoside transacetylase, since suboptimal concentrations of IPTG or other, less effective inducers produced normal kinetics of induction of both enzymes. Similarly, puromycin in suboptimal but inhibitory concentrations did not delay the onset of thiogalactoside transacetylase synthesis.

Since none of the present experiments involved direct measure- ments of the levels of mRNA or other intermediates in protein synthesis, interpretations in terms of detailed mechanisms are necessarily tentative. The most critical link between the data and their meaning is the molecular basis of enzyme-forming capacity, as defined in the introductory section. We conclude that it does not represent an enzymically inactive polypeptide precursor of thiogalactoside transacetylase, since the time of thiogalactoside transacetylase appearance was not determined by the differential rate of thiogalactoside transacetylase synthesis.

For the reasons outlined above, we assume that enzyme-forming capacity represents a specific mRNA and that the appearance of enzyme-forming capacity (i.e. mRNA) results from its synthesis de ?U)VO (i.e. gene transcription), rather than by activation of pre-existing messenger or inhibition of its breakdown.

With these important limitations in mind, the present studies suggest several interesting conclusions. First, they indicate that transcription of the z gene takes place before that of the a gene and, further, that translation of the z mRNA begins before transcription of the a gene is completed. If all the genetic infor- mation in the lac operon is coded on a single strand of mRNA (4, S), then the Zuc enzymes are synthesized on a complex of DNA- growing mRNA-ribosomes, as suggested previously (16, 17). If the mRNAs for /3-galactosidase and thiogalactoside transacetyl-

by guest on Novem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Issue of October 10, 1966 D. H. Alpers and G. M. Tom&s 4443

ase are separate molecules, then the existence of such a complex is not implied.

Perhaps the most surprising finding is that the transcription of the z gene can be uncoupled from that of the a gene. When protein synthesis was completely blocked, mRNA for thiogalactoside transacetylase was not induced even though messenger activity for fl-galactosidase was readily shown. When protein synthesis was slowed, transcription of the a gene was slowed as well, although mRNA from the z gene again was made normally. The puromycin experiment was the only apparent contradiction to the suggestion that a gene transcription requires protein synt.hesis because suboptimal concentrations of the antibiotic did not delay the appearance of thiogalactoside transacetylase. However, it has been suggested (18) that low concentrations of puromycin increase the rate at which ribosomes move along the messenger. It might be that the speed of ribosome movement along the completed portion of the messenger determines the rate of advance of the RNA polymerase and, therefore, the transcription of the remainder of the a gene. These considerations are diagrammed in Fig. 11.

Our experiments, as well as those of Nakada and Magasanik (14), imply that the z gene can be transcribed in the absence of protein synthesis, but the present studies suggest that a gene transcription requires ribosomal movement on the completed messenger strand. This could indicate that movement of the RNA polymerase along the DNA may be limited to a certain critical distance ahead of the leading ribosome. This critical distance might allow x gene transcription, but would not be suffi-

cient to allow transcription of the beginning of the (y and) a genes. (Since we did not measure galactoside permease activity, the question of whether or not the y gene is transcribed under these circumstances cannot be answered at present.) If ribosomes could only attach to mRNA at specific sites marking the beginning of genes, and these starting regions were not transcribed, ribosomes could not attach to the messenger and the advance of the RNA polymerase would be slowed or completely prevented. Such considerations might be involved in the mechanism of the position effect of amber and o&e triplets in the z gene on the synthesis of galactoside permease and thiogalactoside transacetylase reported by Newton et al. (19).

From the standpoint that the operon functions as a unit, controlled by the repressor-operator interaction, the present experi-

ments are disturbing. If our interpretations are correct, transcription of the z gene requires only a short exposure to inducer, but a longer period of induction is required for a gene transcription. A detailed discussion of this surprising result obviously requires direct examination of mRNA synthesis and will be de- ferred until a later time.

AcknowledgmentsThe authors wish to acknowledge the many stimulating conversations they have had with Drs. B. Ames, M. Gellert, R. Goldberger, N. Kredich, and M. Yarmo- linsky. They wish to thank Dr. A. Kepes for informing them of many of his experiments prior to publication.

REFERENCES

1. ALPERS, D. H., AND TOMKINS, G. M., Proc. Natl. Acad. Sci. U. S., 53, 797 (1965).

2. GOLDBERGER, R. F., AND BERBERICH, M. A., Proc. Natl. Acad. Sci. U. S., 54, 279 (1965).

3. PARDEE, A. B., AND PRESTIDGE, L. S., Biochem. Biophys. Acta, 49, 77 (1961).

4. ATTARDI, G., MAONO, S., ROUVIERE, J., JACOB, F., AND GROS, F., Cold Spring Harbor Symp. Quant. Biol., 28, 363 (1963).

5. GUTMAN, B., AND NOVICK, A., Cold Spring Harbor Symp. Quant. Biol., 28, 373 (1963).

6. HAYASHI, M., SPIEGELMAN, S., FRANKLIN, N. C., AND LURIA, S. E., Proc. iVat1. Acad. Sci. U. S., 49, 729 (1963).

7. FRASER, D., AND JERREL, E. A., J. Biol. Chem., 205,291 (1953). 8. PARDEE, H. B., JACOB F., AND MONOD, J., J. Mol. Biol., 1,

165 (1959). 9. ALPERS, D. H., APPEL, S. H., AND TOMKINS, G. M., J. Biol.

Chem., 240, 10 (1965). 10. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL,

R. J., J. Biol. Chem., 193, 265 (1951). 11. BURSTEIN, C., COHN, M., KEPES, A., AND MONOD, J., Biochim.

Biophys. Acta, 95, 634 (1965). 12. KEPES, A., Biochim. Biophys. Acta, 76, 293 (1963). 13. BUSSARD, A., NAONO, S., GROS, F., AND MONOD, J. Compt.

Rend. Sot. Biol., 250,4049 (1960). 14. NAKADA, D., AND MAGASANIK, B., J. Mol. Biol., 8, 105 (1964). 15. JACOB, F., AND MONOD, J., J. Mol. Biol., 3,318 (1961). 16. STENT, G., Science, 144, 816 (1964). 17. BYRNE, R., LEVIN, J. G., BLADEN, H. A., AND NIRENBERG,

M. W., Proc. Natl. Acad. Sci. U. S., 52, 140 (1964). 18. NOLL, H., Developmental and metabolic control mechanisms and

neoplasia, University of Texas Press, Austin, in press. 19. NEWTON, W. A., BECKWITH, J. R., ZIPSER, D., AND BRENNER,

S., J. Mol. Biol., 14,290 (1965).

by guest on Novem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

David H. Alpers and Gordon M. TomkinsRegulation by Protein Synthesis

Sequential Transcription of the Genes of the Lactose Operon and Its

1966, 241:4434-4443.J. Biol. Chem.

http://www.jbc.org/content/241/19/4434Access the most updated version of this article at

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

http://www.jbc.org/content/241/19/4434.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on Novem

ber 3, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/content/241/19/4434

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;241/19/4434&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/241/19/4434

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=241/19/4434&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/241/19/4434

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/241/19/4434.full.html#ref-list-1

http://www.jbc.org/

sequential transcription of the genes of the lactose operon and … · 2003-02-04 · 4436...

Documents