Metabolism of T4 messenger RNA, host Messenger RNA and ribosomal RNA in T4-infected Escherichia coli B

J. Mol. Bid. (1972) 71, 701-715

Metabolism of T4 Messenger RNA, Host Messenger RNA and Ribosomal RNA in T4-infected Escherichia coli B

E. Cn~o, K. CBE~ES AND D. SUELESSINQEB

Department of Microbiology Wadington Univer&y &hool of Me&k

Xt. Louis, MO. 63110, U&A.

(Received 17 April 1972, and in revised form 24 July 1972)

Rifampicin was added to Escher&h&z coli B cells one minute after baoteriophage T4 infection, and the subsequent degradation of newly labeled T4 messenger RNA, host messenger RNA and ribosomal RNA was followed by DNA-RNA hybridization. T4 and host mRNA decayed exponentially. The half-life of T4 mRNA was 9 to 10 minutes; in contrast, the half-life of host mRNA was 2.2 minutes, equal to that in uninfected cells. Newly synthesized rRNA decayed slightly faster than the T4-specific RNA.

The direction of breakdown of labeled T4 mRNA molecules after rifampicin addition was analyzed by competition hybridization. Appropriate unlabeled competitor RNA’s were extracted from cells infected for short but increasing lengths of time. These competitors were used to follow breakdown of portions of labeled RNA chains that extend from the U-end and require 20,60 or 160 seconds for their synthesis. All decayed at the same rate; the over-all sense of decay was 6’ to 3’.

1. Introduction

Bulk Escherichia wli messenger RNA is unstable and degrades exponentially with a chemical half-life of a few minutes (Gros et al., 1963; Mangiarotti & Schlessinger, 1967; Salser, Janin & Levinthal, 1968). Although there have been many studies of the functional inactivation of mRNA species (Geiduschek & Haselkorn, 1969), the RNA transcribed from the tryptophan operon is the only specific messenger RNA for which chemical decay has been analyzed in detail. Degradation of trp mRNA is in the same sense as transcription and translation, over-all in the 6’ to 3’ direction (Morikawa & Imamoto, 1969; Morse, Mosteller, Baker t Yanofsky, 1969).

The case of viral messengers seems to be more complex. Initial work with T7 phage (Summers, 1970) indicated that T7 mRNA is stable, whereas T4 mRNA has generally been considered unstable (see, for example, Jacob & Monod, 1961). Adetailed analysis of the rate and direction of degradation of T4 mRNA would permit comparison between host and phage mRNA degradation. If the results are consistent with those from the trp operon, a second example would exist from which to generalize about the universality of degradation of mRNA in bacterial cells. If not, the question arise-is the mechanism of viral mRNA degradation the same as E. wli mRNA degradation?

The analysis of T4 mRNA degradation reported here is possible because of the technology previously developed for sensitive DNA-RNA competition hybridization

701

702 E. CRAIG, K. CREMER AND D. SCHLESSINGER

analyses (Bolle, Epstein, Salser & Geiduschek, 1968). Our results indicate that T4 mRNA is rather stable, similar to T7 mRNA, but that its over-all breakdown, like that observed for the trp operon, is exponential and in the 5’ to 3’ direction.

2. Materials and Methods (a.) Bacteria and bacteriophage

Escherichia coEi B was grown at 30°C with shaking on M9 medium (Kellenberger & Schauch, 1957) which contains per liter, 6 g Na,HPO,*2 H,O, 3 g KH2POI, O-5 g NaCl, 1 g NH&l, low3 M-MgSO*, lo-’ M-Call z and 4 g glucose. Exponentially-growing cells (3 x 10s cells/ml.) were infected in the presence of 20 pg n-tryptophan/ml. by adding 1 vol. of bacteriophage T4 in growth medium to 3 or 4 vol. of cells, to give a multiplicity of infection of 7 to 8 phagelbacterium.

In the experiments in which host RNA synthesis is analyzed it is important that phage infection be efficient. We have used two criteria; (1) the efficiency of killing of host cells and (2) the ability to induce /3-galactosidase, to determine the efficiency of infection. The sensitivity of both these criteria as measurements of efficiency of infection has been previously established (Adams, 1959; Kaempfer & M&gas&k, 1967; Kennell, 1968).

To teat for efficiency of infection, phage-infected cultures were quickly centrifuged 1 mm after infection to separate unattached phage from cells. 99.5% of the cells had lost the ability to form colonies.

Phage infection has been shown to have an immediate effect on the synthesis of /& galectosidase (Benzer, 1953). If phage infection is efficient, no detectable synthesis of /&galactosidase occurs when phage and inducer are added simultaneously; whereas, less than 30 set of preinduction is easily detectable (Kennell, 1968, and unpublished results). When phctge and inducer were added simultaneously, we could detect no enzyme synthesis above the basal level present before infection, even when induction was carried out in the presence of 10m3 M-S’,5 cyclic AMP, which has been previously shown to stimulate /3- galactosidase synthesis in cells grown in a medium containing glucose (Perlman & Pastan, 1968). (It should be noted however, that the efficiency and synchrony of infection is not critical for the determinations of T4 RNA, because (1) the determinations are made by specific hybridization assays which are not affected by the presence of RNA from uninfected cells, and (2) the addition of rifampicin prohibits further initiation of T4 RNA molecules, and therefore allows us to look at only those T4 messages initiated in the fist minute of infection.)

