Measurement of the unstable RNA in exponentially growing cultures of Bacillus subtilis and Escherichia coli

J. Mol. Biol.

(1968) 31, 237-266

Measurement of the Unstable RNA in Exponentially Growing Cultures of Bacillus subtilis and Escherichia coli ~VINSTON

SALSER,~’

Jo,,

JANIN$ AND CYRUS LEVINTHAI,

Departmelit of Biology Massachusetts I?lstitute of Technology Cambridge, Muss., ~Y3.d. (Received 11 February

196’7, and in revised form 22 August 1967)

A new technique for measurmg the rate of turnover and quantity of unstable RKA in exponentially growing cells has been developed which escapes the interpretive difficulties of earlier methods. It depends upon the fact that an um stable RXA fraction has easdy measurable effects upon the kinetics of labelling of GTP; before the GTP pool can become fully labelled, unlabelled nucleotides present as GTP or in unstable RKA must be washed into stable RXA. (There is no equilibration with guanine in the medium.) Consequent,ly, if the uptake of label into GTP is rapid, with only the delay predicted from the experimentally measured GTP pool size. then it is possible to csonclude that there is little or no unstable RSA. In Bacillus subtilis, however, the GTP pool is labelled very slo~~ly which. Mith other data, indicat,es a large amount of unstable RSA (go/; of the guanine residues of the cell in unstable KSA). Similar measurements of the rapidity of GTP labelling in Escherichin coli indicate that there is much less unstable RX4 (about 33;,) in this organism. The curve expressing GTP specific activity as a function of time is shown to be the sum of two exponential terms with very different time constants. From the relative sizes of the two exponential terms, it is possible to estimate the rate of turnover of the unstable RIiA. The result is weighted most heavily for the slow componentjs of the decayin g messenger, and thus it sets an upper limit to the true avera,ge decay time for the unstable R?;A. This decay time (upper limit) x3-as l,hree minutes for the unstable RSA in B. subtilis grown at 37°C with a tloubling time of 116 minutes and six minutes when the cells were grown at, 30°C with a doubling timcx of 225 minutes. For E. coli (growing at 3b”C with a doubling time of 66 minutes) the decay time measured in this way was about four minutes. Thus the decay times as measured in these ex1,eriment.s are t,he same order of magnitude as have been observed by measuring the decay of pulselwbelled RKA and the decay of protein synthetic capacity after t,he addit,ion of act,inomycin. The various estimates of the decay rate of messenger R?JA in bacteria arc discussed in t,erlns of the possible thcorrtical ambiguities in the intcr~jretation of each.

1. Introduction Previous attempts to measure the amount of messenger RIVr\ in bacteria have been reviewed by Levinthal, Pan, Higa & Zimmermann (1963). The values obtained ranged from 1 to 90/ of the total RNA of the cell, but it was pointed out that each of 7 Present address: Institut de Biologio Physiro-Chirnique, f Present address: Laboratoire d’Enzyn~&gie, C.N.K.S., 237

Paris, France. Gif-sur-Yvct,tr,

France.

“3s

w

SA 1,s E 1-t. J

JANIS

ASD

(.’

LEVINI’HAI.

the techniques used involved assumptions for which there was no experimental proof, Plausible alternat’ives to many of t,hese assumptions were available which could explain the differences in t,he conclusions reached. In addition, since Bacillus subtilis was used in some types of experiments and Escherichia coli in others, actual differences between the two organisms could also explain some of the different values. A method, which does not, involve t,he use of any metabolic inhibitor and has a set of theoretical assumptions different, from those necessary for interpreting other typcls of experiments had been introduced by GKJS et cd. (196la,b). These workers made use of the fact that the presence of an unstable RKA pool will have a profound effect upon the kinet’ics of uptake of labelled guanine into the nucleotide triphosphate RXA precursor pools. Cells of E. coli (with a metabolic block to prevent conversion of guanine to adenine), were grown in exponential culture (generation time 170 minutes). At time zero [14C]guaninc was added to the medium and at subsequent times the soluble nucleotide pools were isolated by precipitation wit’h barium acetate and alcohol. If all RNA molecules were metabolically stable, then the precursor pools should be labelled in a time similar to that reyuiretl for the entr), of label into RKA. But if several per cent, of the Rh’A is unstable and in rapid equilibrium with the precursor pool, then t.he precursor nucleotide pool would reach maximum specific activity much more slowly- than anticipated from RNA incorporation curves. This was found to be the result and the aut’hors concluded that’ an unstable RNA fraction was present. Two dificulties restrict the value of this t’echnique as used by Gros et al. First, due in part to a large and variable background, the accuracy of measurement of the amount, of unstable RX-4 was limited and no measurement of the rate of RNA turnover was possible (the rat,e of RNA turnover may be computed from the curve showing uptaktx of label into GTP, but, only if these data are sufficientlj, accurate). Second, the label in all soluble nucleotides rather than in GTP alone was measured. Therefore the interpretation of the results depended upon the assumpt,ion that the major nurlcotide labelled was GTP, the direct precursor of Rn’S synthesis, rather tllan, for instance, GDP sugars which could have very different labelling kinctics. In this paper wc report the develolnncnt~ and USCof t,echnical improvcmcnts which greatl) reduce tdlese difficulties. 111adflition to measurement’s of the amounts of unstable RXA in B. subtdis and Ii:. coli, data hay(, been obtained which permit calculation of an upper limit for the true mass average decay time for unstable RKA, for which no estimate has been available. This complements the existing data for the dcacay time of pulse-labelled RNA in actinomycin, which gives a lower limit for t hc mass average decay t,ime of unstable RNA.

2. Theoretical Considerations In the following pages, we derivt> t)hc equations describing the uptake of labelled guanine into GTP and from t.herc into nucleic acid by the cell. These equations show how the shape of the GTP labelling curve is dcpendcnt upon the amount of unstable RNB and rate of t)urnovcr, as well as upon other measurable parameters of cell growth, and t,hty form t,hc basis for all t,he quantitative calculat,ions in the remainder of t’he paper. First’, consider the uptake of radioactive guanine into the GTP pool and from there into the bacterial nucleic acid. At, time zero, the guanine being taken into the cell,

UNSTABLE

RNA

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AND

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230

previously unlabelled, is given a specific activity A, which remains constant thereafter. The symbols P(t) and p(t) will be used to denote (respectively) the size of the GTP pool in pmoles of guanine equivalentlmg of cells and the speoific activity of GTP in pc/pmole, as functions of time after the addition of radioactive guanine. (ct) In the absence of an unstable RNA fraction, the washout of material from GTP is a simple exponential process Two simplified models for the uptake of guanine into nucleic acid are shown in Fig. 1. In both cases it is clear that for the GTP specific activity, ~(0) = 0 and p( co) = A. For a model where it is assumed that there is no unstable RNA (Fig. l(a)), the solution for p(t) at intermediate times is: p(t) = A (I- exp - (a (0) t/ P (0))) (1) where a(0) is the rate of intake of guanine into the GTP pool at time zero when the radioactive guanine is added, in ~moles/minute/unit of cells, and t is the time after this addition. This expression is correct for cells in the steady-state of exponential growth.

Unstable RNA CM)

b A

‘+

c

CTP

-

w

d

Stable nucleic acid Ls)

(b)

FIG. 1. Relationships between the stable and unstable nucleic acids and one of the direct precursors of RNA, GTP, are shown in simplified form. A represents the specific activity of the guanine in the medium, which is changed from zero to a positive value at time zero in the experiments described here, and remains constant thereafter. The small letters a, b, o and cl represent the rates of flow of guanine residues into and out of the GTP and nucleic acid fractions. The letters P, M and S denote the amounts of guanine, in pmoles of guanine equivalent, in GTP, the unstable RNA and the stable nucleic acids.

We shall refer to this type of behaviour as an exponential washout of the unlabelled guanine from the GTP pool. The shape of the curve p(t) is determined by P(O)/a(O), which we shall call the “washout time,” proportional to the amount of material which must be washed out, and inversely proportional to the rate at which material flows through the pool. Such an exponential washout is shown in curve A, Fig. 2. (b) An unstable RNA pool increases the time required for GTP to become labelled Now consider the more complex model shown in Fig. l(b), which includes an unstable RNA fraction. The amount of guanine in t,he unstable RNA fraction is

W.

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SALSER,

J. JANIN

AND

C. LEVINTHAL

denoted by &l(t) in pmoles of guanine equivalentlmg of cells. Its specific activity, as a function of time after the addition of the radioactive guanine, is m(t) in pc/pmnle. The rates of degradation and synthesis of the unstable RNA are b(t) and c(t) pmoles of guanine equivalent/minute/unit of cells. If these rates are very large, then the specific activity m(t) will be equal to p(t) at all times. In such a case the unstable 100 90 xl g

80 70

_”

60

g < 8

40

i!

30

50

20

Mass of cells

FIQ. 2. Theoretical curves showing GTP specific activity plotted against cell mass for several combinations of size and rate of turnover of the unstable RNA fraction, as predicted by the model shown in Fig. l(b). In all of the curves it is assumed that the radioactive guanine is added to the culture when the cell mass is one unit, and that the GTP pool P contains 2.5% of the total guanine residues in the cell (a value chosen to approximate the experimentally determined values in B. subtilis), the remaining 97.5% being in RNA and DNA. The amounts of unstable RNA and rates of unstable RNA turnover (expressed as relative velocities of reactions a and b) are as follows: Curve A: no unstable RNA; curve B: 2% unstable RNA, rate b very large compared to rate a; curzle C: 9% unstable RNA, rate b = rate a; curve D: 9% unstable RNA, rate b = 5-3 rate a.

