J. Mol. Bio2. (1972) 71, 687-699

Different Half-lives of Messenger RNA Corresponding to Different Segments of the Tryptophan Operon of

Escherichia coli

.JES FoRcHnA&fE6ERt, ETHELNOLAND JACKSON: AND CHARLES YANOFSKY

Deparhent of Biological Sciences, Stanford University Stanford, Calif. 94305, U.X.A.

(Received 12 April 1972, and in revised form 7 August 1972)

In genetically derepressed strains (trpR-) of Eschmichia coli which are growing exponentially, messenger RNA regions corresponding to different segments of the trp operon are labeled with different kinetics, suggesting that operator- proximal and distal regions of trp-mRNA have different half-lives. This con- clusion was comirmed by direct measurement of trp-mRNA decay; the half-lives for different mRNA regions at 30°C were found to be 60 seconds for trpE-mRNA, 75 seconds for trpDC-mRNA, and 95 to 116 seconds for trpBA-mRNA. Deletions of genetic segments within the operator-proximal region of the operon reduce the half-life of trpBA-mRNA. Large deletions which place the BA region near the operator reduce the half-life of trpBA-mF%NA to values similar to that of trpE-mRNA in the parental strain. Therefore location in the message rather than primary structure appears to determine the half-life of each mRNA region. Several of the internal deletions have a polar effect on the synthesis of the trpB and trpA polypeptides. However, the reduction in trpBA-mRNA half-life does not appear to be due to polarity because trpBA-mRNA half-life is reduced to the same value in three deletion mutants in which there is a sevenfold dif- ference in polarity. These results are compatible with a model of trp-mRNA degradation in which the initial degradative event occurs near the 5’ end of the mRNA molecule and is followed by over-all degradation in the 3’ direction, with random or non-random delays causing an increase in half-life of about 10% per 1000 nucleotides n-RNA. Our findings are not compatible with a model of normal degradation in which the entire mRNA molecule is the target for the initial degradative event.

Degradation of messenger RNA appears to be initiated by a random event leading to first-order kinetics of mRNA disappearance (Kepes, 1969; Mosteller, Rose & Yanofsky, 1970). In Esctirichia coli the over-all degradation of an mRNA molecule proceeds 5’ to 3’, the same direction as transcription and translation (Morikawa & Imamoto, 1969; Morse, Mosteller, Baker & Yanofsky, 1969). It is not known if all mRNA species in a bacterium have the same half-life; values reported for E. coli mRNA vary between 0.7 and 2.5 minutes at 37°C (Kepes & Beguin, 1966; Leive &

t Permanent address: University Institute of Microbiology, 0&x Farimsgsgede 2 A, DK 1363, Copenhagen K. Denmark.

$ Pmaent address: Department of Human Genetics, The University of Michigan, Ann Arbor, Mich. 48104,U.S.A.

687

688 J. PORCHHAMMER, E. N. JACKSON AND C. YANOFSKY

Kollin, 1967; Pato & von Meyenburg, 1970; Mosteller et al., 1970; Blundell, Craig SC Kennell, 1972). These reports raise the question whether individual mRNA species have different half-lives or whether the reported variation reflects the use of different methods of measuring mRNA decay; e.g. assay of the capacity for translation versus physical existence.

In this paper we report physical half-life differences for mRNA regions correspond- ing to different segments of the trp operon of E. coli. The differences detected suggest that there is a half-life gradient along the operon with the operator-proximal region of trp-mRNA being degraded most rapidly. The dependence of half-life on location in the messenger was investigated further by determining the half-life of the most operator-distal mRNA region, trpBA-mRNA, in strains with deletions of various lengths in the operator-proximal segment of the operon. The Cmlings obtained suggest that location in the message rather than primary structure of the mRNA region being attacked is primarily responsible for the characteristic half-life of each region of the trp-mRNA molecule.

2. Materials and Methods

(a) Bacterial strains

Most of the strains used in this study were derived from Escherichia coli strain Al9, RNase I-, met-, X- (X) (Gesteland, 1966), where X stands for an unknown growth requirement. The strain was made cysB- by transduction with phage Pl grown on W3110 cysB- (Lennox, 1955; Yanofsky & Lennox, 1959). It was also made met+, X+ by transduction with Pl grown on W1485 to give strain A19 RNase I- cysB- (X); an isogenic strain, A19 RNaee I- cysB- trpR- (h) was constructed from this strain by transduction with phage grown on W3110, trpR-. From these two a set of isogenic trpR+ and trpR- strains was constructed having either the intact trp operon or one of several internal deletions in the operon (Jackson & Yanofsky, 19723). The extent of the internal deletions in the strains used here is indicated in Fig. 1.