(b) Preparation of labeled RNA

[5-3H]Uridine (26 Ci/m-mole) and [2-14C]uracil (52 mCi/m-mole) were obtained from Schwarz-Mann Bioresearch, Inc. 3H-labeled uridine was added to infected bacteria to a concentration of 25 @i/ml. Initial experiments were done using [3H]uracil. However, we found that at the time of infection uracil uptake was poor, showing a 2-min lag before a maximum rate of incorporation was reached, compared to less than a 30-set lag with [3H]uridine. Stable RNA was labeled by growing bacteria for at least 2.5 generations with an initial concentration of O-1 &i [14C]uracil/ml. Cell metabolism was inhibited by pouring cultures over 2 vol. of ice containing 0.01 M-sodium cyanide. The cells were centrifuged, washed and resuspended in a buffer containing 0.01 as-TriseHCl (pH 7.5) and 0.01 M-

MgSO,, and lysed by treatment with 1% sodium dodecyl sulfate at 66°C for 1 mm. RNA was extracted 4 times at 66°C with phenol saturated with the same buffer. The samples were dialyzed overnight against 0.001 as-Tris*HCl (pH 7.5) containing O-001 x-MgSO,, and then treated with 10 pg DNase/ml. (electrophoretically pure, Worthington Biochemi- cals) for 1 hr at 37“C, followed by treatment with 50 pg pronase/ml. for 1 hr at 37°C. The RNA was then phenol-extracted 4 more times before dialysis against 0.01 x SSC (SSC is 0.16 a6-NaCl, O-016 M-sodium citrate) according to the method ofKennell & Kotoulas (1968).

(c) Preparation of competitor RNA’s Competitor RNA’s were prepared from cells grown in small (300 ml.) fractions in

vigorously shaking 2-1. flasks to ensure rapid mixing of the added phage. The preparation

RNA IN T4-INFECTED ESCHERICHIA COLI B 703

of RNA was essentially the same as for labeled RNA; a final ethanol precipitation was carried out to concentrate the RNA, which was redissolved at a concentration of 4 to 6 mg/ml. The DNase and pronase steps are of critical importance for preparations of competitor RNA’s RNA not treated in this way gave high baokground levels (counts bound in absence of T4 DNA) in hybridization reactions, which were probably due to E. coli DNA contaminating the preparations. All of the RNA’s used here gave background hybridization values of 5 20 cts/min

(d) Preparation of phage and phage DNA Phage T4 were purified by polyethylene glycol precipitation (Yamamoto, Alberts,

Benzinger, Lawhorne t Treiber, 1970), followed by equilibrium sedimentation in a CsCl step gradient (Thomas & Abelson, 1966). DNA was prepared by 4 phenol extractions with gentle shaking at room temperature, followed by ethanol precipitation and “spooling” out of the DNA. The DNA was resuspended in a low-salt buffer (O-001 M-NaCl, 0.001 M- EDTA, O*OOl M-TrissHCl, pH 7.6) (Bolle et al., 1968).

T4 DNA was denatured by heating in a boiling water bath for 10 min at a concentration of 200 pg/ml., followed by rapid cooling in an ice bath. The purity of the DNA was shown by the fact that labeled RNA from uninfected cells hybridized negligibly ( < 0.05%).

(e) Use of inhibitors Rifampicin, a gift from Dr P. Sensi, Gruppo Lepetit, S.p.A. Milano, Italy, was used at a

concentration of 300 pg/ml. in infected and 200 pg/ml. in uninfected cultures. Rifampicin was made up at 6 mg/ml. in 60% ethanol. Proflavine, obtained from Sigma Chemical Co., was used at a concentration of 60 pg/ml. It was made up freshly before use at 2 mg/ml. in 0.005 M-Tris-HCl, pH 7.5.

These concentrations of rifampicin and proflavine were chosen after control experiments were performed which determined the degree of inhibition of RNA synthesis after addition of inhibitor. At the concentrations used, rifampicin reduced the incorporation of [3H]- uridine into acid-insoluble material 96% in uninfeoted cultures, and 92% in infected cultures in 6 mm. Proflavine at 60 pg/ml. reduced incorporation 97% in 3 mm.

(f) Hybrkdization procedum (i) Bactetiophage T4

The hybridization procedure is basically the one followed by Belle et al. (1968). RNA and denatured T4 DNA were annealed at 66°C in 0.26 ml. of 6 x SSC in small stoppered tubes. The hybridization reaction was allowed to continue for 4 hr; using RNA labeled from 0 to 5 min after infection the reaction is 98% complete in 3 hr. DNA was used at a concn of 40 pg/ml. (10 pg/tube). This concentration of DNA is high enough to prevent lowering of hybridization efficiency by concentration dependence (Kennell & Kotoulas, 1968); that is, the reaotion is maximal over the 600-fold range of RNA/DNA inputs (0.1 to 50) used in these experiments. In all cases, the input C3H]RNA was between 1 and 2 pg of RNA, as determined by optical density at 260 mn assuming 43 pg/optioal density unit.

After the incubation period, the samples were diluted to O-76 ml. with water and digested at 37°C for 15 min with 12 pg pancreatic RNase/ml. (freed of DNase activity by heating at 80°C for 10 min). Samples were then put on ice and diluted to 4 ml. with buffer (0.6 ~-Kc1 in O-01 M-Tris*HCl, pH 7.3) and filtered through nitrocellulose filters (Schleicher & Schuell, B6). The filters were soaked in the buffer for at least 3 hr before use. Only those filters which became wet uniformly (that is, sank immediately when placed in buffer and showed no dry blotches) were used; lots of alters that became wet unevenly gave irre- producible results. The samples were washed with 80 additional ml. of buffer, followed by 3 ml. of 80% ethanol; they were then dried and counted in a liquid-scintillation counter.

The competition hybridization experiments were carried out in the same manner, except that at time zero different amounts of unlabeled RNA from cells infected for various lengths of time (from 20 to 150 set) were added, aa indicated in the text. Competitor RNA’s were used at concentrations between 0.2 to 2 mg/ml. (50 to 500 pg/sample tube).

Table 1 and Figure 1 show sample data for a typical T4 hybridization competition experiment. Labeled RNA was extracted from cells harvested 1.3 min after phage infection.


TABLE 1

Hybridization of 3H-labekd 274 RNA in the presence of unlabeled competitor RNA’s

Competitor RNA (mg/ml.)

0.3 0.4 0.6 1.0 1.4 2.0 Competitor w % cts/ o/O cts/ ’ % cts/ % cts/ yh cts/

RNA min camp. min camp. mm camp. min camp. min camp. min camp. hyb. hyb. hyb. hyb. hyb. hyb.

