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Peptide chain growth of β-galactosidase in Escherichia coli
J. Mol. Biol. (1968) 35, 165-173
Peptide Chain Growth of p-Galactosidase in Escherichia coil F. LACROUTEt AND G. S. STENT
Virus Laboratory and Del~artmen$ of Molecular Biology University of Galifornia Berkeley, Galifornia 94720, U.S.A. (Received 7 November 1967, and in revised form 29 March 1968) An experiment was carried out to test the notion that regulation of fl-galacimsidase synthesis in Es~herichia coli involves an inducer-dependent growth period of the enzyme protein. The results of this experiment, in which the time elapsed between laying down an N]Ka-terminal threonine of the fl-galactosidase polypeptide and its appearance in the intact enzyme molecule was measured directly in both maximally and submaximally induced cultures, provided strong evidence against an inducer-dependent growth period. For it was found that, in both maximally and 30% maximally induced cultures, the enzyme growth period is 80 seconds. This constant enzyme growth period indicates a peptide chain growth rate of 15 amino acids per second, a figure which is in reasonable agreement with an estimate of the average peptide chain growth rate per ribosome in the same bacterial strain. 1. I n t r o d u c t i o n Two general, antithetical possibilities can be considered to explain regulatory variations in the differential rate of synthesis of inducible or repressible bacterial enzymes: the variable over-an rate of synthesis of a particular enzyme could reflect: (1) simultaneous growth of a variable number of its polypeptide chains under an invariant peptide chain growth period; or (2) simultaneous growth of an invariant number of its polypeptide chains under a variable peptide-chain growth.period. B y peptide-chain growth-period is meant here the time elapsed between the start of synthesis of a peptide chain and its appearance in the complet~ enzyme molecule. Both of these possibilities are compatible with Jacob & Monod's operon (1961) model of regulation of bacterial protein synthesis, although this model is usually discussed in terms of only the first of these possibilities. Indeed, either of these possibilities could obtain, whether the primary site of control is at the level of transcription of the DNA or at the level of translation of the messenger RNA. An appendix to this paper briefly describes two feasible regulatory models, one involving transcriptional and the other translational control, which do incorporate the element of variable peptidechain growth-period, instead of the canonical notion of variability in the number of growing polypeptide molecules. The experiments to be presented here are directed ¢~ the question of whether in the regulation of fl-galaetosidase synthesis in Escherich/a coli the presence of the t Present address: Laboratoire de g6u6tique physiologique, Ins~itu't de Botanique, 8 Goethe, Strasbourg, l~rance. 165
rue
166
~F, L A C R O U T E AND G. S. S T E N T
galaetoside inducer influences the peptide-chain growth-period of the enzyme molecules. More precisely, these experiments were designed to ascertain whether the time elapsed between laying down of the chain initial NH2-end of the galactosidase polypeptide and the completion of the intact enzyme molecule bears an inverse relation to the inducer concentration. For this purpose, the time elapsed between the laying down of the NH~-termlnal threonine residue of a fl-galactosidase polypeptide chain and the appearance of t h a t chain in the complete, tetramerie enzyme protein was measured directly a t optimal a n d subgp~imal inducer concentrations. This second experiment provided strong evidence against regulation b y means of a n inducer-dependent growth period of fl-galactosidase synthesis, since the growth period of fl-galactosidase molecules was found to be 80 seconds, whether over-all enzyme synthesis proceeds a t 30% or a t 100% of the maximally induced rate.
2. Materials and M e t h o d s (a) E. eoli strains The permeaseless strain 800U(y-) was provided by C. Willson. (b) Bavaria1 growth Bacteria were grown in M9 minimal medium (Anderson, 1946) containing 0.5% glycerol as carbon and energy source, and supplemented with additional growth factors according to the needs of the various experiments. The generation time of strain 300U in this medium is 80 mln. The cultures were maintained at 37°C and aerated by vigorous shaking. (c) Re~¢~n~s M4 Buffer contains 0.01 •-Tris buffer at p H 7.4, 0'01 ~-MgC12, 0.2 M-!NIaC1, 0.8 ml. 2-mercapteethanol/l, 14C- and all.labeled threonine and leucine were purchased from Schwarz Bioresearch Inc., Orangcburg, N.Y.
(d) Pu~e-~Seli~ I n order to rnlnlmi~.e metabolic conversion of exogenous [14C]- and [SH]threouine atoms into isoleucine and other amino acids during pulse-labeling of the fl-galactosidasc protein, radioactive threonine was presented to the bacterial cultures in the presence of a mixture of non-radioactive amino acids. This mixture provided a final concentration in the M9 growth medium of 2/~g threonine/ml., 2/zg glutamic acid/ml., 2/~g isoleucine/ml., 4 ~g leucine/ml, and 4/zg glycine]ml. We are indebted to I. Zabin for recommending this procedure to us.