RNA fraction merely acts as an extension of the GTP pool and increases the washout time, which becomes (M(0) + P(O))/a(O), Curve B in Fig. 2 shows the behaviour of p(t) if P(0) = 2.5% and M(0) = 2.0% of the total guanine incorporated into the cells and if the unstable RNA turns over very rapidly. Larger amounts of unstable RNA would produce proportionally greater increases in the washout time of equation (1). M(0) + P(0) may thus be measured by determining the kinetics of uptake of label into the cells and into the GTP pool. Of course, it is also possible to isolate and determine directly the amount of,GTP in the cells and this may be subtracted from the value obtained for M(0) + P(0) to give the size of t’he unstable RNA fration M(0). (c) The shape of the GTP label&g

curve depends upon the rate of turnover RNA

of unstable

There is a simple exponential washout of unlabelled material from GTP only if the GTP and unstable RNA are in instantaneous equilibrium with each other. When the unstable RNA turns over slowly its specific activity lags behind that of GTP. The differential equations for the general case are solved below and it is shown that’, in general, there is an initial rapid increase in the specific activity of GTP, followed by a much slower continued approach to the final specific activity. Mathematically,

UNSTABLE

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IN

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AND

E. coli

241

these two phases correspond to two exponential terms in the general solution for p(t) which is : p(t) = A[1 - U exp (- t/tJ -(l -U) exp (-t/t,)] (2) where U, tl and t, are constants which are determined directly from the experimental data as described in the derivation below. Curves A and B of Fig. 2, which hsve already been mentioned, represent special cases of this general solution in which there is only one exponential term. Curve C shows the result when P(0) = 2+5%, M(0) = 9% of t,he guanine incorporated by the cell and the rate of intake of guanine from the medium, u(t), equals the rate of flow of guanine, b(t), due to the breakdown of unstable RNA. It may be seen that this curve is the sum of the two exponential components of equation (2). These two components may be explained qualitatively as follows. The guanine taken into GTP via path a (Fig. l(b)) will be fully labelled very quickly. The input to GTP from the breakdown of unstable RNB (path 6) will have specific activity m(t) which is initially zero and only slowly approaches A. Consequently, bhe specific activity of the input will be initially A u(O)/(a(O) + b(0)). The specific activit,yr p(t) will rapidly rise toward the specific activity of this input (account ing for the fast initial rise). At the same time, label is entering unstable RNA and starting to appear in its breakdown products so that. the material entering GTP via pathway b becomes slowly labelled. This accounts for the slow increase of p(t) toward its final value (the slow second component of curve C). The size of the initial component will be roughly A a(O)/(a(O) + b(O)), and measurement of this value permits the computation of the rate of turnover of the unstable RNA. Finally, curve D of Fig. 2 shows the predicted behaviour based upon the same assumptions as curve C except for a faster turnover rate of the unstable RNA (b(t) = 5.3 a(t)). As a result, the magnitude of the fast component is decreased. This curve is very similar to actual data which we report below for B. subtilis. The slow washout is due to the large (9%) unstable RNA fraction. (d) Restrictions upon the mathematical derivation The following assumptions are made in the derivation of the analytical expression describing the behaviour of the model presented in Fig. l(b). (a) The cells are in steady-state growth; that is, all the components increase at an exponential rate. (b) The guanine nucleotide pools other than GTP are either small relative to GTP or they rapidly equilibrate with GTP and may therefore be treated, in kinetic arguments, as part of the GTP pool. (The path of RNA breakdown and conversion to GTP will not affect the behaviour of the model.) (c) There is no exchange with the guanine of the medium by the nucleotides of the cell which are in equilibrium with GTP. (d) In the first part of the derivation it is assumed that all unstable RKA molecules have the same decay time. (e) There is only one pool of GTP in the cell, which serves as the immediate precursor for the incorporation of guanine into all molecules of RNA, regardless of whether that guanine comes from outside the (guanine requiring) cells or from breakdown of unstable RNA. It is shown in Results that these experiments comply with conditions (a), (b) and (c). Condition (d) is probably not met and the mathematical treatment is extended to t,he ca,seof 1~unstable RNA pools in the second part of the derivation. It seems likely

242

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SALSER,

J. JANIN

AND

C. LEVINTHAL

that condition (e) is met in these experiments, i.e. that there are no private pools of GTP in bacterial cells. Derivatioln of the label&g kinetica for GTP The derivation of equation (2) is as follows: P(t), M(t), the sizes (in pmoles of guanine equivalent) of the GTP fraction, and the stable nucleic acid fraction. The rates a(t), b(t), c(t), and d(t) as described in Fig. l(b) guanine transferred/minute/mg of cells. They are related following conservation equations :

and s(t) are, respectively, pool, the unstable RNA are in terms of pmoles of to the pool sizes by the

dW

- db = a(t) + b(t) -c(t) -d(t) = (a(0) + b(0) -c(O) --d(O)) egt = KP(0)eX’, ‘9

=c(t)-b(t)

=(c(O) -b(O)) eKt=KM(0)eKt,

(3)

-$-=d(t)=d(0)eKt = KS(0)eKt where K is the time constant of the exponential growth of the cells. At time zero, the guanine being taken into the cell, previously unlabelled, is given a specific activity A, which remains constant thereafter. As mentioned previously, p(t) and m(t) are the specific activities of GTP and unstable RNA as functions of time after the addition of radioactive guanine. Equations analogous to equation (3) may be written to express the conservation of the radioactivity in the exchange between each pool as a function of the time : Wt) x p(o)

= - (40) + b(O)) p(t) + b(0) m(t) + a(o

(4)

Equation (4) is solved for m(t) and differentiated to obtain expressions for m(t) and dm(t)/dt. These are substituted into equation (5) to give an expression not involving m(t) :

(6) a(O)KA 4WW -, = M(O)P(O) + P(O) The general solution for such a second-order differential p(t) = A[1 -U

exp( -t/tl)

--(1-

equation is:

U) exp( -t/t,)]

(7)

UNSTABLE

RNA

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AND

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243

which is the enalyticaJ solution for the model, subject to the assumptions described earlier. The constants t, and t, are the two roots of the quadratic equation

To evaluate the constant of integration U, we may take advantage of the fact that it must satisfy the boundary condition at t = 0 :

(9) This condition gives : u

_ -

ev1t2

7

v2

-

tlw)

W(O)

’

(10) I--u=

4w1t2 @l -

(e) Applying

-

tam

t2m)

*

the equations to actual data

Equations (7), (8) and (10) give a complete description of the behaviour of the GTP specific activity in our model during a labelling experiment. They permit the calculation of the experimentally measurable values t,, t, and U from the parameters a(O), b(O), P(O), N(O) and K. The inverse expressions are essential, however, to enable us to solve for P(0) and b(0) using the experimental data. By manipulation of t&e solutions of equation (8), it may be shown that: M(O) =

a2(0)P(O)t, + a2(0)P(O)t, - a(0)P2(O) - a3(0)t,tz > P(O)- a(O)Kt,t, b(0) =

Jm)

(P(O) --K4wlt2) 4ow2

(11)

(12)

where KN(O)/a(O) is the proportion of unstable RNA in the cell, and b(O)/M(O) is its decay constant. Consequently, it is necessary only to measure the growth rate K, the total incorporation of guanine (which gives a(O)), the total size P(0) of the pool of GTP (plus GDP, which is found experimentally to be in rapid equilibrium with GTP) and the shape of the GTP incorporation curve in order to determine the size of the messenger fraction and its decay t’ime. (f) Accuracy of the method As will be seen later, all these numbers can be determined experimentally with good accuracy. The value of t, which we define as the time constant of the most rapid component of equation (7) cannot be determined directly by measuring the slope of the uptake curve, but its value is strongly dependent upon another parameter, U, the magnitude of the rapid component of equation (7) which is not otherwise

244

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SALSER,

J. JANIN

AND

C. LEVINTHAL

used in expressions (11) and (12). U can be measured with good accuracy, and to take advantage of this, the following auxiliary equation, from equation (lo), is introduced :

t, = t, u/e (m/w)

) tz + u - 11

(13)

Now we shall question the accuracy with which M(0) and b(O), the size and rate of breakdown of the unstable RNA fraction, can be determined by this method. It is illustrative to simplify the equations by substituting some real values typical of experiments with B. subtilis and discarding the terms which make very small contributions to the answers. Such values are a(O) = 97 and b(0) = 514 (expressed as micromoles/minute/ml. of culture with an optical density of unity at a waveIength of 540 mp), P(0) = 360, M(0) = 1680 (ex p ressed as pmoles/ml. of culture with o.D.~$~ = l), X = 0.006 minute-l, t, =0*571 minute and tz = 23.9 minutes. This gives approximate relationships which will show upon which measurements the determination of M(O) and b(0) depend most strongly. A new term is introduced which has the advantage of being readily measurable from experimental data. It is the area between the curve v(t) and the final value p( co), as shown in Fig. 2, and is obtained from the integral : W

P( a) -p(t) i

dt = Area

A

= WV’(O) k+ WWW+W)P(O) W)W+W)WW. Neglecting terms which contribute expressions :

(14)

less than 4% of the answer gives the following

M(0) N (Area) a(0) -P(O)

(15)

and likewise from (12) and (13) M(O) WWW) b(0) etU a(O)QJ ’ 2

(16)

Expression (15) indicates that the accuracy of the M(0) measurement is almost directly proportional to the accuracy with which the experimental data define the area between the curve p(t) and its asymptotic value p( a). This is true, first because a(0) can be computed very accurately from the rate of incorporation of radioactive guanine into acid-precipitable material. Second, since P(0) is about one-third the size of ill(O) in B. subtilis under the conditions used here, a 30% error in P(0) will produce oniy a 10% error in M(0). The second of these expressions shows the measurements which contribute most strongly to the accuracy of the determination of b(O), the rate of breakdown of the unstable RNA. This expression is dominated by its first term M(0)/t2U. The accuracy of this determination is limited most in these experiments by the precision of measurement of U, the size of the rapid component of PW

UNSTABLE (g) Extetion

of the treatment

RKA

IN

B. aubtilis

AND

“-1-G

E. col,i

to remove the condition that all unstable same decay time

RNA

species have the

It is unrealistic to assume that the unstable RNA fraction can be represented by one decay constant. In cells treated with chloramphenicol, it has been shown (Nierlich, Salser & Levinthal, manuscript in preparation) that the data are consistent with two unstable fractions with decay times of 4 and 25 min. Other experiments (Salser St Barett, manuscript in preparation) indicate that the messenger RNA molecules for different proteins in normal cells have substantially different decay times. The longest decay time observed in actinomycin treated B. subtilis was about 8 mm, while the shortest decay times, as indicated by the decay of pulse-labelled RNA in actinomycin, cannot be much less than 1 min (Levinthal, Keynan & Higa, 1962 ; Fan, Higa & Levinthal, 1964). Consequently, a more accurate model would be similar to that pictured in Fig. l(b), each having a distinct decay but would involve a number, n, of unstable RNA fractions, time. By steps analogous to those used in deriving equations (5) and (6), it may be shown where the variables m (t) and M(t) are now subscripted with i to denote that they refer to the i-th unstable RNA fraction: i I=1