20sset 5799 14.5 5085 24.5 6061 26.7 4977 26.4 4861 27.9 4698 30.2 60-53~ 2310 66.7 2046 69.6 1428 78.9 1083 73.4 766 88.6 656 90.2

150~se0 636 90-6 419 93.8 312 96.4 217 96.2 168 97.7 149 97.8

[sH]RNA was extracted from cells harvested at 1.3 min in the experiment described in detail in the legend to Fig. 2. The labeled RNA was hybridized to T4 DNA in the presence of increasing amounts of the indicated competitor RNA%. The unlabeled competitor RNA’s were extracted from cells infected for 20 set, 60 set and 160 sec. The input of radioactive RNA in each sample tube was 23,800 cts/min; 6746 cts/min were hybridized in the absence of any competitor. For each level of competitor, the cts/min hybridized and y0 competition are listed (see text).

[sH]uridme was added simultaneously with phage; rifampicin 1 min later. Cold competitor RNA’s were extracted from infected cells 20 set (20-SW competitor), 60 set (60.set competitor) and 150 set (150~see competitor) after phage infection. The input of [3H]RNA to each hybridization tube was 23,800 cts/min. The average cts/min hybridized in four trials in the absence of competitor RNA was 6746 cts/min and is defined as the cts/min in total T4 RNA. The cts/min hybridized and the y. competition at each competitor concentration is given in Table 1. The O/e competition is calculated as

cts/mm hybridized without competitor -cts/min hybridized with competitor

cts/min hybridized without competitor x 100.

mg CompeMor RNA /ml

( 0 i

I/rig Competllor RNA

(b)

FIG. I. Competition of 3H-labeled RNA by unlabeled competitor RNA’s. The [sH]RNA from cells harvested at 1.3 min in the experiment described in detail in the legend to Fig. 2 was hybridized to T4 DNA in the presence of RNA from cells infected with T4 for 20 set (20~se0 competitor; -m-m--); 60 set (60-set competitor; -O--O--); and 160~set competitor; --A-A-).

(a) Direct plot of hybridization competition data from Table 1. The percentage of hybridizable counts competed at each competitor concentration is plotted uer8uB the concentration of competitor RNA per ml. of reaction mixture. The hybridization in the absence of the competitor was carried out in quadruplicate.

(b) Reciprocal plot of hybridization competition data. The data of (a) are replotted as the percentage of hybridizable counts veraus the reciprocal of the concentration of the competitor RNA. The percentage of the 3H-labeled T4 RNA which is 20-, BO- or 160~set RNA is determined by the extrapolation of the line to the ordinate.

RNA IN T4-INFECTED ESCHERICRIA CO&I B 706

In Figure l(a) the ye competition is plotted vereTBu8 the mg of competitor added. The percentage of [3H]RNA which can be competed by a specific competitor tends toward a maximum at high concentrations of competitor. To permit an exact determination of the plateau value, the data are replotted ae the percentage [3H]RNA competed ver8u8 l/mg competitor concentration per ml. (Fig. l(b)). A straight line can be drawn through the points to the ordinate (Lava&5 & De Hauwer, 1968). The intercept is defined as the fraotion of 20-set (31%), 60-set (94%) and 150-set (1OOo/o) RNA present in the RNA sample (Table 1).

The amount of [3H]RNA in each ml. of culture at the time of harvest is estimated from the acid-insoluble 3H cts/min present per ml. culture (Fig. 2(a)) at that time (334,000 cts/min/ml.). In this case, the input in the hybridization reaction was 23,800 ctsjmin,

Tmw (mm) Time (rn~n)

Fro. 2. Synthesis and degradation of RNA made early in T4 infection. At time zero phage T4 and 2.6 mCi [3H]nridine were added to a 100.ml. culture. At 1 minute rifampicin (300 pgplml.) was added. At various timas O.l-ml. samples were taken directly into cold 6% trichloroacetic acid and 5 to IO-ml. fractions of the cell3 here harvested. The RNA was purified for hybridization to T4 and E. edi DNA.

(a) Total radioactive material precipitable with cold 6% trichloroacetic acid. (b) EachpointgivestheamountoflabeledT4RNA(-~-~-),E.wZirRNA(--~--~--),

and E. coli non-rRNA (-A-A-), per ml. of culture. This value is derived from the percentage of counts which hybridize to the ape&a type of DNA and the amount of r3H]RNA/ml. cells, which is derived from the number of acid-insoluble 3H cts/min at the different times after infection.

(c) The data in (a) are replotted on semi-log scales. The maximum value (at 26 min for E. coli RNA; 3.6 mm for T4 RNA) is defined as 100 %. The percentage of [3H]RNA remaining is the number of cts/min/ml. at any time minus the number of counts per ml. in “stable RNA”, RNA present at 60 min after infection (29,000 cts/min for E. coli mRNA; 34,000 cts/min for T4 RNA), divided by the number of unstable counts per ml. (116,000 ots/min for 1. wli mRNA; 230,000 cts/min for T4 RNA) times 100. -A-A-, E. coli non-rRNA; --O--O-, T4 RNA.

which is equivalent to 0.072 ml. of culture. Therefore, each ml. of culture contained 6746 cts/min hybridized/O*072 ml. or 94,000 cts/min/ml. of hybridizable T4 RNA. 31% of the hybridizable T4 RNA was competed by 20-set RNA; therefore, each ml. of culture had 29,000 cts/min of 20-set RNA. Each ml. of cells had 79,000 cts/min of 60-set RNA (94%) and 94,000 cts/min of ISO-set RNA (100%).

The percentage of the 3H label in 20-, 60- and 150-set RNA experimentally obtained from cells harvested 80 set after infection is equal to the value expected, assuming that

706 E. CRAIG, K. CREMER AND D. SCHLESSINGEK

most of the radioactivity is in mRNA molecules whose synthesis was initiated sometime during the tist minute of infection. In a control experiment, BO-set competitor RNA was shown to compete lOOo/o against labeled RNA extracted from cells harvested 60 set after infection (data not shown).