(e) fl-Gahwtosidas~ assay A modification of the assay procedure of Pardce, Jacob & Monod (1959) has been used. The assay medium, maintained at 28°C, contains 0"01 ~-NaC1, 0.02 ~-MgSO4, 0.1 r~-Tris at p H 7.4, 0.1 ~t-mercaptoethanol and 0-003 ~-ONPGt. The reaction is stopped b y addition of 0.5 M-NaaSOs and the intensity of color developed measured at 420 mt~. I n order to assay the enzyme content of intact bacteria, the cells are treated with toluene and 0.01% Triton XI00 detergent prior to addition of the ONPG substrate. (f) Part/a/l~u~iflcation oJ fl.ga~actosidase The enzyme purification method used is one suggested by I. Zabin as being suitable for the small quantities of fl-galactosidase recoverable in the present experiments. An E. coli culture containing about 1"5 × 1011 cells and pulse-labeled with [aH]- or [14C]threonine is centrifuged; the pellet is resuspended in 2-5 ml. M4 buffer; and the resuspension is treated for 10rain in a Raytheon sonic oscillator. The sonicate is centrifuged f o r 30rain at t Abbreviations used: ONPG, orthonitrophenylgalatoside; D1VP, dinitrophenyl.; IPTG, isopropylthiogalactoside.
~-GALACTOSIDASE PEPTIDE
CHAIN GROWTH
167
16,000 g. Of the supernatant fluid, 2 ml. are layered on top of a sucrose gradient increasing linearly from 5 to 20% sucrose in M4 buffer. This gradient is then centrifuged in a Spinco SW25 rotor at 25,000 rev./min for 20 hr. Fractions of about 1.35 ml. each of the gradient are then collected and their fl-galactosidase activity and SH or x4C content assayed. A typical result is shown in Fig. 1. The 3 fractions containing the peak enzyme activity and amounting to about 50% of the total enzyme activity of the gradient are now p u t on a D E A E Sephadex A50 column (1 cm diameter, 24 cm in height), washed with 50 ml. M4 buffer, and eluted b y means of a linear gradient of NaC1 in M4 buffer, increasing from 0.2 M to 0.8 M. The elution rate is maintained at 20 ml./hr b y means of a Sigma m o t o r precision pump. Fractions of 2-5 ml. each are collected in tubes containing 1.25 ml. NaC1free M4 buffer and assayed as soon as possible for their ~-galactosidase activity. The 3 peak activity fractions, corresponding to about 600//0 of the tot~tl enzyme activity recovered, are pooled and represent the partially purified ~-galactosidase enzyme on which the subsequent analysis is performed. |
"'
|
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i ,,v,,,,,,,,,
!
1
/ /
5
15 I0 Fraction number
@
20
Fio. 1. Distribution of fi-galactosidase activity and SH.labeled protein in fractions collected upon sucrose density-gradient sedimentation of a crude extract of bacteria grown in the presence of 2 × 10 -s ~-IPTG (maximal induction) and pulse.labeled for 210 see with [sH]threonine. - - O - - O - - , jg-Galactosidase activity; - - O - - O - - , radioactivii~y. The progress of enzyme purification was monitored b y measuring the ratio of ~-galaetosidase activity to [SH]- or [x~C]threonine radioactivity at various stages. I n those fully induced cultures which h a d been allowed to assimilate the labeled threonine for 210 see (the longest time in the present experiments), the final degree of purification ranged from 28- to 47-fold relative to the initial specific activity in the crude extract. For the 30o//o maximally induced cultures, the corresponding final degree of purification was 62-fold. Since these calculations are based on radioactivity introduced b y pulse-labeling, and since the fl-galactosidase polypeptide is larger t h a n the average protein, t h e y are undoubtedly over-estimates of the actual degree of enzyme purification. (g) NHg4erm~naZ rad~oactiv~y measurement I n order to assay the amount of SH- or 14C.labele d threonine present in the NHz-term~nus of ~-galactosidase protein, Sanger's (1945) fluorodinitrobenzene method, as described b y Fraenkel-Conrat, Harris & L e v y (1955), was used. To 10ml. of an ice-cold solution containing the partially purified, radioactively labeled ~-galactosidase, 3 m g of yeast RI~TA is added as a carrier, and the protein is precipitated b y addition of 1 ml. 50°//0
168
F. L A C R O U T E AND G. S. S T E N T
trichloroacetic acid~ The precipitate is collected b y eentrifugation at 16,000 g for 30 min and the supernatant liquid (which contains less than 1 ~o of the radioactivity) is discarded. The pellet is m~xed with 0.3 ml. of a 5% solution of sodium bicarbonate and with 2.5 ml. of a 5~/o solution of fluorodlnitrobenzene in 95°/o ethanol. This m ~ t u r e is incubated for 2 hr at room temperature, before 3 ml. of ether are added. The precipitate is centrifuged, the supernatant fraction is discarded and the pellet is resuspended in 4 ml. of ether. A second centrifugatlorL is now undertaken, after which the pellet is resuspended in a mixture of 1 ml. ~-HC1 and 4 ml. of ether. The aqueous phase of this suspension is separated and extracted once more with 4 ml. of ether. Now 1.1 ml. of concentrated HC1 and 0"I ml. of a 10% solution of Casamlno acids are added to~the ~xtracted aqueous phase, which is heated briefly to 100aC to bring all material into solution. This solution is transferred t o a new tube, to which a 1.5.ml. 6 ~r-HCI washing of the old tube is added. The new tube is sealed under vacuum and its contents hydrolyzed for 4 hr at ll0°C. The contents of the tube are then diluted to 25 ml. with water and extracted 3 times with 5 ml. ether. The ether extracts containing the DNP.amino acids are pooled in a plastic scintillation vial, the ether is evaporated off, 10 ml. of Bray's scintillation fluid is added and both 1~C and 8H radioactivity assayed in a liquid-scintillation spectrometer. The presence of DNP-amlno acids in the samples to be assayed causes some scintillation quenching, and hence a reduction in efficiency of counting 14C and ~H radioactivity. I n order to estimate the amount of quenching, standard amounts of both isotopes were added to each scintillation vial after the first count and the radioactivity of the vial recounted. The first count was then corrected b y the quenching factor inferred from the second count. (h) Measurement of to~.l radioactivity in bct~terialprotein Bacterial cultures having assimilated [14C]- or [SH]threonine were collected on a Millipore filter and washed twice with 10 ml. of ice-cold 5~/o trichloroacetic acid. The radioactivity present on the filters was then measured in a liquid-scintillation spectrometer.