M,(O)

= (Area)

a(0) -P(O)

i I=1

‘F

SW (ml(t)

-p(t))

dt

(17)

0

Equation (17) is analogous to the approximate equation (15), and therefore the last term expresses the size of the error in equation (15). For fractions of RNA which turn over very rapidly, m,, the specific activity of the RNA will always be close top, the specific activity of the GTP pool. Consequently, the error introduced, expressed by this integral, will be very small for such fractions. With longer decay times the value of the integral in the error term of equation (17) becomes larger but it can be shown that the total error is less than 5% as long as the bulk of the unstable RNA has decay times smaller than about 14% of a generation time. Since there is no evidence for unstable RNA having such a long decay time in normal cells, the simple equation (15) will be adequate for this xvork. It may be shown for similar reasons, that the,.accuracy of the measurement presented here for M(0) does not necessarily depend upon our assumption that the decay process is exponential. A model in which an unstable RNA molecule has an allotted lifetime during which it is protected, and is then rapidly degraded, leads to a value for M(0) not differing appreciably from that obtained here. We may further ask whether the estimate of the average value of b(0) will be accurate if there are really several fractions of unstable RNA with different decay times. The errors which may occur are of t)wo sorts. The first is that, if there is a sizable fraction of the unstable RNA with a very long decay time, then the second phase of the GTP labelling from the straight, line expected in the semilog plot of curve p(t) will deviate seriously Fig. 6(b) of the sum of the two exponentials. Such a situation would make it impossible to measure t, unambiguously, but such an error fortunately would be obvious because of this deviation from the theoretical curve. Such deviations have not been observed. The second type of error m-hich might occur in the measurement of b(0) is due to RSA fractions which turn over rapidly, An unstable RiYA fraction with extremely rapid turnover would be in instantaneous equilibrium with GTP and act as as extension of the CTP pool. The p(t) curve would look perfectly normal but would show no evidence of that part of the true a(O) due to this hypothetical fraction. Consequently, if different species of RNA have different decay times, our calculation here will give an answer weighted most heavily for the fractions turning over slowly rather than a mass average decay time. The decay time calculated here will therefore be an upper limit for the possible mass average decay times. For a two-pool model having one pool of 1 -min decay time, one pool of 4-min decay time and a 1 ‘IO-min generation time, the difference between the mass average decay time and the value calculated using equations (ll), (12) and (13) was computed by numerical techniques and found to be less than 30%. A number of trial solutions suggests that this is about the greatest difference which could be obtained with a 1-min minimal decay time, but the difference can be further increased by assuming shorter decay times.

24G

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LEVINTHAL

3. Materials and Methods (a) Strains B. subtilis, strain 295, was obtained from Dr Robert Guthrie, Children’s Hospital, by X-ray treatment of Buffalo, N.Y. It requires exogenous guanine and was produced American Type Culture Collection no. 6061 derived from the Marburg strain. B. subtilis W23 Sms T- H- is a derivative of W23 SmR which requires thymidine and histidine. It was obtained from Dr Frank Rothman, Brown University. E. coli R25’7, requires guanine or xanthine. It was obtained from Dr Boris Magasanik of M.I.T. and originally came from the collection of Dr Joseph Gotts of the University of Pennsylvania Medical School. Added broth or histidine counteract the toxic effects of adenine or hypoxanthine. E. coli T-, U-, arg-, pro-, trp- met(16 T- TAU Bar), originally from Hanawalt, was obtained from Dr Ronald Rolfe,.St. Louis University School of medicine. (b) Culture

conditions

The Tris-glucose minimal medium used in these experiments contained 0.08 M-NaCl, 0.02 M-KCl, 0.02 M-NH&~, 0.12 M-Tris, 10e3 M-MgCl,, 0.0025 M-Na,SC,, 2 x 10m4 M-Cd&, 2 x 10e6 M-!hC12, 0.2% glucose, 7 x 10M4 M-KH,PO,, pH 7.5 with HCl. Additional supplements were added as required to satisfy the metabolic requirements of the bacteria, or as described in the individual experiments. Cultures were grown in rotary shakers at 37 or 3O”C, depending upon the growth rate desired. When desired, the culture medium was changed by centrifuging the cells for the minimal time necessary, discarding the supernatant and carefully wiping off any excess medium around the pellet with tissue paper before resuspending in warm medium. B. subtilis was stored as spores and a fresh inoculum used for each experiment. E. coli was maintained on slants with a complete medium. Cultures were grown, tested for growth requirements, and several dilutions made such that at least one culture would be in exponential growth at the right concentration on the following day when the experiment was performed. The growth of this culture was followed by measuring the optical density at 540 rnp in a Zeiss PMQII spectrophotometer for more than one doubling time prior to the beginning of the experiment. Cultures were not used unless at least one doubling had occurred without any change in the growth rate. (c) Experiments

with preconditioned

media

The preconditioned medium used in one experiment with B. subtilis was prepared by growing B. subtilis W23 Sma in it to an O.D .640 of 0.3. The medium was collected by filtration and supplemented with the necessary 14C and 3H radioactive and unlabelled guanine immediately before it was added t)o the culture of B. subtilis strain 295 growing in medium with [3H]guanine at the same specific acbivity. Hypoxanthine, a precursor of IMP, was used to supplement the medium in place of adenine. It was found to be equally effective in suppressing the conversion of labelled guanine to adenine nucleotides. The cells were grown at 30 instead of 37°C so that growth would be slower (generation time? 325 min) facilitating the taking of samples at early times. ay For the experiments with E. co&, preconditioned medium was prepared in the same with one important exception. The guanine used for the experiments with E. coli (but 7not in any of the experiments with B. subtilis) was supplied by Schwartz and had been prepared by hydrolysis of GMP. We have observed that this [r4C]guanine preparation contained about 0.5% [14C]guanosine and that, when this preparation is added directly to a culture containing unlabelled guanine, there is preferential utilization of the added radioactivity for a very short time, presumably until the small amount of guanosine is exhausted. To avoid such effects in the experiments reported here, the medium containing t The decay time is the time to decrease to l/e of the original value. It is 1.44 times the half-life and equals the amount of RNA divided by its rate of breakdown. The average decay time can be used when there may be several unstable RNA fractions and is the total amount of unstable RNA divided by the total rate of breakdown. The generation time is the time for a culture to increase by a factor of e and is I.44 times the doubling time.

UNSTABLE

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the [14C]- and [3H]guanine was preconditioned by incubating it with E. coli R257 for 0.6 generation time. These cells were removed by filtration just prior to the addition of this conditioned medium to the main culture. (d) Preparation

of nucleotides

f or electrophoreais

For the isolation of soluble nucleotides and measurement of their radioactivity, we have used the techniques developed by Nierlich & Vi&netter (manuscript in preparation) with several modifications. This modified technique is described briefly as follows. IO-ml. samples of labelled culture were pipetted into 5 ml. of 0.76 M-perchloric acid chilled in an ice bath. The perchloric acid contained a carrier mixture of m&belled nuoleotides including GMP, GDP, GTP, guanosine, frequently AMP, ADP and ATP and, in some experiments, GDP-mannose, IMP and XMP. The amount of each unlabelled carrier nucleotide was either 0.10 or 0.16 pmole in most experiments. Care was taken that the samples did not remain in perchloric acid long (unless stored frozen at - 20°C) or at elevated temperature, because control experiments showed that significant amounts of 5’-GMP and 2’, 3’-GMP appeared from degradation reactions if the samples were warmed to room temperature briefly. The samples were filtered on Millipore filters of 0.46 p pore size and the perchloric acid-soluble material absorbed onto 20 mg of acid-washed (Lipkin, Talbert & Cohn, 1954) Darco G50 activated charcoal. The charcoal with absorbed nucleotides was washed on a cellulose acetate membrane filter of 0.30 p pore size (Gelman Co.) with 5 ml. of 0.001 MHCl. The nucleotides were eluted with 4 ml. of 95% ethanol-l M-NH,OH-H,O (66: 10: 133, by vol.) pH 11.8. The eluate was dried by a stream of filtered air in a 37°C bath, taking care not to blow small droplets out of the drying tube. Evaporation to dryness took about 30 mm and control experiments indicated no detectable breakdown of GTP during this procedure.

(e) Electrophoresis The samples were applied to the electrophoresis paper by the method of Popowicz (1962) on small strips out from the origin. Electrophoresis was carried out on a 36-in. cold plate apparatus in 0.06 M-sodium citrate buffer at pH 4.1 or 2.6. A pH of 4.1 was chosen to give the best separation of GTP, ATP, GDP and ADP. When it became obvious that labelling of the adenine nucleotides was completely inhibited, the pH 2.6 system was used because it allows separation of 5’-GMP from XMP, IMP, 2’3’-GMP and deoxy-GMP. Considerable improvement of the cooling of the electrophoresis paper was obtained by using Mylar sheet (3-mil thickness) instead of polyethylene sheet for electrical insulation between the paper and the metal plate. The temperature differential between the electrophoresis paper (as measured by a thermistor probe) and t,he cooling water did not exceed 5°C even with power levels of 5 w/in .z Wicks of a triple thickness of electrophoresis paper were used to conduct the current from the buffer tanks to the cold plates,. Endosmosis barriers were not necessary and, in fact, were found to constitute high-resistance elements which generated heat sufficient to fuse the Mylar sheet at the high power levels routinely employed. Electrophoresis was carried out at 5300 v for 2 hr, monitoring the progress of the separation with an ultraviolet lamp. The temperature of the paper wasmaintained near 35°C because it was found that adsorption to t,he electrophoresis paper at lower temperatures causes some smearing of the GTP peak. After the electropherograms were dried, they were counted in a Vanguard Geiger automatic strip scanner, in order to determine the position and shape of the radioactive peaks. Comparison of these data with the ultraviolet absorption of the known carrier compounds was used as one criterion of the purity of the isolated compounds. The peak sizes were integrated digitally and these were used as a preliminary estimate of the amount of radioactivity in each compound. The recovery of each compound was measured by eluting it from the electropherograms, making the eluate 0.1 N in HCl, and measuring the optical absorption of each recovered carrier nucleotide from 3200 to 2100 A in the Cary recording spectrophotometer. The