(ii) Escherichia coli

Published procedures were used to hybridize labeled RNA to E. coli DNA trapped on filters (Kennel1 & Kotoulas, 1968). Hybridization experiments were carried out at an RNA/DNA ratio of l/1000, a ratio at which DNA sites for all RNA species are in excess; 200 pg of DNA were used for each filter. In the case of competition experiments, a 20-fold excess of ribosomal RNA (4 rg per assay) was used ; it was purified from ribosomes washed three times in buffer containing 0.01 iw-Tris.HCl (pH 7.5) and 10m5 M-MgSO,. In all cases, the input [3H]RNA contained more than 3000 cts/min, and the efficiency of hybridization of ribosomal RNA in the absence of competitor was greater than 5596.

(g) Other techniques

Published procedures were used to induce and measure /Y-galactosidase (Hogness, Cohn & Monod, 1955). 3’,5’-cyclic AMP was obtained as the sodium salt from Sigma Chemical Company.

3. Results To measure synthesis and degradation of new RNA in infected cultures, [3H]uridine

was added followed 1 minute later by rifampicin to inhibit further initiation of RNA synthesis (Lill, Lill, Sippel & Hartmann, 1969; see section (c) (i) below). Increase and subsequent loss of trichloroacetic acid-precipitable counts was then assayed. In the first minutes after infection, new RNA includes host mRNA and rRNA as well as T4 RNA (Landy & Spiegelman, 1968). The level of each class of RNA was followed by specific RNA-DNA hybridization.

(a) Half-life of RNA synthesized early in 1’4 infection

(i) Bulk RNA

Figure 2(a) shows the incorporation into total RNA of [3H]uridine added at the time of infection. Label continues to accumulate for about 2.5 minutes, reaches a peak and begins to decrease, very rapidly at first, then more slowly.

(ii) TCspecijk RNA

The amount of T4-specific RNA present at increasing times after rifampicin addition was determined by hybridization of labeled RNA to purified T4 DNA. T4 RNA accumulated until 3.5 to 4.5 minutes after infection, then decreased slowly; at about 50 minutes, a plateau value of 15% of the maximum was reached (Fig. 2(b) and unpublished results at later times). This plateau level of T4-specific stable RNA may reflect T4-specific tRNA species, at least some of which are transcribed early (Hsu, Foft t Weiss, 1967). In Figure 2(c) the loss of T4-specific RNA is plotted on a logarithmic scale. T4 RNA degraded exponentially with a half-life of 9.5 minutes.

(iii) Escherichia coli RNA

The amount of E. coli ribosomal and messenger RNA present at any time was determined by hybridization of [3H]RNA to purified E.coli DNA at an RNA/DNA input ratio where DNA sites are in excess for all RNA species (l/1000). Ribosomal RNA is defined as the [3H]RNA competed away in hybridization samples by a 20-fold excess of cold ribosomal RNA (i.e. 20 pg/pg input RNA). The rRNA synthesized

RNA IN TI-INFECTED ESCHERICHIA COLI B 707

immediately after infection (Fig. 2(b)) accumulates for up to 3 minutes and then begins to decrease. By 30 minutes after infection, 33% of the sH.labeled rRNA is degraded.

Non-ribosomal E. coZi RNA is defined as that RNA not competed by excess cold rRNA. The amount of “non-rRNA” is maximum about 2 minutes after infection and then decreases rapidly, reaching a plateau value at about 9 minutes. The plateau value, 15% of the maximum, probably represents tRNA (Maalee & Kjeldgaard, 1966; Norris & Koch, 1971), which along with mRNA, is not competed by ribosomal RNA. The unstable non-ribosomal RNA, presumptively mRNA, degrades exponentially with a half-life of 2 minutes (Rig. 2(c)).

(b) Ghparison of T4 RNA decay after rijampicin and projhine addition

Rifampicin rapidly inhibits RNA synthesis as measured by a decrease in ability of oells to incorporate [3H]uridine into aoid-insoluble material; however, we observe a low level of residual synthesis, about 6% of maximum, in infected oells, even 14 minutes after addition of label (data not given). It is important to know that the residual synthesis does not affect the determination of either the half-life (part (a) ) or the direction of degradation (part (c) ). Figure 3 shows the loss of acid-insoluble cts/min

-& 0% (0) IOO- 4 \ “0 ~~‘t

50-4 \, l \ 1

P 25- I “\, \ ‘.

% \ Oo” i .- .E

E \

L. d

IO- I o O\o, l

1 5 I IO 1 15 1 20 I E 30 I - -2 m. 100 (b)

50-

25-

Time (min 1

FIG. 3. Degradation of [3H]RNA in the presence of different inhibitors of RNA synthesis. At zero time either 1.5 ml. medium or 1.6 ml. medium containing T4 phage was added to a 4-ml. culture of E. c&i B. At 2 or 14min, 3 c(Ci [3H]uridiie/ml. were added.

(a) Rifampioin: 1 min after [3H]uridiie addition rifampicin was added to stop further initiation of RNA synthesis. The breakdown of RNA was followed as in Fig. 1. -A-A-, Uninfeoted; -a-@-, early T4, uridine added at 2 min; -O-O-, late T4, uridine added at 14 min.

(b) Proflavine: at 4 min proflavine was added to stop further RNA synthesis. The breakdown was followed ES in Fig. 2(c). -A-A-, Uninfected; -O-O--, TQ-infected, uridine added at 2 min.


in infected and uninfected cells after inhibition of RNA synthesis by rifampicin. The unstable RNA of uninfected cells was degraded with a half-life of 2 minutes. The unstable RNA from infected cells labeled at 2 and 14 minutes had half-lives of 10 and 8.6 minutes, respectively. (The RNA labeled 25 to 35 minutes after infection is 90% T4-specific as determined by hybridization experiments. Only 5% of the RNA hybridizes to E. coli DNA 6lters which have been shown, using RNA from uninfected cells, to have an efficiency of hybridization of 50% at an RNA/DNA ratio of l/1000.)