3. R e s u l t s I n order to test the notion of an inducer-dependent growth period of the fi-galactosidase polypeptide chain, the growth period was measured b y following the l~]n~tics of appearance of a radioactive label in the NH2-term~nal threonine of intact fl-galactosidase molecules (Brown, Koorajian, K a t z e & Zabin, 1966) upon adminiStration of labeled threonine to growing E. cell in m a ~ m a l and s u b m a ~ m a l states of induction. I n these experiments three parallel cultures of strain 300U(y-) were grown for more t h a n three generations in Mg-glyeerol medium, one in the absence of I P T G (non-induced), one in the presence of 5 × I 0 - S M - I P T G (30~/o, m a x i m a l induction), and one in the presence of 2 × 10 -3 ~ - I P T G ( m a m m a l induction). At this point, [SH]threonine was added to the non-induced culture and [14C]threonine was added to the two induced cultures. (In some experiments, the distribution of the label was reversed, so t h a t the non-induced culture received [14C]threonine and the induced cultures [3H]threonine.) Incorporation of radioactive material was stopped a t various later times b y chl]Hng and adding 0-02 M-sodium azide to samples of these cultures. Figure 2(a) presents the ~]netics of incorporation of radioactive threon~ne into the total acid-insoluble fraction of these bacteria in one such experiment. As can be seen, the total a m o u n t of radioactive threonine incorporated rises linearly with time over the 210-second labeling period. However, the extrapolation to zero incorporation of the curves appears to intersect the time abscissa slightly before zero time, a feature t h a t has been observed in all the experiments to be summarily reported in Figure 2(b). The mean extrapolation to zero incorporation of these experiments was five seconds, which time m u s t represent a period of residual incorporation following the
fl-GALACTOSIDASE PEPTIDE I
I
t
(
I
CHAIN GROWTH |
/d
Ca)
!
.J
¢/ 60
120
3,0
2'5 ,~ 8
0
0
169
2"0
180
Seconds of labeling o
/ I 7
(b)
.g_
3"5 ~ /
-0
I
I
30
60
I" /t I
I
I
!
&
!
90 120 150 180 2i0 240 Seconds of labeling
FzG. 2. Pulse-labeling of fl-galactosidaes with radioactive threonlne in submaximaUy and maximally induced E. colt cultures. Panel (a): incorporation of radioactive threonine into total acid-insoluble protein of bacteria. Panel (7o): corrected radioactivity of DN'P-threonine recovered from partially purified fl-galactosidase protein, per unit of enzyme activity after various periods of labeling.
Symbol
Induction
• Q O •
maximal m~Y~mal 30% of maximal ma~irnel none
×
Label of threonine in ex~oerimenf~al
Label of threonine in non-induced control culture
SH x4C ~4C 14C
14C SH SH SH SH
culture
--
chilling and sodium azide addition to the samples. Hence in the kinetic analysis of the remainder of this experiment, a constant correction of five seconds was added to the measured reaction times. The fl-galactosidase enzyme was extracted and purified from these various culture samples. Its activity and the amount of radioactive threonine present in the NH2termini of the purified enzyme were then determined, according to the procedures described in the Materials and Methods section. The degree of purification of fl-galactosidase protein achieved by each extraction was monitored by working up a mixture of a non-induced and an induced culture, one labeled with [SIll- and the other
170
F. LACROUTE
AND
G. S. S T E N T
with [14C]threonine_~f accurately known specific radioactivity for identical reaction times. The ~-galactosidase NH~-terminal threonlne recovered from this mixed extract was then counted separately for its content of 8H and 14C isotope. Now since the non-induced culture contained negligible quantities of ~-galactosiclase, the radioactivity recovered corresponding t o its isotope represents NH2-termlnal threonlne of proteins contaminating the ~-galactosidase isolated from the induced cultures. This conta"ruination must be subtracted from the NH2-termJnal threonine recovered bearing the isotope of the induced Culture, i~ order to gain a true measure of the labeling of the NH~-termlnl of the ~-gal~ctesidase protein. This correction Varied from 4 to 10~/o of the total NH2-termlnal threon~ne, depending on the experiment. (The level of contamination of NH~-term~nal threonine is considerably lower than the level of threonlne contamination of the whole ~-galactosidase protein (which varied between 30 and 40~/o), since most of the contaminating proteins do not, apparently, carry threonlne as their NH2-termlnal amino acid.) The corrected amount of NH~terminal radioactive threonine recovered from the induced cultures was divided by the amount of fl-galaetosidase enzyme activity found in the same purified preparation, in order to determine the specific degree of labeling per unit of enzyme activity present in the growing bacterial cultures. The results of these analyses are presented in Figure 2(b). It can be seen there that after an initial lag, the specific radioactivity of the fl-galactosidase NH2-terminal threonine begins to rise linearly with time, so that after 200 seconds of labeling about 3% of the final, steady-state radioactivity (cf. below) has been attained. Most important, i~ can be seen that the I~inetics of NHz-terminal labeting of the 8ubma~iraally induced culture are virtually indistinguishable from those of the maximally induced vultures. The extrapolation of these linear incorporation kinetics to zero labeling yields an average lag