248

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AlUD

C. LEVIXTHAL

radioactivity of the eluted sample was then determined and corrected for losses using the measured recovery of the carrier nucleotides. When samples are eluted from commonly employed electrophoresis papers, such as Whatman 3MM, there is frequently a high background of ultraviolet-absorbing material which may be further increased by traces of pyridine absorbed on to the paper. To avoid these difficulties, several precautions were taken, which involved very little extra effort and gave uniformly low ultraviolet absorption in control samples. An acid-washed paper (Whatman 3HR) was further washed with 0.01 M-EDTA, then xvith distilled n,at,er and then dried. This paper was used for the electrophoresis. Just before the samples were to be cut out and eluted, the strips were rolled and the roll inserted into the top of a beaker in the bottom of which there was 20 ml. of concentrated solution of KH,OH. This “column” was placed under vacuum so that) all pyridine was removed from the dry roll of electropherograms by gas chromatography as the ammonia passed through the short, column of rolled electrophoresis paper. Ammonia is known to displace pyridine and pyridine absorption spectra were never observed in blanks cut from strips treated in this manner, akhough they were common in our laboratory without such treatment. The ultraviolet-absorbmg spots were then cut out and trimmed very closely, leaving only a thin st,rip protruding from each side of the spot for elution purposes. The ultraviolet-absorbing background of appropriate blanks was subtracted from the absorption of the samples using the fact that in control series the magnitude of the background was found, as expected, to be l)roportional to the weight of the paper eluted. With these precautions, the optical absorption of the blanks was normally less than 0.1 at 2550 A (and longer wavelengths) while that of the samples was greater than 0.6 at 2550 A. The recoveries were normally quite uniform in all of the samples of a particular nucleotide from the same experiment. For example, in the experiment sho\vn in Figs 5 to 7, t,he specific activity curve for GTP and GDP was det,ermined in t)hree separate \vays. The first two used the recoveries calculated from the recovered ultraviolet absorbing material at 2740 and 2540 K. The average deviation of the two s&s of data obtained in this \\‘a~ 11as only 20,;. The third calculation used the radioactivity in each peak as measuretl by the Vanguard strip scanner, without any correction for recovery differences between t,he individual samples. In this experiment, at least, the recovery was so uniform that these uncorrected points fit the same theoretical curve specified by the results of the first t.n-o methods and their reliability appeared to be roughly similar as judged by the size of the fluctuations about this theoretical curve. In some experiments, a double-label technique .was used, [3H]guanine being present, m the medium in uniform specific activity before and after the addition of [14C]guanine to the medium. The specific activity of the GTP with respect to 14C was then determined by measuring the rat,io of the 14C and 3H in isolated GTP. For this purpose, the amounts of 3H and 14C in each sample were determined by counting in a scintillation counter using known standards to compute the ratio of efficiencies in each channel. In such experiments, the recovery of the individual carrier nucleotides was measured in only a few samlllrs in order to determine the sizes of the individual nucleotide pools. In control experiments with [‘V]GTP (Schwartz), it. was found that the breakdou II of’ to (:RlP GTP to GDP during the recovery procedure was about 5’;. and the breakclo\vn was less than 196. The recoveries of GMP, GDP and CTP were 79 and 727: in a cont’rol experiment with labelled nucleotides, but the recovery of GTP was nearer 50’$& in the kinetic experiments reported here. The percentage recovery was always quite reproducible among the samples from a particular experiment. (f) Purity

of the isolated

GTP

The establishment of the purity of the GTP peak is crucial for the interpretation of the results which will be presented. This was investigated in several M ays. In one exlleriment one set of samples was separated by electrophoresis at pH 4.1, wElile pH 2.0 (chosen to give the greatest separation of guanine nucleotides from xanthine and inosine nucleotidcs, which run near the guanine nucleotides at pH 4.1) was used for a parallel set of samples. The kinetics of labelling observed for the GTP peak were the same in both cases. GTP peaks isolated by pH 4.1 electrophoresis were re-run at high pH in borate buffer to separate

UNSTABLE

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249

GTP from deoxy-GTP. In no case was there evidence for contamination of the GTP peak with other compounds: the radioactivity peak was always symmetrical and coincided well with the ultraviolet absorption of the authentic GTP added as marker (see Fig. 4(a) and (b) ). The amount of deoxy-GTP was not large enough to be detected and is certainly less than 10% of the amount of GTP. The purity of the GTP peak is not surprising: its high charge to mass ratio gives it a higher electrophoretic mobility in these systems than would be expected for most of the compounds to which guanine might be converted.

(g) Acid-precipitable The total acid-precipitable digestion with NaOH were (1960).

mdiouctivity

radioactivity and that remaining determined essentially as described

acid-precipitable after by Roodyn & Mandel

(h) Chel-niculs Corporation (10 [S-14C]Cuanine-HCl was obtained from the New England Nuclear Inc. (30 &pmole). [8-3H]Guanine-HC1 was +/pmole) or from Schwartz Laboratories, obtained from Nuclear Chicago Corporation. Volk Radio-chemicals, or Merck, Sharpe & Dohme of Canada Ltd. ‘Y!-labelled GTP, GDP and GMP used to determine recoveries for the isolation procedures were obtained from Schwartz Laboratories, Inc. 5’-GMP,-GDP,-GTP,-AMP and-IMP were obtained from the California Corporation for Riochemical Research, 5’-GDP and guanosine were obtained from Schwartz Laboratories, Inc., guanine and adenine from Sigma Chemical Company, and B/-AMP, xanthine and hypoxanthine from Pabst Laboratories. GDP-mannose, sold by Sigma Chemical Company, was the gift of Andrew Wright.

4. Results (a)

Guanine

nucleotide pools and their interconversions

In order to use the model shown in Fig. l(b), it is necessary to use conditions where labelled guanine is not converted to adenine nucleotides, as in prototrophs of B. subtilis and E: coli, and to show that there are no guanine-containing compounds other than RNA present within the cell in amounts large enough to disturb the kinetics of entry of guanine into GTP. Furthermore, it is desirable to ensure that the externally supplied guanine is used as the sole source of guanine to the exclusion of de novo synthesis. In the following paragraphs it is shown that these conditions have been achieved. The pathways of de nova synthesis of IMP are well known and have been reviewed by Magasanik (1962). IMP serves as the branch point for the synthesis of adenine and guanine nucleotides, and also as an intermediate in all interconversions between the two. The main features of these pathways are shown in Fig. 3. The conversion of guanine to adenine nucleotides was prevented by supplying unlabelled adenine in high concentration to the medium. The conversion of this adenine t,o guanine, as well as the de nova synthesis of guanine, were prevented by using mutants which cannot convert IMP to GMP. To determine the sizes of the guanine nucleotide pools, cells of B. subtilis 295 were labelled for one doubling time in the presence of unlabelled adenine (10 pg/ml.). The size of the aggregate guanine nucleotide pools was estimated as 2.3 to 2*6o/o of the guanine residues in the nucleic acids and soluble nucleotides of the cell. GTP accounted for about 70 to 80% of these soluble nucleotides, GDP for about 10% and the remainder was distributed between GMP, GDP sugars, XMP, IMP and two small unidentified peaks. The radioactivity profile, uncorrected for recovery, is shown in Fig. 4(a).

250

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C. LEVINTHAL

dAMPedADP.=*dATP-wDNA

P‘

XMP ,I

FIG. 3. Pathways

u ATP

~

de nova

L

synthesis I\“ILII. SYNTHESIS -r--s.

of interconversion

of the purine

nucleotides.

Reactions whose pathways are still speculative are indicated by dotted lines. For instance, GTP is required for in vitro peptide synthesis; however, present data do not clearly differentiate between a need for catalytic amounts or stoichiometric breakdown to GMP or GDP. Reactions which are well established by biochemical data sre indicated by solid lines.‘Reactions established not only by enzyme isolation but also by the behaviour of mutants which lack the reaction are indicated by double lines. The evidence for many of these reactions has been reviewed by Magasanik (1962). The diagram represents evidence from 8 variety of different bacteria. We have found it useful in designing experiments and our data do not conflict with any of the pathwdys shown. The strains of bacteria. used in this work contained mutational blocks in the pathways leading from IMP to GMP as shown in the diagram. Abbreviations used: SAMP, adenylosuccinate; GDPX, GDP-sugar.

Figure 4(b) gives similar data for E. coli R257. The acid-soluble nucleotides were estimated as 1.5 to 1.7% of the total guanine residues in the cells. Of these acid-soluble nucleotides more than 70% were in GTP and 15% in GDP. A large part of the remainder is an unidentified compound running ahead of GTP in the position expected for a tetraphosphate of guanosine. (A smaller amount of such a compound was also observed frequently in the acid-soluble pools of B. subtilis.) The other compounds, especially GMP, are present only in very small amounts in E. COG. These results indicate first that our conditions successfully prevent the conversion of guanine to adenine nucleotides because no radioactivity was observed in ATP which is present in large amount,s in cells grown in this way. Second, we see that GMP, GDP sugars and other compounds, large amounts of which might disturb the kinetics of entry of label into GTP: are present only in small quantity. There are appreciable amounts of GDP, but the kinetics of labelling of GDP have been found in several experiments to be almost exactly the same as the kinetics of GTP labelling. This indicates that GDP and GTP are in very rapid equilibrium. Because of the small size of the GDP pool and its rapid equilibrium with GTP, we may consider it mathematically as part of the GTP pool in the model of Fig. 1 to correct for the small error it would otherwise cause.

UNSTABLE

5000

-

4000

-

RNA

IN

B. subtilis

AND

E. coli

251

7.:

v) 2 3000 6 T 3 :: .o zoooB CL XMP IO00 t

20

40 Distance

SO

60

from

oryn

(cm)

(a) I

I

I

I

I

40

50

I GDP -

IO hg1n

20 Distance

30 from ongln

(cm)

(b)

FIQ. 4. Guanine-labelled

nucleotides

of B. subtilti

and E. COG.