(b) Media and growth conditiona Bacteria were grown with shaking at 30°C in a New Brunswick gyrotory water-bath.

The minimal medium employed (Vogel & Bonner, 1956) was supplemented with glucose

--- p/E

-==

-=I

plDl0

-p/BAfS

: @A EC8 trpA EC/7 trpA EC20 @A ED// lrpAE4/ irpd DC71

FIG. 1. The trp operon. The operon is drawn to scale and the regions of the operon carried by the three transducing

phages ptE, ptDl0 and ptBA15 are indicated with solid bars. The internal deletions used in this study (Jackson & Yenofsky, 19725) are shown with open bars

in relation to known markers (Yanofsky, Horn, Bonner & Stasiowski, 1971). Uncertainty with regard to endpoints is indicated with dashed lines.

HALF-LIVES OF trp-mRNA REGIONS 689

O-4%, and 40 pg of L-tryptophan/ml., where indicated. In thii medium all strains have a mass doubling time of 96 min f 8 min.

(c) RNA extraction

Bacteria were labeled with [sH]uridine as described; when they reached an optical density of 80 to 120 (Klett Summerson filter no. 64) corresponding to 6 x lo* bacteria/ml. 20 or 25 ml. of culture were poured into a crushed-ice mixture containing 0.01 x-NaNs and 200 pg chlorampheniool/ml. After centrifugation (10,000 rev&in, 6 min, O’C), the pellet was washed once in 0.01 M-TrisHCl (pH 8-l), 0.01 M-NaNs, 0.006 M-MgSOI, 0.06 M-KC& 200 cog chloramphenicol/ml., and centrifuged again. The pellet was then resuspended in 2.5 ml. of the same buffer and 375 pg of lysozyme and 50 pg of DNase were added, After freezing and thawing 3 times the bacteria were lysed with O*4o/o sodium dodecyl sulphate, swirled well, and the solution acidified with 0.1 vol. of 1 M-acetate buffer (pH 6.1). The procedure for RNA extraction (For&hammer & Kjeldgaard, 1967; Lindahl & For&hammer, 1969) included treatment with DNase and pronaee as inter- mediate steps in the phenol extraction and alcohol-precipitation procedure. The RNA concentration was calculated on the basis of an extinction coefficient at 260 nm of 20 mg-l cma, and the specific activity of total RNA extracts was calculated from two 20-g. samples taken from the cuvette, dried on nitrocellulose filters and counted in a toluene-based scintillation mixture, using a Packard liquid scintillation spectrometer.

(d) Meeaenger RNA-DNA hybrkiimtion

The preparation of DNA from various phages has been described by Imamoto & Yanofsky (1967) and Morse & Yanofsky (1969b). The bacteriophages used include iAh-be0 (from N. Franklin) and i”h- @OptE (from J. P. Gratia); and i?h-?OptDIO and ptRAl6, described by Rose, Mosteller’ & Yanofsky (1970) and Morse & Yanofsky (19693). The segments of the trp operon carried by the phages are indicated in Fig. 1. The hybridiza- tion technique was essentially that of Gillespie & Spiegehnan (1965); 5 pg of alkali- denatured DNA was immobilized on each nitrocellulose filter, and hybridization was carried out in 0.01 M-Trie’HCl (pH 7.2), 0.5 M-KC& 0.001 zr-EDTA at 66’C for 44 hr. RNA was dissolved in the same buffer at a concentration of 70 pg/ml. Each hybridization vial contained 1 DNA filter and 500 4. of the RNA solution. The precise concentration of RNA was calculated from the specific activity of the RNA and the counts in two IO-$. samples. The efficiency of hybridization of trp-mRNA was estimated as described by Landy t Spiegehnan (1968); under our conditions the efficiency was 93%. trp-specific mRNA is then calculated as the difference between ots/min hybridized to DNA from the transducing phages and from the non-transducing parental phage.