time of 75 seconds, to which the 5-second correction due to residual incorporation after stopping bacterial growth must still be added. I t thus appears from these results that the time elapsed between laying down the NH2-termmal threonine and completion of the finished protein molecule, i.e. the growth period of the ~-galactosidase polypeptide, is 80 seconds, whether synthesis proceeds under conditions of ma~rn~l induction or of 30~/o of mammal induction. An additional experiment was carried out in which the specific activity of the NH2-term~nal threonlne was measured after a 260-second labeling period, in both maximally and subma~mally induced cultures. Although the two single time-points obtained in that experiment do not permit extrapolation to determine the polypeptide growth period, they do confirm the main result of Figure 2, in that after 260 seconds the fl-galactosidase protein in the subma~mally induced culture had re~ched the same specific radioactivity in i~s NH2-termlnus as the ma~mally induced culture. In order to assess the reliability of the determination of NH2-termlnal labeling of ~-galactosidase protein, control experiments were carried out in which fully induced and non-induced cultures were labeled with 1~C- and 8H-labeled threonine for a period of one hour. The ~-galactosidase was then extracted and purified from a m ~ u r e of these long-term labeled cultures and the ratio of the corrected amount of NH~terminal threonine label to total threonlne label in the enzyme was determined. The specific NH2-term~nal activity per unit of enzyme activity represents half the steadystate labeling level and the ratio of NH2-termlnal to total radioactivity the reciprocal of the number of threonine residues per ~-galactosiclase polypeptide. This ratio was found to be 0"95~/o. Since it has been reported (Craven, Steers & Anfinsen, 1965) that
fl-GALACTOSIDASE P E P T I D E CHAII~ GROWTH
171
the ~-galactosidase polypeptide chain contains 65 threonine residues, the expected ratio would be 1"5~/o. This discrepancy between observe~ and expected values probably reflects the partial destruction of DNP-threonine during subsequent acid hydrolysis of the protein, which has been reported to amount to as much as 50~/o of the t o t ~ (Brown et a/., 1966). With this proviso, it would appear that the present experiments confirm the previous rel~ort (Brown et al., 1966) that threonlne/8 the NH2-terminus of E. col~fl-galactosidase. 4. D i s c u s s i o n
The equality of the growth.times of the fl-galactosidase polypeptide in ma~mally and subma~imally induced cultures reported here thus appears to rule out the possibility that in the subma~imally induced culture the differential rate of enzyme synthesis is only 30~/o that of the fully induced culture, because the growth period of the enzyme in the former is three times that in the latter. This finding does not, of course, eliminate altogether the notion of regulation of fl-galactosidase synthesis by an inducer-dependent variation in the specific peptide-chain growth-period. For example, it could still be supposed that there exists a "regulatory" polypeptide (coded ~ by a stretch of genetic material operator-proTimal to the codon specifying the NH~-termlnal threonine of the mature enzyme) the growth period of which does bear an inverse relation to the inducer concentration. This polypeptide could represent either the putative "trtm", formylmethionyl NH2-terminus of the polypeptide, which is cut away upon maturation of the enzyme protein, or a separate protein coded for in the operator region. The existence of such an operator-pro~m~l regulatory polypeptide would also explain the stimulatory effect of trimethoprim inhibition on the differential rate of fl-galactosidase synthesis in submaximally induced cultures (Lacroute & Stent, 1968). Our attempts to reveal the existence of such a regulatory peptide by means of joint chromatography on DEAE cellulose columns of [14C]- and [3H]amluo acid-labeled extracts of induced and non-induced cultures, slm~lar to the procedure followed by Kolber & Stein (1966), did not give any positive results, however. The present measurement of 80 seconds for the steady-state growth-period of the fl-galactosidase protein is in excellent agreement with an earlier indirect estimate of 80 to 90 seconds by Kepes & Beguin (1966) for the growth period of the first enzyme molecules appearing upon addition of inducer to a previously non-induced culture. Since the fl-galactosidase polypeptide contains about 1170 amino acid residues (Craven et a/., 1965), one may reckon from its 80-second growth period that its rate of peptide chain growth is 1170/80 ~ 15 amino acids per second. This estimate of fl-galactesidase chain growth rate is in excellent agreement with Maal~e & Kjeldgaard's (1966) estimated growth rate of 16 amino acids per second per ribosome for the average protein of Salmonella. However, it is 40~/o higher than the value of 10.5 amino acids per second per ribosome determined by Schlcif (1967) for the growth of the average protein of the very same E. coli strain under the very culture conditions of the present experiments. In order to reconcile the present finding with that of Sehleif, one might suppose either that the fl-galactosidase polypeptide grows at a rate 4 0 ~ higher than that of the average E. coli protein, or that under the growth conditions of these experiments 40~/o of all ribosomes do not happen to be engaged in protein synthesis at any one instant.