As a control, proflavine was used as an alternative inhibitor of RNA synthesis. Proflavin inhibits elongation of RNA chains by binding to DNA (Hurwitz, Furth, Malamy & Alexander, 1962); in phage-infected cells it caused a rapid inhibition of incorporation of [3H]uridine into RNA (greater than 98% in 4 min; data not shown). Using proflavine, a half-life for unstable RNA of 2-2 minutes in uninfected cells and 9.6 minutes in infected cells was obtained (Fig. 3(b)). The half-lives of RNA in cells treated with proflavine and rifampioin are essentially the same; thus, the residual synthesis seen in the presence of rimfapicin causes no artifactual increase in our half- life determination.

(c) Direction and rate of degradation of early T4 messenger RNA molecules

(i) Prin&le of experiment

We have extended the results above to determine the direction of degradation of mRNA molecules. For such experiments, one must be able to (1) synchronously initiate the synthesis of a class of mRNA molecules and (2) selectively inhibit the initiation of RNA chains without preventing elongation. Moreover (3) the RNA molecules must be of a sufficient length to permit detection of segments within molecules, and (4) the techniques available must be sensitive enough to assay these segments quantitatively.

The first condition is easily met by infection at a high multiplicity of phage, which results in synchronous initiation of the mRNA’s transcribed from a number of early promoters (Bolle et al., 1968).

There is substantial evidence that in vitro, rifampicin inhibits the initiation of new RNA chains (Lill et al., 1969; diMauro et al., 1969), but allows elongation (Gold $ Schweiger, 1970). Extensive experiments on the expression of the tryptophan operon have shown that the mode of action of rifampicin is the same in vivo as in vitro

(Mosteller & Yanofsky, 1970). We have also done control experiments under our conditions which show that rifampicin can be used to inhibit selectively the initiation of RNA synthesis (see Fig. 4), and probably does not affect the rate of degradation of mRNA (Fig. 2). Thus, the second condition is satisfied.

Concerning the third condition, T4 early RNA is composed of sizeable chains of RNA, some requiring as long as 5 minutes to be synthesized, the synthesis of which is initiated immediately upon phage infection (Brody, Sederoff, Belle & Epstein, 1970; Witmer, 1971; Black t Gold, 1971).

The condition most difficult to satisfy was the differential detection of portions of t,he messenger molecules. To determine direction of degradation of mRNA molecules, we have used competition hybridization to assay for the presence of different segments of labeled T4 RNA molecules. Non-radioactive RNA which corresponds to increasing lengths of the early mRNA’s was tested to see the extent to which it could compete away the labeled early mRNA. The competitor RNA’s were prepared from cells which

RNA IN T4-INFECTED l!MCHERICHIA COLI B 709

were infected for very short, but increasing lengths of time (20 set, 60 set, 160 set) in the absence of any inhibitor of RNA synthesis. The T4-specific RNA extracted from cells harvested 20 seconds after infection (20~set competitor) consists of the 6’- proximal segments of those T4 molecules initiated from 0 to 20 seconds after infection. The 60-set competitor corresponds to the 5’ segments which require up to 60 seconds to be synthesized. The 60-set competitor thus contains all the 20-SW RNA as a subclass; the MO-set competitor contains both the 60-set and 20-seo RNA as subclasses.

25 I I I I I 2 3 4 5

I/mg competitor RNA

FIQ. 4. Synthesis of RNA after addition of rifampicin. A lo-ml. oulture was infected with T4 at zero time. At 1.5 min, 300 pg rifampicin was added/ml.; at 3 min [3H]uridine was added. Cells were harvested at 6 min, the RNA purified and hybridized to T4 DNA in the presence of increasing amounts of 60-set and 160~set competitor RNA%. The data are plotted aa in Fig. 1 (b); -O-O-, 60-sea competitor; -A-A-, 160-set competitor.

These experiments were based on the idea that an excess of cold 20-set competitor RNA would prohibit the hybridization to T4 DNA of the labeled 6’ segment of the molecule which requires 20 seconds to be synthesized. The number of counts which the 20-set competitor prevents from hybridizing equals the number of counts in the 5’ segment.

A control experiment was designed to test the ethciency of shut-off of early T4 messenger RNA synthesis by rifampicin (condition 2) and the sensitivity of the competition hybridization method (wndition 4). Rifampioin was added to a culture one minute after phage infection. The culture was labeled with [sH]uridine from 3 to 6 minutes after infection. None of the label could be competed by BO-seo competitor RNA (Fig. 4), indicating that RNA initiation had indeed been inhibited, since the sequences of RNA made in the first l-5 minutes of infection are still being transcribed at these later times in infected cells not treated with rifampicin (Brody et al., 1970). The incorporated label enters chains which continue to elongate, because as expected, the 150~see RNA can compete away part of the labeled RNA.

(ii) Determination of the over-all direction of early T4 messenger RNA.

The two extreme possible modes of T4 early mRNA degradation, 5’ to 3’ and 3’ to 6’, should be readily distinguished by competition hybridization experiments. If over-all degradation is in the 3’ to 5’ sense one should see a decrease in the amount of RNA competed by 160-set RNA before a decrease in that competed by 60.~~ RNA, and a decrease in 60-set RNA before a deorease in 20-set RNA. If degradation is in the 5’ to 3’ sense and jeopardy begins at the time of synthesis, 20-set RNA should begin to decrease before the 60-set RNA, and 60-seo RNA before the 160.set RNA.