(e) Tryptophan eylnt?&aee ae8aya A 225-11. culture was grown exponentially for 3 generations at 30°C. At an optical

density of 80 (Klett Summerson), 120 ml. were rapidly cooled in centrifuge bottles placed in ice, and 60 ccg chloramphenicol/ml. was added. The remaining culture was used for RNA extraction. After centrifugation (10,000 rev./min, 10 min, 0°C) the pellet was washed in O8So/o NaCl, and resuspended in O-5 ml. 0.1 rd-Tris*HCl (pH 78). The cells were disrupted with a Bronson Sonifier, and debris removed by centrifugation (10,000 rev./min 10 min, 0°C). Extracts were then assayed for tryptophan synthetase /la and c( activities according to Creighton t Yanofsky (1970). Protein concentration was determined by the method of Lowry, Rosebrough, Farr & Randall (1961).

C3H]uridine (25 Ci/m-mole) was purchased from New England Nuclear, Boston, Maas. Membrane filters (13 mm, type B6) were from Schleicher & Schuell Company, Keene, N.H. The DNase employed was the electrophoretioally purified RNase-free grade from Worthington Biochemical Corporation,

690 J. FORCHHAMMER, E. N. JACKSON AND C. YANOPSKY

3. Results

(a) The ratio of fH] ur id ine counts incorpora&d into different regions of trp-mRNA varies with the length of the pulse period

In the following experiment it was observed that in a repressor-negative (trpR-) culture growing exponentially in the presence of [3H]uridine, different regions of trp-mRNA are labeled with different kinetics. This would not be expected if RNA polymerases are randomly distributed along the trp operon in the bacterial population, and if mRNA half-life is the same for mRNA regions complementary to different segments of the operon.

A culture of E. coli A19 trpR- was labeled with [3H]uridine for different periods, and RNA was extracted and hybridized to DNA from different transducing phages, each of which carries approximately 30% of the operon (Figure 1). Sufficient [3H]uridine was added to ensure unchanged incorporation for 10 to 15 minutes after the last sample was taken (determined in preliminary experiments). In short labeling periods, the incorporation of [3H]uridine into trpE-mRNA, trpDC-mRNA and trpBA-mRNA reflects the rate of synthesis if degradation of labeled mRNA is negligible. Figure 2 shows that the mRNA regions complementary to the three different segments of the operon were labeled to approximately the same extent during short pulse periods, suggesting that each region is transcribed at the same rate.

When the period of incorporation of E3H]uridine was prolonged, however, an increasing fraction of the counts accumulated in mRNA complementary to the inter-

130

, , , , , ( ,

0 I 2 3 4 5 6 7 8 9 IO II Time of[3H]uridine incorporoton !mln)

FIQ. 2. Ratios of [3H]uridine incorporated into different segments of the trp-mRNA as a function of the length of the labeling.

A 226-ml. culture of strain A19 trpR- was grown exponentially in minimal medium + 0.4% glucose at 30°C. At O.D. = 100 (Klett Summerson, filter 54), 4 samples of 26 ml. each were pipetted into minimal medium + [3H]uridine, 600 &i (spec. act. 4 G/m-mole) at 8 ti81 concn of 2.7 pM, and incorporation was stopped as indicated between 30 and 120 sec. Four more 25-ml. samples received [3H]uridine, 600 PCi (spec. act. I.5 Ci/m-mole) at 8 fin81 concn of 8.8 pM, and these were stopped 8fhr 3 to 11 min of incorporation. RNA was extracted and hybridized to DNA from plE, @tolO, ptBAI5 and the parental non-transducing phage. Cts/min speci6o for tip-mRNA were 360 to 2000 above background (91 to 326 ots/min). The ratio of counts in trpE-mRNA to counts in tipDO-mRNA is indicated (-A--A--) 88 well as the ratio of counts in IrpJ%‘-mRNA and WpBA-mRNA (-+-+-). Each point represents the average of values obtained with dupkate samples minus background values.

HALF-LIVES OF trp-mRNA REGIONS 691

mediate and distal segments of the operon (when compared to the proximal trpE segment). Since with time the specific activity of mRNA approaches that of the pyrimidine pool (Winslow & Lazzarini, 1969; Pato & von Meyenburg, 1970), at late times incorporation can be taken as a measure of the total amount of mRNA present when synthesis and degradation of radioactive mRNA are balanced. Since the short and long pulse ratios are not identical, it appears that the rates of degradation of the three mRNA regions are different.