Peptide Chain Growth of p-Galactosidase in Escherichia coil F. LACROUTEt AND G. S. STENT
Virus Laboratory and Del~artmen$ of Molecular Biology University of Galifornia Berkeley, Galifornia 94720, U.S.A. (Received 7 November 1967, and in revised form 29 March 1968) An experiment was carried out to test the notion that regulation of fl-galacimsidase synthesis in Es~herichia coli involves an inducer-dependent growth period of the enzyme protein. The results of this experiment, in which the time elapsed between laying down an N]Ka-terminal threonine of the fl-galactosidase polypeptide and its appearance in the intact enzyme molecule was measured directly in both maximally and submaximally induced cultures, provided strong evidence against an inducer-dependent growth period. For it was found that, in both maximally and 30% maximally induced cultures, the enzyme growth period is 80 seconds. This constant enzyme growth period indicates a peptide chain growth rate of 15 amino acids per second, a figure which is in reasonable agreement with an estimate of the average peptide chain growth rate per ribosome in the same bacterial strain. 1. I n t r o d u c t i o n Two general, antithetical possibilities can be considered to explain regulatory variations in the differential rate of synthesis of inducible or repressible bacterial enzymes: the variable over-an rate of synthesis of a particular enzyme could reflect: (1) simultaneous growth of a variable number of its polypeptide chains under an invariant peptide chain growth period; or (2) simultaneous growth of an invariant number of its polypeptide chains under a variable peptide-chain growth.period. B y peptide-chain growth-period is meant here the time elapsed between the start of synthesis of a peptide chain and its appearance in the complet~ enzyme molecule. Both of these possibilities are compatible with Jacob & Monod's operon (1961) model of regulation of bacterial protein synthesis, although this model is usually discussed in terms of only the first of these possibilities. Indeed, either of these possibilities could obtain, whether the primary site of control is at the level of transcription of the DNA or at the level of translation of the messenger RNA. An appendix to this paper briefly describes two feasible regulatory models, one involving transcriptional and the other translational control, which do incorporate the element of variable peptidechain growth-period, instead of the canonical notion of variability in the number of growing polypeptide molecules. The experiments to be presented here are directed ¢~ the question of whether in the regulation of fl-galaetosidase synthesis in Escherich/a coli the presence of the t Present address: Laboratoire de g6u6tique physiologique, Ins~itu't de Botanique, 8 Goethe, Strasbourg, l~rance. 165
rue
166
~F, L A C R O U T E AND G. S. S T E N T
galaetoside inducer influences the peptide-chain growth-period of the enzyme molecules. More precisely, these experiments were designed to ascertain whether the time elapsed between laying down of the chain initial NH2-end of the galactosidase polypeptide and the completion of the intact enzyme molecule bears an inverse relation to the inducer concentration. For this purpose, the time elapsed between the laying down of the NH~-termlnal threonine residue of a fl-galactosidase polypeptide chain and the appearance of t h a t chain in the complete, tetramerie enzyme protein was measured directly a t optimal a n d subgp~imal inducer concentrations. This second experiment provided strong evidence against regulation b y means of a n inducer-dependent growth period of fl-galactosidase synthesis, since the growth period of fl-galactosidase molecules was found to be 80 seconds, whether over-all enzyme synthesis proceeds a t 30% or a t 100% of the maximally induced rate.
2. Materials and M e t h o d s (a) E. eoli strains The permeaseless strain 800U(y-) was provided by C. Willson. (b) Bavaria1 growth Bacteria were grown in M9 minimal medium (Anderson, 1946) containing 0.5% glycerol as carbon and energy source, and supplemented with additional growth factors according to the needs of the various experiments. The generation time of strain 300U in this medium is 80 mln. The cultures were maintained at 37°C and aerated by vigorous shaking. (c) Re~¢~n~s M4 Buffer contains 0.01 •-Tris buffer at p H 7.4, 0'01 ~-MgC12, 0.2 M-!NIaC1, 0.8 ml. 2-mercapteethanol/l, 14C- and all.labeled threonine and leucine were purchased from Schwarz Bioresearch Inc., Orangcburg, N.Y.
(d) Pu~e-~Seli~ I n order to rnlnlmi~.e metabolic conversion of exogenous [14C]- and [SH]threouine atoms into isoleucine and other amino acids during pulse-labeling of the fl-galactosidasc protein, radioactive threonine was presented to the bacterial cultures in the presence of a mixture of non-radioactive amino acids. This mixture provided a final concentration in the M9 growth medium of 2/~g threonine/ml., 2/zg glutamic acid/ml., 2/~g isoleucine/ml., 4 ~g leucine/ml, and 4/zg glycine]ml. We are indebted to I. Zabin for recommending this procedure to us.