The synthesis and degradation of regions of T4 early mRNA molecules are shown in two similar experiments (Fig. 5(a) and (b)). [sH]U ri d ine was added 6 seconds before infection; the bacteria were infected at zero time and rifampicin was added at 1 minute. Samples were removed at various times, and the radioactive RNA extracted and hybridized to T4 DNA. The amount of 20-, 60- and 150~set RNA was determined by

Fro. 6. Degradation of sub&wee of early T4 mRNA. Samples of RNA prepared from cells treated ae deacoribed in Fig. 2 were hybridiid in the preeenoe and absence of different competitor RNA’s The data for each sample of [SH]RNA and each type of competitor were plotted a~ in Fig. l(b). The percentage of *H corresponding to the competitor used wae determined and the COITW pending &e/min/ml. of culture preoept et the time the sample ww taken (from type of data presented in Fig. 2(a)). The d&a are plotted by setting the greatest number of cts/min in each [3H]RNA present at any time equal to 100%. -m-m-, ~O-S~C RNA; --O--O--, 60-set; -- A -- A --, 160~set; -e-e--, total T4 RNA (total counta hybridizable in the absence of competitor).

(a) RNA samples were taken from 1.6 to 8 min after infection. 100% equals 23,600 cts/min/ml. at 1.6 min for 20-eec RNA; 101,600 cts/min/ml. at 2.6 min for BO-set RNA; 136,000 cts/min/ml. at 3.6 for 160-1~ RNA; 160,000 cts/min/ml. at 6 min for total T4 RNA. (The 1.3-min points are from data presented aa cm example in Material and Methoda.)

(b) RNA samples were taken from 1.6 to 18 min after infection. 100% equela 80,000 cts/min/ml. at 3 min for 60+x RNA; 146,000 cts/min/ml. at 4 min for 160~eec RNA; 166,600 cts/min/ml. at 6 min for total T4 RNA.

competition hybridization experiments similar to those described above. The amount of eaoh [aH]RNA per ml. of culture was calculated as described in Materials and Methods.

In the experiment shown in Figure 5(a), RNA samples were taken at 30-second intervals between l-5 and 8 minutes. All samples of RNA were hybridized in the presence and absence of 20-set, 60-set and 150~set competitors. The results were plotted as the percentage of each RNA subclass remaining wrBuB time after infection.

RNA IN T4-INFECTED ESCHERICHIA COLI B 711

20-set RNA reached a maximum value by 1.5 minutes and began to decrease by 2 minutes; 60-set RNA reached a peak by 2-5 minutes and was decreasing by 3 minutes. 150~set RNA peaked at 3.5 minutes and began to decrease by 4 minutes. Total T4 mRNA peaks at about 4.5 minutes and decreases by 6 minutes. Clearly, the RNA made first after infection starts to degrade before that made later, which is consistent with an over-all 5’ to 3’ sense of degradation.

In the experiment shown in Figure 5(b) samples were taken at later times after infection. By 5 minutes after infection there was no further increase of T4-specific RNA in the culture. By 18 minutes 15% of the T4 RNA (55% of the 150~set RNA and 59% of the 60-set RNA) was degraded. The rates of degradation of the different sub-classes of RNA are about equal, and very similar to the rate of degradation of bulk early T4 mRNA. Analyses of 20-set RNA were not carried out at times later than 2 minutes after infection. At later times 20-set RNA represents only about loo/ of the radioactive RNA; the competition hybridization analysis is not sensitive enough to measure the loss of 10% of the labeled RNA quantitatively.

4. Discussion

(a) Stability of T4 RNA

From our data we obtain a half-life of 8 to 10 minutes for both early and late T4 mRNA at 30°C a value only slightly higher than earlier estimates. Several laboratories obtained values of 3 to 4 minutes at 37”C, which is equivalent to 5.5 to 7.5 minutes at 30°C (Greene & Kern, 1967; Bose & Warren, 1967; Freisen, 1969) for the halflife (functional or chemical) of T4 messages. From results of pulse-chase and competition hybridization experiments, Bolle et al. (1968) estimated an upper limit for the half-life of the RNA species synthesized in the first 5 minutes of infection of about 8 minutes. These values are from 2.5 to 3.5 times greater than the half-life of E. wli mRNA. The emphasis in earlier cases was on the fact that T4 mRNA was unstable, not the fact that it is significantly more stable than host mRNA.

Support for the idea of greater stability of T4 mRNA compared to host mRNA can be found in other types of experiments. Adesnik & Levinthal(l970) detected discrete size classes of T4 messenger molecules as bands on acrylamide gels. In these experiments, where relatively long labeling times (6 min) were used and molecules were in various stages of synthesis at the beginning of labeling, the ability to detect complete messenger species indicates that the half-life must be at least as long as the time required for synthesis. At 25°C 5 to 6 minutes are required to transcribe a molecule of lo6 molecular weight (Bremer $ Yuan, 1968). Discrete species as large as 1.5 x 10s daltons were found; these molecules would require about 8.5 to 9 minutes to transcribe at 25°C. To be able to detect any label in discrete bands after a 6 minutes labeling, the half-life of this RNA molecule must be at least 8 to 9 minutes.

(i) Synthesis (b) Synthesis and stability of host RNA

Using the data presented in Figure 2(b) and the efficiency of hybridization of our T4 and E. wli systems one can estimate that 53% of the RNA labeled in the first 2 minutes of infection is T4-specific. The efficiency of hybridization, 55% for E. wli RNA and 60% for T4 RNA, was determined in control experiments using RNA with label only in E. wli or T4 RNA. Therefore, after correcting for the eflticiency of


hybridization, at 2 minutes after infection 367 x lo5 cts/min/ml. are found in T4- specific RNA, 2.0 x lo5 cts/min/ml. in E. coli non-ribosomal RNA, and 1.17 x lo5 cts/min/ml. in ribosomal RNA, or a total of 68 x lo5 cts/min/ml. in RNA. This determination agrees with the value of 7.0 x lo5 cts/min of trichloroacetic acid- precipitable 3H at 2 minutes. 63% of the host RNA made is messenger and transfer RNA; 37% is ribosomal RN-4.

We find a rapid inhibition of host RNA synthesis after phage infect’ion, 90% of the RNA which is pulse-labeled from 3 to 4 minutes after infection is T4specific. This rapid shut-off after infection of E’. coli B with T4 is in agreement with results reported previously (Landy & Spiegelman, 1968; Adesnik & Levinthal, 1970).