(a) Culture of B. subtilti, strain 295, exponentially growing in guanine (1.5 rg/ml.), and adenine (20 pg/ml.) supplemented minimal medium was labelled with [SV1*C]guanine (final specific activity 5 me/m-mole). For the sample shown, the cells were labelled for 100 min and prepared as described in Materials and Methods, using the pH 4.1 electrophoresis. GDPM is GDP-mannose. (b) E. coli labelled for 42 min in the experiment shown in Fig. 8 were prepared as described in Materials and Methods, using the pH 2.6 electrophoresis system. At the same time, the relative recoveries of the GMP, GDP and GTP were measured and used to compute the relative amounts of label originally present in GTP and GDP, as given in the text. In both cases the bulk of the radioactivity is found in GTP and GDP, bot,h substances being well separated from other I~~belled compounds.

G

252

W.

SALSER,

J. JANIN

AND

C. LEVINTHAL

The other nucleotide compounds of guanine which are present in even smaller amounts are neglected in Fig.1 because they are not present in amounts sufficient to cause an appreciable lag in the entry of labelled guanine into GTP. The quantitative validity of this last approximation is directly substantiated by other experimental findings. Specifically, some label appears in GTP (Figs 6 and 8) so rapidly as to rule out lags due to the passage of guanine through any undetected pool of appreciable size on the pathway to GTP. If GTP could be labelled by direct, two-way, exchange with the guanine in the medium in addition to the flow from the medium through GTP into nucleic acid, then our estimates of the amount of unstable RNA would be too low. Such exchange was investigated by transferring cells from medium with radioactive guanine to medium wit,h unlabelled guanine and measuring the amount of label released to the medium and the amount of trichloroacetic acid-soluble label within t’he cells (unpublished data). No release of label into the medium was detected for either B. subtiZis or E. coli, showing that once nucleotide precursors have been t)aken into the cell and phosphorylated they are no longer free to exchange with t,he medium. (b) Measurement

of soluble nucleotide

pool expansions guanine

after the addition

of external

Cells might be expected to respond to guanine newly added to their medium by taking it in and utilizing it with accompanying changes in guanine nucleotide pool sizes and in the patterns of synthesis of purine nucleotides. It would not be surprising if other changes, less easily foreseen, were to occur, and, if these required the synthesis of enzymes, several minutes might elapse before the cell had fully settled down to its new pattern of growth. Such processes might have profound but not easily understood effects upon the initial kinetics of incorporation of label into RNA and t,he soluble nucleotide pools. The possible effects of such departures from exponential growth have frequently been ignored (McCarthy & Bolton, 1964; Buchwald & Britten, 1963). To avoid such difficulties, we have used mutants blocked in de novo synthesis of guanine and have attempted to avoid changes in external guanine and adenine (or hypoxanthine) concentration at the time of addition of label. It was nevertheless possible to imagine t,hat a small increase inguanine concentration at the time of addition of label might result in a pool expansion which would rapidly draw a small amount of labelled guanine into the GTP pool. Since some of the most important data presented here consist of just such a rapid initial uptake of label into GTP, which we interpret in an entirely different way, it was necessary to rule out the possibility that our results could be due to such departures fromexponential growth. Such control experiments were carried out by two independent methods. Our conclusion from these experiments is first that there is an appreciable pool expansion when guanine is added to B. subtilis growing without guanine (B. subtilis W23 SmR which can grow in t,he absence of guanine was used in this experiment). Once the cells are growing in the presence of even a small amount of guanine, however, large increases (from two- to tenfold) in the external guanine concentration do not cause further appreciable pool expansions in either B. subtilis W23 SmR or R. subtilis 295, t,he guanine auxotroph used in these experiments. Consequently, the initial rapid uptake of label which we observe cannot be due to pool expansion. The detailed evidence for these conclusions is presented in an appendix to bhis paper.

UNSTABLE

RNA

IN B. aubtilis

AND

253

E. coli

(c) Measurement of the amount of unstable RNA and its rate of turnover in B. subtilis The uptake of radioactivity into GTP in B. subtilis 295 was measured. Adenine was supplied to prevent conversion of guanine to adenine nucleotides, and no radioactivity could be detected in ATP. Several precautions were taken to ensure that the cells were growing exponentially during the labelling. The culture was maintained in a state of exponential growth (generation time 170 minutes) for 16 hours prior to use. Cells were transferred from this exponential culture with excess guanine to the same medium with 3 pg of guanine per ml. Before adding the labelled guanine, one-third of a generation of additional growth was allowed for return to exponential growth after the centrifugation and change in guanine concentration. To start the labelling, an equal volume of warm aerated medium containing [14C]guanine (15 pg/ml.) was added to the cells. Samples (10 ml.) were withdrawn from the culture at intervals and put into 5 ml. cold 0.75 M-perchloric acid with added

L@P1i 0 20 IO.0 I I 3 0.9E 0.8s 0.7t3

I

I

I

I

I

I

40

60

80

100

120

140

I

I

I

I

I

I

(b)

+7Jy

/PI Pf+ 0.3

1 -90

I -60

I -30

I 0

I 30

I 60

I 90

I _ 120

Time (min) Fra. 5. (a) The uptake

of [14C]guanine (-O-O--). The uptake is compared of 168 min. The filled triangles represent should describe the uptake into such (b) The growth of the culture before 540 mp. The cells were centrifuged and resume exponential growth after any the labelling was started by adding an as described in the text.

into trichloroacetic acid-precipitable material is shown with that expected for cells growing with a generation time points on the theoretical curve k (exp (t/168) - 1) which cells. and during the experiment is plotted as optical density at resuspended in new medium at - 98 min to allow them to perturbation due to the centrifugation. At zero minutes, equal volume of 37% medium containing labelled guanine

264

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JANIN

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C.

LEVINTHAL

carrier nucleotides. The nucleotide pools were separated and isolated by electrophoresis and measured as described above. The perchloric acid-precipitable radioactivity from the same samples was measured and used to compute the rate of incorporation of guanine into the nucleic acids of the cells (Fig. 5(a)). The growth of the cells (Fig. 5(b)) was followed carefully before and during the labelling, in order to determine the generation time and to detect any departures from exponential growth. In computing the specific activities of the various nucleotide pools, it was assumed that the pool sizes (Figs 5 and 6) increased with t,he exponential growth of the cells. The recovery of GTP and GDP was reproducible : the values obtained from integration of the strip scanner output, uncorrected for recovery, fit the same experimental curve as did the independently determined values, corrected for recovery, obtained from the eluted peaks. The reliability of the two sets of data also appeared to be roughly similar, as judged by their fluctuations about the same theoretical curve. The average values are graphed in Fig. 6(a) and (b) and were used for subsequent calculations. 400

0

,

,

,

,

I

I

I

II

IO

20

30

40

,

50

,

,

I

11

60

70

Time

,

80

I

I

Time 11

hn)

90

I

I

1

‘.

I20

130

140

11

100 HO

I

I

(mtn)

FIG. 6. Exponentially growing cells of B. subtilis, strain 295, were grown at 31i’C in a Tris-glucose medium supplemented with adenine (20 pgjml.) and guanine (3.0 rg/ml.). The generation time was about 168 min under these conditions. At time zero, radioactive guaniue was added as described in the text. Samples were taken and their GTP isolated by electrophoresis of the perchloric acid-soluble fraction. The measured growth of t,he cells was used to compute the specific activity of the GTP as ppmoles of labellod GTP/ml. of culture laf an optical density (540 ml*) of 1.0. These data are plotted in (a) on a linear scale, and in (b) on a semilogarithmic scale. The solid lines are the theoretical curves for a single unstable RNA pool which gave the best fit to the data, and correspond to an unstable RNA pool which contains 9s’, of the total guanine residues of the cell and whose rate of breakdown is 5.3 times as great as the rate of net synthesis of RXA hy the cells. As shown in the theoretical section, the accuracies of the estimates of the amouut ofuustable RKA and its rate of breakdown are, respectively, proportional to the accuracy with which the area between the curve and its asymptotic value may be determined in (a) and the accuracy with which the second component in (b) may be extrapolated t,o time zero.

The results of this experiment are shown in Figs 5 to 7. Figure 6(a) shows the specific activity of GTP plotted on a linear scale. The solid line represents the theoretical curve corresponding to 9% unst,able RKA whose rate of synthesis is 5.3 times as great as the rate of net RPiA synt,hesis (decay time of 3.87 minutes without correction for the possibility of unstable RNA fractions with more rapid than average turnover). Figure 6(b) shows the same data plotted as the final specific act)ivity minus the specific activity at time t on a semi-logarithmic scale. It may be seen that) the data fit very

UNSTABLE

R?iA

IN

B. subtilis

AND

E.

coli

25.;

well a theoretical curve which is the sum of two exponential components and can therefore be approximated by two straight lines on such a plot. Very similar data were obtained for GDP, establishing that GDP is in rapid equilibrium with GTP. This is the result which would be expected if the products of RNA breakdown, as well as nucleotides newly synthesized, flow through GDP into the GTP pool (see also Appendix, section (b)). The main features of the guanine distribution and its rate of conversion under the conditions of this experiment are shown in Fig. 7. The most striking features of the pattern are the large amount of unstable RNA, which is consistent with the earlier determinations (Levinthal et al., 1962) using actinomycin, and the rapid rate of turnover of this unstable RNA fraction. Stable nucleic acid 16,300~~moles

Unstable RNA 1680 /ipmoles

O.D.540

O-D540

86 ,u+moles

514 ppmoles 0.D.540 Exogenous quanine

97ppmoles -0.D.540 ml”

CMP - 15 /~/~moies ~ O.D.540 ~

-

ml”

0 D.540

GDP 28 ,v~moles-330 ___ 0. D.540 _

I 1

ml”

CTP I*pmoles 0. D.5S0

%l:+ compounds -40 /I/lmoles

7. The results of the experiment shown in Figs 5 and 6 are presented here as pool sizes FIG. and flow rates computed from the data. The rate of intake of guanino into the cell was 27 ppmoles guanine/min/ml. of CUhre at 0.D.5*0 = 1. The rate of breakdown of material from unstable RNA w&s 514 /.+moles guanine equivalent/min/ml.of culture at 0.D.540 = 1, while t,he rate of synthesis of stable RNA was about one-sixth as great. The unstable RNA accounted for about 9.0% of the guanine residues in the cells. About 2.6% of the guanine residues were in the soluble nucleotide pools. 70 to 80% of this 2.6% wa sin GTP, with the remainder distributed among GDP, GMP and other compounds approximately as shown. The generation time of the cells was about 168 min.