(e) fl-Gahwtosidas~ assay A modification of the assay procedure of Pardce, Jacob & Monod (1959) has been used. The assay medium, maintained at 28°C, contains 0"01 ~-NaC1, 0.02 ~-MgSO4, 0.1 r~-Tris at p H 7.4, 0.1 ~t-mercaptoethanol and 0-003 ~-ONPGt. The reaction is stopped b y addition of 0.5 M-NaaSOs and the intensity of color developed measured at 420 mt~. I n order to assay the enzyme content of intact bacteria, the cells are treated with toluene and 0.01% Triton XI00 detergent prior to addition of the ONPG substrate. (f) Part/a/l~u~iflcation oJ fl.ga~actosidase The enzyme purification method used is one suggested by I. Zabin as being suitable for the small quantities of fl-galactosidase recoverable in the present experiments. An E. coli culture containing about 1"5 × 1011 cells and pulse-labeled with [aH]- or [14C]threonine is centrifuged; the pellet is resuspended in 2-5 ml. M4 buffer; and the resuspension is treated for 10rain in a Raytheon sonic oscillator. The sonicate is centrifuged f o r 30rain at t Abbreviations used: ONPG, orthonitrophenylgalatoside; D1VP, dinitrophenyl.; IPTG, isopropylthiogalactoside.
~-GALACTOSIDASE PEPTIDE
CHAIN GROWTH
167
16,000 g. Of the supernatant fluid, 2 ml. are layered on top of a sucrose gradient increasing linearly from 5 to 20% sucrose in M4 buffer. This gradient is then centrifuged in a Spinco SW25 rotor at 25,000 rev./min for 20 hr. Fractions of about 1.35 ml. each of the gradient are then collected and their fl-galactosidase activity and SH or x4C content assayed. A typical result is shown in Fig. 1. The 3 fractions containing the peak enzyme activity and amounting to about 50% of the total enzyme activity of the gradient are now p u t on a D E A E Sephadex A50 column (1 cm diameter, 24 cm in height), washed with 50 ml. M4 buffer, and eluted b y means of a linear gradient of NaC1 in M4 buffer, increasing from 0.2 M to 0.8 M. The elution rate is maintained at 20 ml./hr b y means of a Sigma m o t o r precision pump. Fractions of 2-5 ml. each are collected in tubes containing 1.25 ml. NaC1free M4 buffer and assayed as soon as possible for their ~-galactosidase activity. The 3 peak activity fractions, corresponding to about 600//0 of the tot~tl enzyme activity recovered, are pooled and represent the partially purified ~-galactosidase enzyme on which the subsequent analysis is performed. |
"'
|
"" "" '"
i ,,v,,,,,,,,,
!
1
/ /
5
15 I0 Fraction number
@
20
Fio. 1. Distribution of fi-galactosidase activity and SH.labeled protein in fractions collected upon sucrose density-gradient sedimentation of a crude extract of bacteria grown in the presence of 2 × 10 -s ~-IPTG (maximal induction) and pulse.labeled for 210 see with [sH]threonine. - - O - - O - - , jg-Galactosidase activity; - - O - - O - - , radioactivii~y. The progress of enzyme purification was monitored b y measuring the ratio of ~-galaetosidase activity to [SH]- or [x~C]threonine radioactivity at various stages. I n those fully induced cultures which h a d been allowed to assimilate the labeled threonine for 210 see (the longest time in the present experiments), the final degree of purification ranged from 28- to 47-fold relative to the initial specific activity in the crude extract. For the 30o//o maximally induced cultures, the corresponding final degree of purification was 62-fold. Since these calculations are based on radioactivity introduced b y pulse-labeling, and since the fl-galactosidase polypeptide is larger t h a n the average protein, t h e y are undoubtedly over-estimates of the actual degree of enzyme purification. (g) NHg4erm~naZ rad~oactiv~y measurement I n order to assay the amount of SH- or 14C.labele d threonine present in the NHz-term~nus of ~-galactosidase protein, Sanger's (1945) fluorodinitrobenzene method, as described b y Fraenkel-Conrat, Harris & L e v y (1955), was used. To 10ml. of an ice-cold solution containing the partially purified, radioactively labeled ~-galactosidase, 3 m g of yeast RI~TA is added as a carrier, and the protein is precipitated b y addition of 1 ml. 50°//0
168
F. L A C R O U T E AND G. S. S T E N T
trichloroacetic acid~ The precipitate is collected b y eentrifugation at 16,000 g for 30 min and the supernatant liquid (which contains less than 1 ~o of the radioactivity) is discarded. The pellet is m~xed with 0.3 ml. of a 5% solution of sodium bicarbonate and with 2.5 ml. of a 5~/o solution of fluorodlnitrobenzene in 95°/o ethanol. This m ~ t u r e is incubated for 2 hr at room temperature, before 3 ml. of ether are added. The precipitate is centrifuged, the supernatant fraction is discarded and the pellet is resuspended in 4 ml. of ether. A second centrifugatlorL is now undertaken, after which the pellet is resuspended in a mixture of 1 ml. ~-HC1 and 4 ml. of ether. The aqueous phase of this suspension is separated and extracted once more with 4 ml. of ether. Now 1.1 ml. of concentrated HC1 and 0"I ml. of a 10% solution of Casamlno acids are added to~the ~xtracted aqueous phase, which is heated briefly to 100aC to bring all material into solution. This solution is transferred t o a new tube, to which a 1.5.ml. 6 ~r-HCI washing of the old tube is added. The new tube is sealed under vacuum and its contents hydrolyzed for 4 hr at ll0°C. The contents of the tube are then diluted to 25 ml. with water and extracted 3 times with 5 ml. ether. The ether extracts containing the DNP.amino acids are pooled in a plastic scintillation vial, the ether is evaporated off, 10 ml. of Bray's scintillation fluid is added and both 1~C and 8H radioactivity assayed in a liquid-scintillation spectrometer. The presence of DNP-amlno acids in the samples to be assayed causes some scintillation quenching, and hence a reduction in efficiency of counting 14C and ~H radioactivity. I n order to estimate the amount of quenching, standard amounts of both isotopes were added to each scintillation vial after the first count and the radioactivity of the vial recounted. The first count was then corrected b y the quenching factor inferred from the second count. (h) Measurement of to~.l radioactivity in bct~terialprotein Bacterial cultures having assimilated [14C]- or [SH]threonine were collected on a Millipore filter and washed twice with 10 ml. of ice-cold 5~/o trichloroacetic acid. The radioactivity present on the filters was then measured in a liquid-scintillation spectrometer.