During exponential growth the following equationt is valid for any labile molecular species whose mean-life is short compared to the generation time of the bacterium: amount = rate of synthesis times mean-life. Applying this equation to the three regions of mRNA studied gives: mean-life of trpE-mRNA = 0231 x the mean-life of trpDC-mRNA and 0.61 x the mean-life of trpBA-mRNA. This experiment was re- peated three times and the observed ratios for mean-lives of trpE-mRNA and trpBA-mRNA were: 051, 0.57 and 0.74 (trpDC-mRNA was not measured in these experiments). In a different strain (W3110, ha,-, trpR-) the trpE-mRNA: trpBA- mRNA was found to be 050. Together, these observations indicate that during exponential growth trpE-mRNA is more labile than trpDC-mRNA, which is more labile than trpBA-mRNA.

(b) Degradation of three regions of trp-mRNA folhing repression

In order to substantiate the conclusions of the previous experiments an A19 trpR+ strain was depressed with /I-3-indolyl acrylic acid (IA) and its trp-mRNA was uniformly labelled with [3H]uridine. The culture was then repressed with excess tryptophan and degradation of different regions of tv-mRNA was followed.

tin exponentially growing bacteria the amount of any molecular species whose mean-life is short compared to the generation time of the cells, is equal to the rate of synthesis times the mean-life of that molecule. Thus, when the relative amount, (A), of two mRNA species and their relative rate of synthesis, (B), are known, their relative mean-life is A/R. In Figure 2 the initial incorporation of [3H]uridine into trpE-mRNA, tipDO-mRNA and trpBA-mRNA reflects the rate of synthesis, if degradation of labeled mRNA is neglected. During the tlmt minute the specific activity of the UTP- and CTP-pools increases rapidly (Winslow & Lazzarini, 1969; Pato BE von Meyenburg, 1970) 80 that approximately 76% of the label found will be incorporated during the last 30 seconds. At the termination of 30 and 60 second pulses an mRNA molecule with a half-life of 60 seconds should retain Borne 80 to 90% of the pyrimidine incorporated during the pulse. The incorporation into mRNA in the 30 and 60 second pulses can therefore be taken a8 an arbitrary measure of the rate of transcription, and the values obtained can be multiplied by the length of the different segments of the trp operon carried by the three transducing phages (Fig. 1). The ratio of radioactivity in mRNA hybridized to DNA of ptE and ptDlU was 116% and the cor- responding ratio for ptE and ptBA15 DNA’s was 112% (Fig. 2). Both ratios may be slightly low, since trpl-mRNA was found to be more labile than the more distally located segments, but the error is probably less than 10% because the degradation expected during the pulse would be small. When the period of labeling was increased, these ratios were found to decrease until they reached final values of ptE/ptDIO = 91% and ptE/ptBAl5 = 6So/o (averages of the values found after 5, 8 and 11 min of labeling). The cts/min present in the different mRNA segments at later times can be taken a~ a relative measure of the amount of mRNA since the specific activity of the pyrimi- dine pools changes little after the first few minutes of incorporation (Winslow & Lazzarini, 1969; Pato & von Meyenburg, 1970).

Thus, our data (Fig. 2) permit the following calculations:

(4 mean-life of trpl-mRNA 91 mean-life of trpDC-mRNA

= - = 0.81 and 116

(b) mean-life of trpE-mRNA 60 mean-Me of trpBA-mRNA = 112

= 0.61.

692 J. FORCHHAMMER, E. N. JACKSON AND C. YANOFSKY

4000

FIG. 3. Degradation of trp-mRNA after repression. A 225ml. culture of strain A19 trpR+ was grown exponentially at 30°C as in Fig. 2. At

O.D. = 100 /3-3-indolyl acrylic acid was added at 20 pg/ml. to derepress the tqn operon. Two min later [sH]uridine, 6 mCi (spec. act. 1.5 Ci/m-mole) was added, and after 8 more min tryptophan (100 pg/ml.) was added, and this defined zero time of repression. Samples of 26 ml. were taken, RNA extracted and hybridized as in Fig. 2. trpE-mRNA cts/min (-+-+-), tr@C-mRNA (-A-A-) and &@A-mRNA (-O-O-). Each point represents the average of values obtained with duplicate samples minus background values.