(ii) 8tability

The shape of the [3H]RNA curve in Figure 2(a) is different from that expected for exponential decay of a class of RNA molecules with identical half-lives. The curve is a composite of the rapidly degrading E. coli mRNA and the more slowly degrading T4 and ribosomal RNA. The initial drop in trichloroacetic acid-precipitable counts is very rapid. This initial loss of counts is due mainly to the degradation of the E. coli mRNA, which loses 65% of its counts between 25 and 5 minutes while the T4-specific RNA and rRNA lose only 15 and 25% of their counts. Clearly the host mRNA labeled in the first minute of infection is degraded with a half-life of about 2 minubes, which is the same as the rate of degradation in uninfected cells.

The analysis of the degradation of host mRNA is complex, because the molecules labeled during the experiment are a mixture of messages initiated after infection and those which are being synthesized at the time of infection. However, at the time degradation can begin to be measured, one can estimate from the exponential rate of degradation that about 60% of the label is in chains that had not begun to be degraded at the time of infection. Therefore, the initiation of chemical degradation of host mRNA molecules occurs at the same rate in T4-infected and in uninfected cells.

(c) Direction of degradation of T4 mRNA

It is clear from Figure 5 that the first T4 RNA synthesis in infection is the first to be degraded. Interpretation of this result relies heavily on the assumption that most early RNA species require more than 1 minute for synthesis. Gradient analyses of RNA labeled in infected cells show that most early T4 RNA (> 70%) is in quite high molecular weight RNA which would require 3 to 4 minutes for its synthesis at 30°C (Brody, Gold & Black, 1971; Witmer, 1971). This does not eliminate the possibility that there is a significant amount of small molecular weight RNA made; however, no bulk early T4-specific RNA has been found sedimenting slower t’han 8 s (including the mRNA that codes for internal protein 1 (Black & Gold, 1971), which is known to appear in the first minute of infection). At least 1 minute would be required to transcribe an RNA molecule of this size. Therefore the fact that 20-set RNA begins to degrade before 60-set RNA shows that the first part (5’ end) of early T4 RKA molecules start to be degraded before the part made last (3’ end).

The exact time at which the amount of label in different segments of RNA reaches a maximum and then begins to decline is dependent both on t$he time required for synthesis of the segment and on the rate of initiation of degradation. The times after inhibition of initiation of RNA synthesis at which the subclasses reach maximum

RNA IN T4-INFECTED ESCHERICHIA UOLI B 713

values are about the same as the times of synthesis (28sec RNA at 30 set; 60-set RNA at 90 set; 150-set RNA at 150 set; total T4 RNA at 3.5 mm). The fact that the peak times are as long as the times required for synthesis indicates that the rate of initiation of degradation is slow. Total T4 RNA reaches a peak at about 4.5 minutes after the addition of rifampicin ; therefore, the average early T4 messenger RNA in these cultures requires 3.5 minutes to be synthesized and lasts about 9 minutes.

(d) Comparison of viral and host RNA’s

The results of experiments on mRNA presented here are interesting for two reasons: (1) the over-all sense of degradation of the phage messenger RNA is the same as that of an E. coli messenger RNA, the trp RNA; and (2) two messenger molecules whose stabilities differ sixfold, both show exponential kinetics of decay and an over-all direction of degradation in the 5’ to 3’ sense.

Host mRNA made early after T4 infection is very unstable with a half-life of 2 minutes while T4-specific RNA molecules are attacked 4 to 5 times more slowly. A similar but more complete analysis (Marr $ Yanofsky, 1971) gave similar results in T7-infected cells; trp mRNA decays with a half-life of 90 seconds in both infected and uninfected cells, whereas T7 mRNA has a half-life between 6 and 20 minutes. These results indicate that mRNA molecules have unique properties, structural or confor- mational, which at least in part determine their stability, since two molecules in the same cell are degraded at very different rates.

Furthermore, in the literature, T7 mRNA has been shown to be relatively stable, while T4 mRNA has been considered unstable. For example, in experiments in which actinomycin D was used to stop RNA synthesis, T7 mRNA decayed with a half-life of about 15 minutes (Summers, 1970, Figs 3 and 4). ‘However, these values are not so different from the half-life of 9 to 10 minutes for T4 mRNA reported here. It may be a general phenomenon that phage RNA molecules are relatively stable compared to host mRNA’s, with R17 RNA being an extreme case of stability (Hattman & Hofschneider, 1967). This stability would be likely to be an asset to the infecting virus, for a longer lifetime allows a greater translation yield per mRNA molecule.

It may be of interest that the instability of the ribosomal RNA made immediately after infection is comparable to that of T4 mRNA, with a 50% loss after 9 minutes. Instead, in experiments with cells with prelabeled ribosomal RNA (data not shown), pre-existing ribosomes were stable.

In another reported case, certain E. ccli strains broke down newly formed ribosomal RNA during amino-acid starvation, again at about the same rate seen in T4-infected cells (Craig, Schlessinger & Gurgo, 1972). Furthermore, in cases where translation is blocked by mutational lesions or antibiotic action, bulk mRNA is stabilized to a comparable extent (Craig, 1972). Thus, in the known cases when newly formed rRNA becomes destabilized or mRNA is “stable”, half-lives approach the same range of values observed for T4 and T7 mRNA (9 to 15 min). It is not clear if this is coinci- dental, or if a similar mechanism may regulate breakdown in these various cases.

From our in vivo data, the exact mechanism of degradation in any of these cases can of course not be determined. As has been pointed out for the trp operon (Morikawa & Imamoto, 1969; Morse et al., 1969), a mechanism involving a 5’ exonuclease can not be distinguished from random cleavage by an endonuclease followed by the degradation of fragments in the 3’ to 5’ direction. However, for the second case, as Marr & Yanofsky have pointed out (1971), an increase in stability as great as that seen here


demands an elimination of susceptible sequences or conformations from the stabilized message, to prevent the continuous endonucleolytic attack required. It is much easier to envisage a unique difference in structure or conformation at or near the 5’ end which inhibits a rate-limiting step required for initiation of chemical degradation; a similar suggestion has come from studies of mRNA breakdown in the RNase V assay system (Mangiarotti, Schlessinger t Kuwano, 1971).