3. R e s u l t s I n order to test the notion of an inducer-dependent growth period of the fi-galactosidase polypeptide chain, the growth period was measured b y following the l~]n~tics of appearance of a radioactive label in the NH2-term~nal threonine of intact fl-galactosidase molecules (Brown, Koorajian, K a t z e & Zabin, 1966) upon adminiStration of labeled threonine to growing E. cell in m a ~ m a l and s u b m a ~ m a l states of induction. I n these experiments three parallel cultures of strain 300U(y-) were grown for more t h a n three generations in Mg-glyeerol medium, one in the absence of I P T G (non-induced), one in the presence of 5 × I 0 - S M - I P T G (30~/o, m a x i m a l induction), and one in the presence of 2 × 10 -3 ~ - I P T G ( m a m m a l induction). At this point, [SH]threonine was added to the non-induced culture and [14C]threonine was added to the two induced cultures. (In some experiments, the distribution of the label was reversed, so t h a t the non-induced culture received [14C]threonine and the induced cultures [3H]threonine.) Incorporation of radioactive material was stopped a t various later times b y chl]Hng and adding 0-02 M-sodium azide to samples of these cultures. Figure 2(a) presents the ~]netics of incorporation of radioactive threon~ne into the total acid-insoluble fraction of these bacteria in one such experiment. As can be seen, the total a m o u n t of radioactive threonine incorporated rises linearly with time over the 210-second labeling period. However, the extrapolation to zero incorporation of the curves appears to intersect the time abscissa slightly before zero time, a feature t h a t has been observed in all the experiments to be summarily reported in Figure 2(b). The mean extrapolation to zero incorporation of these experiments was five seconds, which time m u s t represent a period of residual incorporation following the
fl-GALACTOSIDASE PEPTIDE I
I
t
(
I
CHAIN GROWTH |
/d
Ca)
!
.J
¢/ 60
120
3,0
2'5 ,~ 8
0
0
169
2"0
180
Seconds of labeling o
/ I 7
(b)
.g_
3"5 ~ /
-0
I
I
30
60
I" /t I
I
I
!
&
!
90 120 150 180 2i0 240 Seconds of labeling
FzG. 2. Pulse-labeling of fl-galactosidaes with radioactive threonlne in submaximaUy and maximally induced E. colt cultures. Panel (a): incorporation of radioactive threonine into total acid-insoluble protein of bacteria. Panel (7o): corrected radioactivity of DN'P-threonine recovered from partially purified fl-galactosidase protein, per unit of enzyme activity after various periods of labeling.
Symbol
Induction
• Q O •
maximal m~Y~mal 30% of maximal ma~irnel none
×
Label of threonine in ex~oerimenf~al
Label of threonine in non-induced control culture
SH x4C ~4C 14C
14C SH SH SH SH
culture
--
chilling and sodium azide addition to the samples. Hence in the kinetic analysis of the remainder of this experiment, a constant correction of five seconds was added to the measured reaction times. The fl-galactosidase enzyme was extracted and purified from these various culture samples. Its activity and the amount of radioactive threonine present in the NH2termini of the purified enzyme were then determined, according to the procedures described in the Materials and Methods section. The degree of purification of fl-galactosidase protein achieved by each extraction was monitored by working up a mixture of a non-induced and an induced culture, one labeled with [SIll- and the other
170
F. LACROUTE
AND
G. S. S T E N T
with [14C]threonine_~f accurately known specific radioactivity for identical reaction times. The ~-galactosidase NH~-terminal threonlne recovered from this mixed extract was then counted separately for its content of 8H and 14C isotope. Now since the non-induced culture contained negligible quantities of ~-galactosiclase, the radioactivity recovered corresponding t o its isotope represents NH2-termlnal threonlne of proteins contaminating the ~-galactosidase isolated from the induced cultures. This conta"ruination must be subtracted from the NH2-termJnal threonine recovered bearing the isotope of the induced Culture, i~ order to gain a true measure of the labeling of the NH~-termlnl of the ~-gal~ctesidase protein. This correction Varied from 4 to 10~/o of the total NH2-termlnal threon~ne, depending on the experiment. (The level of contamination of NH~-term~nal threonine is considerably lower than the level of threonlne contamination of the whole ~-galactosidase protein (which varied between 30 and 40~/o), since most of the contaminating proteins do not, apparently, carry threonlne as their NH2-termlnal amino acid.) The corrected amount of NH~terminal radioactive threonine recovered from the induced cultures was divided by the amount of fl-galaetosidase enzyme activity found in the same purified preparation, in order to determine the specific degree of labeling per unit of enzyme activity present in the growing bacterial cultures. The results of these analyses are presented in Figure 2(b). It can be seen there that after an initial lag, the specific radioactivity of the fl-galactosidase NH2-terminal threonine begins to rise linearly with time, so that after 200 seconds of labeling about 3% of the final, steady-state radioactivity (cf. below) has been attained. Most important, i~ can be seen that the I~inetics of NHz-terminal labeting of the 8ubma~iraally induced culture are virtually indistinguishable from those of the maximally induced vultures. The extrapolation of these linear incorporation kinetics to zero labeling yields an average lag