(d) Use of conditioned

medium and double-label

techniques

Hartwell & Magasanik (1964) observed that tenfold dilution of a culture of B. subtilis into new medium interfered with the induction of histidase. When dilution medium in which B. subtilis had previously grown (preconditioned medium) was used, the results appear to be normal. Even though the dilution used here for the addition of label was only twofold, we thought it worth while to repeat the experiment described above, using preconditioned medium in order to be certain that there was no deviation from exponential growth. In addition, the double-label technique was used, as described in Materials and Methods, with [3H]guanine present throughout the experiment and [14C]guanine added at time zero. This simplifies the determination of GTP specific activit,y and makes it independent of any assumptions concerning the behaviour of GTP pool size during the experiments. The cells were grown at 39°C instead of 37°C. Otherwise, except as noted in Materials and Methods, the techniques used were the same as in the previous experiment. Again the data showing the labelling of GTP rould hc fitted very well by a theorebical curve which was thr sum of t,wo exponential

256

W.

SALSER,

J. JANIN

AND

C. LEVINTHAL

components. The parameters of equation (7) were varied to give the least squares fit to this data, giving the following result: p(t) = 1 - 0.89 exp (-t/49)

- 0.11 exp (-t/1*2).

These parameters, in addition to the size of the guanine-soluble nucleotide pools as determined by measuring the radioactivity and recovery of the individual compounds, the rate of incorporation of [14C]guanine into nucleic acid, and the generation time, measured directly from the increase in optical density, were used to compute the amount of unstable RNA and its turnover. It was found that the data again correspond to 9*Oo/ounstable RNA, whose average decay time (without correction for the possibility of unstable RNA fractions with very rapid turnover) is O-016 generation time (6.2 minutes)?. (e) Measurement

of the unstable RNA

in E. coli

One of the chief aims in developing this technique was the comparison of the amounts of normally unstable RNA in B. subtilis and E. coli. We now describe the results obtained with a guanine-requiring strain (R257) of E. coli which cannot convert 1M.P to XMP (Fig. 3). The cells were grown in the presence of hypoxanthine (20 pg/ml.) to inhibit conversion of guanine to adenine nucleotides. No conversion was detected. Histidine (20 pg/ml.) and vitamin B, (5 pg/ml.) were also supplied. The cells were grown at 3O”C, with a generation time of 96 minutes. The 3H, 14C double-label technique was used, as described in Materials and Methods. Cells were grown overnight in 10 pg/ml. [3H]g uanine. They were centrifuged and resuspended in the same medium with a guanine concentration of 6 pg/ml. and the same 3H specific activity. Further growth of O-6 of a generation time was allowed, to ensure the reestablishment of exponential growth after the centrifugation. Then an equal volume of preconditioned medium was added to the culture having the same guanine concentration and 3H specific activity as the growing culture, but also labelled with l*C in the 8 position of the guanine. Samples were taken after this time in order to determine the uptake of 14Cinto the guanine nucleotide compounds. The uptake of [14C]guanine into the GTP pool is shown in Fig. 8(a) where the continuous line is the theoretical curve for p(t) = 1 - 0.55 exp ( - t/74) -0.45 exp ( -t/0*77) which gives the least squares fit of the experimental points. The two exponential components of the uptake are clearly seen in the semilogarithmic plot of Fig. 8(c). Using the parameters determined in this way, as well as direct measurements of the generation time, rate of incorporation of labelled guanine into total nucleic acid and measurement of the soluble nucleotide pool size, the amount and average decay time of the unstable RNA were computed. The unstable RNA contains about 3.4% of the total guanine in RNA or 3% of the total guanine residues contained in the cell (DNA represents about 10% and the soluble nucleotide pools l*S% of the botal guanine residues). The average decay time (without correction for the possibility of unstable RNA fractions with very rapid turnover) is four minutes and the rate of breakdown of the unstable RNA, b(0) is only 0.9 as great as the net intake of guanine into the cells, a(0). The kinetics for GDP are also shown (Fig. 8(b)) and the data fit the same curve as for GTP, confirming our assumption that GTP and GDP equilibrate quickly. t See footnote

on p. 246.

UNSTABLE

RNA

IN

B.

subtilis

AND

5 IO Time after addition of [14Clguanlne (mln)

E.

coli

257

15

FIG. 8. The uptake of [14C]guanine into the GTP and GDP pools of E. coli. Conditioned medium containing [l%]guanine was added at time zero. Both media contained the same [zH]guanine specific activity, so that the ratio of 14C to 3H provided a convenient measure of the specific activity of GTP with respect to nucleotides taken into the cell after time zero. This fact was used t,o compute the fraction of each pool labelled with I%, which is plotted above. Figure 8 (a) shows t’he labelling of GTP along with the theoretical curve which gave the best fit to the data. The broken curve shows the expected result if E. co.% had a 9% unstable RNA pool with the rapid turnover found in B. aubtilis. The results for E. coli correspond to a much smaller unstable RNA pool (3.0 to 3.4%) and a rate of synthesis of unstable RNA slightly less than the rate of synthesis of stable nucleic acid. In (b) the uptake of label into GDP ( x ) is presented for comparison to show that it has very similar kinetics, and must therefore be in rapid equilibrium with GTP( 0). In (c) the data of (a) are replotted on a semilogarithmic scale so that it is possible to see the accuracy of determination of the constant U, the interception of the extrapolated second component with the vertical axis, upon which depends the accuracy of the measurement of the rate of turn. over of the unstable RNA.

The broken curve of Fig. 8(a) shows the labelling of GTP in B. subtilis, corrected for the different generation times of the two organisms. The washout of u&belled material clearly takes much longer in B. subtilis, showing conclusively that there is a much larger amount of material in equilibration with GTP in B. subtilis. This large difference between the amount of unstable RNA found in B. subtilis (9%) and in E. coli (3%) is very striking. To verify further this finding, we have recently developed an independent technique for measuring the amount of RNA tihich is normally unstable in exponentially growing cells. This method is described briefly below.

258

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(f) Independent measurements of the amount of unstable RNA When labelled nucleotide precursor is added to a growing culture, the unlabelled nucleotides present in the unstable RNA and the nucleotide pools will be chased into the stable nucleic acids. Consequently, there will be a lag in the incorporation of labelled nucleotides into stable nucleic acid. The size of this lag will be a measure of the amount of material in the unstable RNA and soluble nucleotide fractions. While it is difficult to separate stable from unstable RNA, it is quite easy to isolate DNA by the method of Roodyn & Mandel (1960). The pathway of deoxycytidine synthesis from uracil supplied in the medium is known (Magasanik, 1962) to involve conversion of UTP to CTP. The work of Nierlich et al. (manuscript in preparation) establishes that the intermediates in the pathway from CTP to dCTP are present in very small amounts. Consequent,ly, the kinetics of uptake of radioactive deoxy-CTP into DNA should be the same as those of CTP into stable RNA. By measuring the lag in incorporation of radioactivity from [14C]uracil into the deoxycytidine of DNA, it is therefore possible to measure the amount of material in unstable RNA and the soluble nucleotide pools. Such experiments have been carried out, using thymine-requiring strains of B. subtilis and E. coli to prevent conversion of [14C]uracil to thymine. The amount of unstable RNA in B. subtilis as measured by this method was between 8 and 11.7o/o of the nucleic acid of the cell. The data for E. coli showed that its unstable RNA fraction was about three times smaller than that observed in B. subtilis, in complete agreement with the other results reported here. (The experimental procedure and the complete mathematical treatment on which these conclusions are based are contained in Salser, 1966.)

5. Discussion (a)

What is the rate of breakdown of unstable RNA ?

Previous estimates of the rate of breakdown of messenger RNA have involved the use of actinomycin D or other drugs which inhibit RNA synthesis. Unstable RNA labelled in a short pulse was found to be degraded with a decay time of one minute in actinomycin-treated cells of B. subtilis grown at 37°C. The synthetic capacity for the histidase of B. subtilis (Hartwell & Magasanik, 1963) was found to disappear at a slower rate ; 3.6 minutes decay time at 37°C. Higa (1964) found that hhe initial rate of decrease in the rate of protein synthesis after treatment of B. subtilis wkh actinomycin was from 2.5. to 4*1-fold slower than the initial rate of breakdown of the unstable fract’ion of pulse-labelled RNA. Schaechter, Previc & Gillespie (1965), found that the decay time for polyribosomes in actinomycin-treated B. megaterium was fivefold longer than the decay time of pulse-labelled RNA. Using a very different set of techniques which depend upon different assumptions, we have concluded that the average decay time at 37°C is three minutes or less. It is significant that techniques so diverse and depending upon different assumptions give answers which are so similar. However, there are differences which can be explained in a number of ways. First, our medium, chosen to suppress conversion of guanine to adenine nucleotides, gave longer generation times than have been investigated in the actinomycin decay experiments. Higa (1964) found however t,hat the actinomycin decay time at 37°C is the same in a variety of media with generation times ranging from 47 to 100 minutes. For the purpose of further discussion wc will

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assume that this can be extended to our medium with a 170 minute generation time. It is difficult to predict the relationship of the decay time for protein synthesis to the other decay times. The protein synthesis decay time may be affected by a changing messenger/ribosome ratio during the decay and, moreover, will be weighted in favour of the decay times of those messengers which are translated most efficiently. Consequently, at the present time, we cannot see any way of assessing the significance of the decay times for protein synthesis except to remark that they are rather close to the decay times measured in this work. The difference between the actinomycin decays of pulse-labelled RNA and the decay times measured from GTP labelling in this work are more interesting and we will discuss below two possible reasons for the difference. (i) Messenger RNA’s

have a wide range of decay times

It is necessary to use short pulses of label in the actinomycin decay experiments. With longer labelling times, the accuracy of the measurements deteriorates because of the increase in the relative amount of label in stable RNA. This practical restriction on the decay time measurements with actinomycin will lead to large errors where messenger RNA molecules have a wide range of turnover times. The pulse label will preferentially enter those RNA molecules which have the fastest rate of turnover. Consequently, assuming for the moment that the decay times are not affected by the actinomycin, the actinomycin measurement will be weighted in favour of the shortest decay times. On the other hand, the studies of nucleotide labelling kinetics reported here give a figure for the average decay time whose weighting due to the heterogeneity of unstable RNA decay times will be in the opposite direction in favour of the longest decay times. From this it follows that the correct average decay time should lie between the values obtained here, using GTP labelling kinetics, and those obtained with actinomycin. Moreover, a large difference in the values obtained by the two independent methods would suggest that there is, in fact, considerable heterogeneity in the decay times for different messengers. In fact, Salser (1966) and Salser & Barrett (manuscript in preparation) have shown that the functional decay times for the messenger RNA molecules for different proteins of B. subtilis do seem to be different. This observation was made by measuring the labelling of specific protein bands separated by acrylamide gel electrophoresis after actinomycin was added to the culture. These measurements indicate that the functional decay times range from almost nine minutes to less than 2.2 minutes. However, these measurements cannot be taken as an indication of the absolute decay rates of the messenger RNA molecules, since there is no reason to assume that ribosomes function with equal efliciency in an excess and in a relative absence of messenger RNA. However, it does seem reasonable to conclude that these measurements suggest a range of about fourfold in the decay rates for different messengers. It seems clear therefore that part of the difference between the two measurement,s, by different techniques, of the unstable RNA decay times, is due to the fact that different unstable RNA molecules have. different decay times. Our data (Salser & Barrett, manuscript in preparation) on the heterogeneity of functional (protein synthesis) decay times for different proteins are not sufficiently complete to say whether this explanation is quantitatively adequate to account for the differences observed.