These experiments were motivated by discussions with Larry Gold. One of us (E. C.) was supported by Training grant TOl-Al-00257, and the work was supported by National Institutes of Health grant, no. CA12021.

REFERENCES

Adams, M. H. (1959). In Bacteriophages, p. 137. New York: Interscience Publishers Inc. Adesnik, M. & Levinthal, C. (1970). J. Mol. Biol. 48, 187. Benzer, S. (1953). Biochirn. biophys. Acta, 11, 383. Black, L. W. & Gold, L. M. (1971). J. MOE. BioZ. 60, 365. Bolle, A., Epstein, R. H., Salser, W. & Geiduschek, E. P. (1968). J. Mol. BioZ. 31, 326. Bose, S. K. & Warren, R. J. (1967). Biochemi&ry, 58, 2408. Bremer, H. & Yuan, D. (1968). J. Mol. BioZ. 34, 527. Brody, E. N., Gold, L. M. & Black, L. W. (1971). J. Mol. BioZ. 60, 389. Brody, E. N., Sederoff, R., Bolle, A. & Epstein, R. H. (1970). Cold Spr. Ha& Symp.

Quad. Biol. 35, 203. Craig, E. (1972). Genetics, 70, 331. Craig, E., Schlessinger, D. & Gurgo, C. (1972). J. Back 111, 66. Freisen, J. D. (1909). J. Bact. 97, 313. Geiduschek, E. P. & Haselkorn, R. (1969). An?&. Rev. Biochem. 38, 647. Gold, L. & Schweiger, M. (1969). Proc. Nat. Ad. Sci., Wash. 62, 892. Greene, R. & Korn, D. (1967). J. Mol. BioZ. 28, 435. Gros, F., Dubert, J. M., TissiBres, A., Bourgeois, S., Michelson, M., Soffer, R. & Legault, L.

(1963). Cold Spr. Had. Symp. Quad. Biol. 28, 299. Hattman, S. & Hofschneider, P. H. (1967). J. Mol. BioZ. 29, 173. Hogness, D. S., Cohn, M. & Monod, J. (1955). Biochim. biophys. Acta, 16, 99. Hsu, W. T., Foft, J. W. & Weiss, S. B. (1967). Proc. Nat. Acud. Sci., Wah. 58, 2028. Hun&z, J., Furth, J. J., Malamy, M. & Alexander, M. (1962). Proc. Nat. Acud. Sci., Wash.

48, 1222. Jacob, F. & Monod, J. (1961). J. Mol. BioZ. 3, 318. Kaempfer, R. 0. R. & Magasanik, B. (1967). J. Mol. BioZ. 27, 475. Kellenberger, E. & Schauch, J. (1957). Virology, 3, 256. Kennell, D. (1968). J. ViroZ. 2, 1262. Kennell, D. & Kotoulaa, A. (1968). J. Mol. BioZ. 34, 71. Landy, A. & Spiegelman, S. (1968). Biochemistry, 7, 585. LavallB, R. & De Hauwer, G. (1968). J. Mol. BioZ. 37, 269. Lill, H., Lill, U., Sippel, A. & Hartmann, G. (1969). Lepetit CoZZ. Biol. Med. 1, 55. Maalee, 0. & Kjeldgaard, N. 0. (1966). Control of MucromoEecuZar Synthkx. New York:

W. A. Benjamin. Mangiarotti, G. & Schlessinger, D. (1967). J. dIoZ. Biol. 29, 395. Mangiarotti, G., Schlessinger, D. & Kuwano, M. (1971). J. Mol. BioZ. 60, 441. Marr, B. L. & Yanofsky, C. (1971). Nature New Biol. 234, 168. diMauro, E., Snyder, L., Marino, P., Lamberti, A., Coppo, A. & Tocchini-Valentini, G. P.

(1969). Nature, 222, 535. Morikawa, N. & Imamoto, F. (1969). Nature, 223, 40. Morse, D. E., Mosteller, R. F., Baker, R. & Yanofsky, C. (1969). Nature, 223, 43. Mosteller, R. D. & Yanofsky, C. (1970). J. Mol. BioZ. 48, 525. Norris, T. E. & Koch, A. L. (1971). Bach Proc. p. 164. Perlman, R. L. & Pa&an, I. (1968). J. Bid. C&m. 243, 5420.

RNA IN T4-INFECTED ESCHERIOHIA COLI B 715

Salser, W., Janin, J. & Levinthal, C. (1968). J. Mol. B&Z. 31, 237. Summers, W. C. (1970). J. Mol. BioZ. 51, 671. Thomas, C. A. & Abelson, J. (1966). In Procedures in Nucleic Acid Reeearch, ed. by

G. L. Cantoni & D. R. Davies, p. 553. New York: Harper 8z Row. Witmer, H. J. (1971). J. BioZ. Chem. 216, 5220. Yamamoto, K. R., Alberts, B., Benzinger, R., Lawhorne, L. & Treiber, G. (1970). Virology,

40. 734.

Note added in. proof: The proposal that many phage specific mRNA species show greater chemical stability than host mRNA is further supported by the observations of Hayaehi & Hayashi (1970) with 4X 174, and of Janish et al. (1970) (with M13); in these cases individual mRNA molecules can be purified from infected cultures 10 minutes or more after their synthesis.

REFERENCES

Hayashi, Y. & Hayashi, M. (1970). Cold Spr. Harb. Symp. Quant. BioZ. 35, 171. Jrtnish, F. J., Jacob, E. & Hofschneider, T. H. (1970). Nature, 227, 59.

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Metabolism of T4 messenger RNA, host Messenger RNA and ribosomal RNA in T4-infected Escherichia coli B