time of 75 seconds, to which the 5-second correction due to residual incorporation after stopping bacterial growth must still be added. I t thus appears from these results that the time elapsed between laying down the NH2-termmal threonine and completion of the finished protein molecule, i.e. the growth period of the ~-galactosidase polypeptide, is 80 seconds, whether synthesis proceeds under conditions of ma~rn~l induction or of 30~/o of mammal induction. An additional experiment was carried out in which the specific activity of the NH2-term~nal threonlne was measured after a 260-second labeling period, in both maximally and subma~mally induced cultures. Although the two single time-points obtained in that experiment do not permit extrapolation to determine the polypeptide growth period, they do confirm the main result of Figure 2, in that after 260 seconds the fl-galactosidase protein in the subma~mally induced culture had re~ched the same specific radioactivity in i~s NH2-termlnus as the ma~mally induced culture. In order to assess the reliability of the determination of NH2-termlnal labeling of ~-galactosidase protein, control experiments were carried out in which fully induced and non-induced cultures were labeled with 1~C- and 8H-labeled threonine for a period of one hour. The ~-galactosidase was then extracted and purified from a m ~ u r e of these long-term labeled cultures and the ratio of the corrected amount of NH~terminal threonine label to total threonlne label in the enzyme was determined. The specific NH2-term~nal activity per unit of enzyme activity represents half the steadystate labeling level and the ratio of NH2-termlnal to total radioactivity the reciprocal of the number of threonine residues per ~-galactosiclase polypeptide. This ratio was found to be 0"95~/o. Since it has been reported (Craven, Steers & Anfinsen, 1965) that
fl-GALACTOSIDASE P E P T I D E CHAII~ GROWTH
171
the ~-galactosidase polypeptide chain contains 65 threonine residues, the expected ratio would be 1"5~/o. This discrepancy between observe~ and expected values probably reflects the partial destruction of DNP-threonine during subsequent acid hydrolysis of the protein, which has been reported to amount to as much as 50~/o of the t o t ~ (Brown et a/., 1966). With this proviso, it would appear that the present experiments confirm the previous rel~ort (Brown et al., 1966) that threonlne/8 the NH2-terminus of E. col~fl-galactosidase. 4. D i s c u s s i o n
The equality of the growth.times of the fl-galactosidase polypeptide in ma~mally and subma~imally induced cultures reported here thus appears to rule out the possibility that in the subma~imally induced culture the differential rate of enzyme synthesis is only 30~/o that of the fully induced culture, because the growth period of the enzyme in the former is three times that in the latter. This finding does not, of course, eliminate altogether the notion of regulation of fl-galactosidase synthesis by an inducer-dependent variation in the specific peptide-chain growth-period. For example, it could still be supposed that there exists a "regulatory" polypeptide (coded ~ by a stretch of genetic material operator-proTimal to the codon specifying the NH~-termlnal threonine of the mature enzyme) the growth period of which does bear an inverse relation to the inducer concentration. This polypeptide could represent either the putative "trtm", formylmethionyl NH2-terminus of the polypeptide, which is cut away upon maturation of the enzyme protein, or a separate protein coded for in the operator region. The existence of such an operator-pro~m~l regulatory polypeptide would also explain the stimulatory effect of trimethoprim inhibition on the differential rate of fl-galactosidase synthesis in submaximally induced cultures (Lacroute & Stent, 1968). Our attempts to reveal the existence of such a regulatory peptide by means of joint chromatography on DEAE cellulose columns of [14C]- and [3H]amluo acid-labeled extracts of induced and non-induced cultures, slm~lar to the procedure followed by Kolber & Stein (1966), did not give any positive results, however. The present measurement of 80 seconds for the steady-state growth-period of the fl-galactosidase protein is in excellent agreement with an earlier indirect estimate of 80 to 90 seconds by Kepes & Beguin (1966) for the growth period of the first enzyme molecules appearing upon addition of inducer to a previously non-induced culture. Since the fl-galactosidase polypeptide contains about 1170 amino acid residues (Craven et a/., 1965), one may reckon from its 80-second growth period that its rate of peptide chain growth is 1170/80 ~ 15 amino acids per second. This estimate of fl-galactesidase chain growth rate is in excellent agreement with Maal~e & Kjeldgaard's (1966) estimated growth rate of 16 amino acids per second per ribosome for the average protein of Salmonella. However, it is 40~/o higher than the value of 10.5 amino acids per second per ribosome determined by Schlcif (1967) for the growth of the average protein of the very same E. coli strain under the very culture conditions of the present experiments. In order to reconcile the present finding with that of Sehleif, one might suppose either that the fl-galactosidase polypeptide grows at a rate 4 0 ~ higher than that of the average E. coli protein, or that under the growth conditions of these experiments 40~/o of all ribosomes do not happen to be engaged in protein synthesis at any one instant.