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molecules may decay abnormally

in actinomycin

Actinomycin is expected to create a class of unfinished RNA molecules which cannot be completed in the presence of the drug and which therefore have an abnormal fate. If the time required to complete individual RNA molecules is longer than the pulse labelling then most of the label incorporated will be in unfinished RNA molecules. The work of several authors (Goldstein, Kirschbaum & Roman 1965; Leive, 1965; 1965; Zimmerman & Levinthal, manuscript in preparation) Alpers & Tomkins, suggest,s t,hat the synthesis time may be as long as one minute per RNA molecule of molecular weight lo6 in cells growing at 37°C. In a very short labelling period then, such as has been used in some of the actinomycin decay experiments in order to minimize the background of label in stable R.NA, we can expect that the bulk of the radioactivity might be found in unfinished ribosomal and messenger RNA molecules. We can only speculate whether incomplete messengers, when their completion is blocked by actinomycin, will code for incomplete proteins or be degraded without participating in protein synthesis. In either case, their rate of breakdown might be abnormal. On the other hand, no large change in the apparent decay time was found (Levinthal et al., 1962) when actinomycin was given after pulse labelling for various lengths of time. Thus it is not likely that any such abnormal decay time affects the estimates from the actinomycin experiments by a large fraction. Experiments by Schaechter & McQuillen (1967) with very short pulse times do show a change in the decay time after the addition of actinomycin. If unfinished ribosomal RNA molecules decay when their completion is blocked, then there would be other consequences. There should be a difference between our measurements of the amount of normally unstable RNA and those measurements using actinomycin. As pointed out by Zimmerman & Levinthal (manuscript in preparation) such unfinished molecules would only change the estimates of the amount of messenger by about 0*5%, which is smaller than the experimental accuracy of either bechnique and certainly an order of magnitude smaller than the amount of abnormally unstable RNA postulated by Acs, Reich & Valanju (1963) and Kennel1 (1964). On the other hand, it has not been recognized in previous discussions that decay of such a small fraction of ribosomal RNA, since it is preferentially labelled in a short pulse, would greatly alter the calculation of the relative rates of synthesis of stable and unstable RNA from measurements of the fraction of label incorporated in a very short pulse which decays in actinomycin (Levinthal et al., 1962). This result will be seriously altered if even so much as 0.5% of the most recently synthesized (partly finished), and therefore, most radioactive, ribosomal RNA molecules are degraded. (b) How much unstable RNA

is present in normal cells?

Messenger RNA is a clearly defined concept, i.e. that RNA which codes for the synthesis of the proteins of the cell. Operational definitions are more difficult, and previous measurements of the amount of messenger RNA have depended upon a variety of properties attributed to messenger. Its sedimentation properties, its presumed nucleotide base composition, its ability to form hybrids with complementary DNA, its ability to attach reversibly to ribosomcs, and it,s suscept,ibilit,g to metabolic breakdown after treatment with actinomycin D, have all been used (Gros et al.,

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1961a,b; Midgley $ McCarthy, 1962; Levinthal et al., 1962; Fan et al., 1964; Bolton & McCarthy, 1963; Hayashi & Spiegelman, 1961; Leive, 1965; Buchwald & Britten, 1963; Roberts, 1964). As pointed out by Levinthal et al. (1963), in each of these oases there are particular difficulties of interpretation which bring the results into doubt. For example, in the calculations using the base ratios of the pulse-labelled RNA (Midgley & McCarthy, 1962), it was necessary to assume that the messenger RNA had a DNA-like base composition. However, only one strand of DNA is transcribed into messenger RNA (Guild & Robinson, 1963 ; Tocchini-Valentini et al., 1963 ; Hayashi, Hayashi & Spiegelman, 1964) and some genes are thought to be transcribed much more frequently than others. Consequently it would not be surprising if this assumption were incorrect, as is suggested by the experimental evidence of Doskocil & Hochmannova (1965). As another example, in the actinomycin work (Levinthal et al., 1962; Fan et al., 1964), it was necessary to assume that actinomycin D stopped RNA synthesis without having any influence on the breakdown of pre-existing RNA. This was challenged by Acs et al. (1963), who asserted that actinomycin D caused the breakdown of a large amount of otherwise stable RNA in cells treated with chloramphenicol. There are a number of reports, many of which have been summarized by Lieve (1965) which indicate that the results of Acs et al. can be accounted for by the continual breakdown and resynthesis of some part of the RNA synthesized during the chloramphenicol treatment. The finding reported here indicates that the total amount of unstable RNA in B. subtilis is about the same as the amount of unstable RNA determined from the actinomycin decay experiments. These results would seem to indicate that the breakdown of RNA observed in the presence of actinoinycin D is not produced by that drug, but is a breakdown which occurs in exponentially growing cells even in the absence of the antibiotic. There is also reasonable agreement between the results we find for E. coli of about 3% of the guanine residues of the cell being in unstable RNA, and that reported by Leive of the unstable fraction measured by actinomycin decay between 1.5 and 3%. The interpretation of our data, as pointed out above, is now mainly dependent upon the assumptions that the observed results are not seriously influenced by either private pools of GTP or by reserves of guanine-containing compounds in equilibrium with GTP but undetected in our experiments (as, for instance, any compound bound to cell material and not extracted by our techniques). We know of no evidence to contradict these assumptions. Some authors (McCarthy & Bolton, 1964; Buchwald Ss Britten, 1963; Roberts, 1964) have invoked models involving private pools. However their data seem to us to be consistent with our model while ojur data are inconsistent with the private pool models which they have proposed (see Salser, 1966). More important is the fact that these assumptions are very different from those made in interpreting earlier results with actinomycin. The agreement of these two methods in showing that about 9% of the guanine residues of B. subtilis are contained in the unstable RNA fraction seems highly significant. Our finding, by the same technique, that the comparable figure for E. coli is only about 3% substanbiates differences found earlier and sometimes attributed to errors of interpretation of the different methods used. The fact that E. coli contains a much smaller fraction of unstable RNA than B. subtilis makes it tempting to speculate

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that, E. coli may make more copies of protein per unstable RNA molecule than does B. subtilis. Even if true, however, this would not necessarily mean that E. coli was more eficient than B. subtilis in terms of the amount of protein produced for a given amount of energy expended in protein synthesis, because unstable RNA synthesis, even when it accounts for most of the RNA synthesis of the cell, as in B. subtilis, only uses a small fraction of the total energy spent in protein synthesis. The fact that RNA turnover, even when rapid, involves only a small part of the total energy spent in the over-all process of protein synthesis is obvious if we divide this process into two parts and look at the energy requirements of each. One part is the synthesis of a messenger RNA codon and requires the energy necessary to rephosphorylate the nucleotide mono- or diphosphate breakdown products of messenger degradation to the nucleotides triphosphate level (3 to 6 high-energy phosphate bonds per codon-net synthesis of RNA would require much more energy for de nova synthesis of the nucleotides). The other part of the process involves the synthesis, activation and incorporation into protein of the large number of amino acids directed by a single codon. Assuming 30 to 60 protein molecules per messenger (results of Levinthal et al., 1962, corrected for a 3-minute average decay time of unstable RNA), 3 high-energy bonds and disregarding the energy for de nova synthesis of the amino acids or the synthesis of ribosomes, give an estimate of from 90 to 180 high-energy phosphate bonds for the amino acids whose incorporation into protein is directed by a single RNA codon. This is 15 to 60 times as much energy as required for the RNA turnover part of the over-all process of protein synthesis. This work is taken in part from theses submitted by one of us (W. S.) to the Massachusetts Institute of Technology for a Ph.D. and by another (J. J.) to The Universite de Paris for a Doctorat de SpBciBlitB. This research was supported by a grant from the National Institutes of Health, grant no. AI-02028. W:‘S. Gould like to express his gratitude to Dr Donald P. Nierlich for his help and valuable suggestions during the early part of this work. REFERENCES

S. (1963). Biochim. biophys. A&u, 76, 68. Acs, G., Reich, E. & Valanju, Alpers, D. H. & Tomkins, G. M. (1965). Proc. Nat. Acad. Sci., Wash. 53, 797. Bolton, E. T. & McCarthy, B. J. (19G3). Proc. Nut. Acad. Sci., Wash. 48, 139. Buchwald, M. & Britten, R. J. (1963). Biophys. J. 3, 155. Doskocil, J. & Hochmannova, J. (1965). Biochim. biophys. Acta, 108, 504. Fan, D. P., Higa, A. & Levinthal, C. (1964). J. Mol. Biol. 8, 210. Goldstein, A., Kirschbaum, J. B. & Roman, A. (1965). Proc. Nat. Acad. Sci., Wash. 54, 1669. Gros, F., Gilbert, W., Hiatt, H. H., Kurland, C. G., Risebrough, R. W. & Watson, J. D. (1961a). Nature, 190, 531. Gras, F., Hiatt, H. H., Attardi, G., Spahr, P. F. & Watson, J. D. (1961b). Cold SIT. Harb. Symp. Quunt. Biol. 26, 111. Guild, W. R. & Robinson, M. (1963). Proc. Nat. Acad. Sci., Wash. 50, 106. Hartwell. L. H. & Magasanik, B. (1963). J. Mol. BioZ. 7, 401. Hartwell, L. H. & Magasanik, B. (1964). J. Mol. BioZ. 10, 105. Hayashi, M., Hayashi, M. N. & Spiegelman, S. (1964). Proc. Nat. Acad. Sci., Wash. 51, 351. Hayashi, M. & Spiegelman, S. S. (1961). Proc. Nat. Acad. Sci., Wash. 47, 1564. Higa, A. (1964). Ph.D. thesis Massachusetts Institute of Technology. Kennell, D. (1964). J. Mol. BioZ. 9, 789. Leive, L. (1965). J. Mol. BioZ. 13, 862. Levinthal, C., Keynan, A. 8: Higa, A. (1962). Proc. Nut. Acad. Sci., Wash. 48, 1631. Levinthal, C., Fan, D. P., Higa, A. & Zimmermann, R. A. (1963). Cold Spr. Hurb. Symp. Quant. BioZ. 28, 183. Lipkin, D., Talbert, P. & Cohn, M. (1954). J. Amer. Chem. Sot. 76, 2871.

UNSTABLE McCarthy, Magasanik, York: Magasanik, Midgley,

RNA

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B. J. & Bolton, E. T. (1964). J. Mol. Biol. 8, 184. B. (1962). In The Bacteria, vol. 3, ed. by I. C. Gunsalus Academic Press. B. & Karibian, D. (1960). J. Biol. Chem. 235, 2672.

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J. E. M. & McCarthy, B. J. (1962). Biochim. biophys. Actu, 61, 696. Popowicz, J. (1962). J. Chromatog. 7, 271. Roberts, R. (1964). In Studies of Macrowwlecular Biosynthesis, ed. by Richard

Roberts,

Carnegie Inst. of Washington. Roodyn, D. B. & Mandel, H. G. (1960). Biochim. bio-phys. Acta. 41, 80. Salser, W. (1966). Ph.D. thesis, Massachusetts Institute of Technology. Schaechter, M., Previc, E. P. & Gillespie, M. E. (1965). J. Mol. Biol. 12, 119. Schaechter, M. & McQuillan, K. (1967). J. Mol. Biol. in the press. Tocchini-Valentini, G. P., Stodolski, M., Aurisicchio, A., Sarnat, M., Graziosi,

F., Weiss,

S. B. 8: Geiduschek, E. P. (1963). Proc. Nat. Acad. Sci., Wash. 50, 935. Weygand, F. & Grosskinsky, 0. A. (1951). Ber. 84, 839. APPENDIX

Measurement of Pool Expansion (a) prom incorporation

of label into RNA

As was mentioned in Theoretical Considerations (preceding manuscript), there should be a lag in incorporation of radioactivity into nucleic acid due to the washout of the unlabelled nucleotides from the acid-soluble pools. However, we have found that there is no detectable lag in the incorporation into acid-precipitable material if guanine is added to cells growing in the absence of guanine, as described in the following experiments. This may be explained by assuming that the addition of the radioactive guanine causes a rapid expansion of the guanine nucleotide pools which draws labelled guanine into the cell very rapidly at first. Such pool expansions are departures from exponential growth which might seriously hamper the interpretation of the results presented above. To avoid this, an effort was made to avoid changes in the external guanine concentration. It was nevertheless interesting to investigate the magnitude of pool expansions which might possibly occur as a result of small shifts in the guanine concentration. We have found that there is no measurable outflow of nucleotides from the cell which would cause equilibration with the guanine of the medium. Consequently, if there is no pool expansion, then the lag in uptake of label into nucleic acid, which is due primarily to the washout of unlabelled nucleotides from GTP and GDP, should equal the number of minutes of net synthesis which can be supplied by these pools. Actually, as shown in Theoretical Considerations, there are two components of the pool washout, and in practice, it is only possible to measure accurately the lag corresponding to the fast component, However, this lag is still proportional to the size of the pool, and we made use of it to determine the dependence of the pool size upon the concentration of external guanine. The results of a typical experiment are shown in Fig. 9. Even where the change in guanine concentration upon addition of the [14C]guanine is small, so that we expect no pool expansion, the measurable lag is much smaller than would be predicted from the known size of the guanine nucleotide pools if all the RNA of the cells were stable. This further verifies the existence of a small rapid component of pool washout and a large slow component, which are the result of the rapid turnover of the large unst’able RNB fraction. The cells (B. subtilis W23 SmR) were grown overnight in guanine-supplemented

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Time kec)

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FIQ. 9. The uptake of [14C]guanine into trichloroacetic acid-preoipitable material is shown. until time zero, The cells, B. subtilti W23 S,a, were growing in guanine of various concentrations, when O-4 pg/ml. [“C]guanine was added to each culture. The concentrations of unlabelled guanine present just prior to this addition were: curz)e A, 0.0 pg/ml,; curwe B, 0.30 pg/ml.; curve C, 0.60 pg/ml.; CUUTZ)~ D, l-2 pg/ml.; and curve E, 2.4 rg/ml. In curve A, with no unlabelled guanine present, there was immediate rapid uptake of label, indicating a pool expansion which drew [14C]guanine quickly into the pool. The other cultures, with pre-expanded pools, showed a lag in uptakewhich was of similar size regardless of the external guanine concentration over the range from 0.3 to 2.4 pg/ml. This suggests that the amount of pool expansion is almost as large with 0.3 pg/ml. of guanine as it is with 2.4 pg/ml. (a) Shows the same data as (b), but with an expanded time scale.

medium to ensure the presence of any enzymes necessary for utlilization of exogenous guanine. The guanine was removed by filtration and the cells were grown for an additional doubling. To different portions of this culture, guanine was then added, at various concentrations (from 0 to 2.4 pg/ml.), and the cells were incubated for 15 minutes. A constant amount of [14C]guanine was then added to each culture. In the control, with no unlabelled guanine, there was no detectable lag in uptake of label, indicating a pool expansion. There was a short lag in all cultures which had unlabelled guanine present when the [14C]guanine was added. These lags were of nearly equal duration regardless of the amount of guanine present in the medium before addition of 14C. We interpret the lag to be proportional to the nucleotide pool sizes since there is no indication that the other relevant parameter, the ratio of unstable to stable RNA synthesis, varied. The conclusion is again, therefore, that the pool size expands upon the addition of external guanine to cells grown in the absence of guanine, but that there is very little further change in the pool size over a large range of external guanine concentrations, from O-3 to 2.4 pg/ml. The experiment was repeated with similar results, in the guanine-requiring strain B. subtilis 295. The control, with no guanine present before the addition of labelled guanine, not only had no detectable lag, but also had an initial rate of incorporation 50% greater

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than a short time later. Such a rapid uptake of label into trichloroacetic acid-precipitable material is the anticipated result (Salser, 1966) if there is a rapid pool expansion when the [l*C]guanine was added to the medium. Because of the rapid turnover of the unstable RNA, the radioacbivity drawn into the cell in such a pool expansion would be rapidly incorporated into RNA. We have used this method to obtain maximum labelling of RNA in very short pulses (2 x 10m3 generation time) which would be ohherwise impractical usage. (b) Direct measurement of pool expansion A number of control experiments was conducted to see if there were changes in the sizes of the guanine acid-soluble nucleotide pools as a result of changes in the external guanine concentration. It was found that the pool sizes for GTP and GDP were unaffected by large changes in the external guanine concentration. Cells of B. subtilis 295, growing exponentially in medium supplemented with adenine and guanine, were filtered and resuspended in a similar medium with 0.8 pg/ml. of [14C]guanine. At a later time, the amount of radioactive guanine was doubled and still later, it was raised to a concentration of 14.6 pg/ml. The nucleotide pools were isolated as in the preceding experiments, using electrophoresis at pH 4.1.

I

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FIG. 10. Cells of B. sub&s 295 were filtered from an exponentially growing culture and resuspended in medium containing 0.8 pg [14C]guanine/ml. At 21.5 mm (indicated by the first vertica.1 line) the concentration of radioactive guanine was doubled. At 40.5 min (second vertical line) the concentration of radioactive guanine was raised to 14.5 rg/ml. The individual nucleotide pools were isolated by electrophoresis and the radioactivity in GTP (-O--O-), GDP (-A--A) and GMP (-O-e-) are shown. The GTP and GDP pools label slowly, as seen in other experiments, and are not appreciably expanded by the further increases in the guanine concentration. The GMP pool, however, seems to label quickly and to undergo further pool expansion with the additional increases in the guanine concentration.

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The results are shown in Fig. 10. They are similar in many respects to those obtained in the earlier experiments with exponential cells. The GTP and GDP pools show the slow uptake of label characteristic of cells with a large amount of unstable RNA. After this slow uptake, the GTP radioactivity shows no further appreciable increase in radioactivity, such as would be expected if there was pool expansion resulting from an increase in external guanine concentration. From this lack of response to an 1% fold change in the external guanine concentration, it was concluded that the GTP pool size is well regulated and independent of external guanine concentration over the large range of values studied. The GDP pool may have expanded slightly in response to the large increase in the guanine concentration, but the data are not accurate enough for such a small increase to be convincing. The data for GMP give a much different picture. There seems to be a substantial pool expansion with each increase in the level of external guanine. This may constitute a control of guanine synthesis, since GMP is known to inhibit the enzyme which converts IMP to XMP (Magasanik & Karibian, 1960). The very rapid labelling of the GMP pool, while GDP and GTP are labelled slowly, suggests that the breakdown of the unstable RNA is not to the nucleotide monophosphate level. If the breakdown products from unstable RNA degradation passes through the GMP pool, then we would expect to find that GMP and all compounds such as IMP and XMP, which might receive from GMP in this system, would be labelled as slowly as GDP and GTP. The guanine used in all of these experiments was prepared by the method of Weygand & Grosskinsky (1951), a method which should not give any by-products with the mobility of GMP in this system. Separation of the small GMP pool from other compounds with small amounts of radioactivity, such as IM.P, was difficult in the pH 4.1 system. This experiment is the only one of many which gave an acceptable separation of these compounds. Figure 4(a), on tb.e other hand, shows one of the worst separations obtained in the GMP region. Because of these very real difficulties, we wish only to emphasize that an experiment of this sort is capable of distinguishing, in principle, between the breakdown of RNA to the monophosphate and that to the diphosphate level.