The pattern of residual synthesis and degradation of trp-mRNA seen in Figure 3 is consistent with previous reports that repression occurs almost instantaneously and that transcription of trpE-mRNA is completed in about 1 minute at 30°C (Rose et al., 1970). trpE-mRNA decays with a half-life of 58 seconds. Transcription of the D gene is complete 1 minute later, and trpDC-mRNA disappears with a half-life of 75 seconds. Finally, transcription of trpB and trpA is complete 3 to 4 minutes after the addition of tryptophan and trpBA-mRNA is degraded with a half-life of 95 seconds. The ratios of the half-lives of trpE-mRNA and trpDO-mRNA, 0.77, and of trpE-mRNA and trpBA-mRNA, 0.61, are close to the ratios calculated for cultures in exponential growth (Figure 2). Figure 3 also shows that degradation and synthesis of trpBA-mRNA are balanced during the first three minutes of repression. This indicates that labeling of trp-mRNA is uniform by the beginning of the repression period. One unfavorable feature of the design of the experiment shown in Figure 3 is that incorporation of [SH]uridine into RNA continues during the repression period. This causes the background counts (labeled RNA hybridized to DNA of the parental non-transducing phage) to increase from an initial value of 220 cts/min (0-024Oh of the cts/min in 35 a RNA) to a final value of 330 cts/min (O*016°~ of the cts/min in 35 pg RNA). This makes measurements obtained near the end of the period of degradation uncertain. Another complication is introduced by transcription initiations occurring at the internal promoter, P2, located within the operator-distal end of trpD (Jackson & Yanofsky, 1972a). P2 initiations are not subject to repression and

HALF-LIVES OF trp-mRNA REGIONS 693

are estimated to ocour at a frequency of approximately 3% of the initiation frequency at the principal promoter, Pl (Jackson & Yanofsky, 1972a).

To reduce these sources of error in subsequent experiments we pulse-labeled during the last minute of synthesis of each region of the messenger and then chased with a lOO-fold excess of unlabeled uridine. Prom the increase in the specific activity of the RNA during the repression period it can be concluded that incorporation of label into total RNA is reduced to less than 10% of the initial rate 2 minutes after addition of the chase. Thereafter, incorporation decreases further. Thus, transcription initiated at P2 will contribute negligible amounts of radioactivity to trp.BA-mRNA during the last 4 to 5 minutes of the degradation experiment. The chase has the additional advantage of reducing by twofold the cts/min of control filters; this is to be compared with the 50% increase in the experiment illustrated in Figure 3.

2000 - (a) .+--y - (“1

l --.

‘.

.

\ . .

\

@BA

.

+ 0

& 50- \,

\

.

,,,,

0 5+ :i, ,, , , ,, ,, (,

0 5 IO

Time offer addition. of tryptophon (mid

FIG. 4. Degr8detion of pulse-labeled Irp-mRNA during repression and in the presence of 8 chase.

A 22%ml. culture of strain A19 trpR+ wae grown, derepreesed for 10 min (fi-3-indolyl acrylic acid 20 &ml.) and repressed (tryptophen 100 w/ml.) 8s in Fig. 3.

In (8) 6 mCi [3H]uridine (8 Ci/m-mole) WBB added at the time of repression (0 min), and 1 min hater cold uridine (121 &ml.) WEB sdded. Semples of 25 ml. each were used for RNA extreotion 8nd hybridization to DNA from ptl, ptRA15 and the p8rental non-tr8naducing phege, to cal- cul8te trpE-mRNA (-+-+-) end trpBA-mRNA (-0-O-) in 36 H RNA.

In (b) 6 mCi [3H]tidine (8 Ci/m-mole) w8a added 3 min after repression followed by cold uridine (121 s/ml.) 1 min later. The &p&4-mRNA ~8s mecasured 88 above (-@-a--).

Figure 4 presents results obtained using the experimental conditions mentioned above. The half-life of trpE-mRNA is estimated as 60 seconds and that of trpBA- mRNA as 114 seoonds. (In a similar experiment a value of 101 seconds was obtained for trpBA-mRNA.) These values are in good agreement with the values derived from the data in Figure 3. In the experiment of Figure 4(a) trpBA-mRNA decay was also measured; as expected from Wgure 3, transcription of the trpB-trpA region continued for about 4 minutes before degradation of trpBA-mRNA was observed. The decay rate is similar to that estimated from Figure 4(b).

694 J. FORCHHAMMER, E. N. JACKSON AND C. YANOFSKY

(c) The s.tability of trpBA-mRNA as a function of the physical distance between the operator and the B and A-genes

The observation of different half-lives for different regions of trp-mRNA raises the following question: are the half-lives of trp-mRNA regions determined by their primary and secondary structure, or is the half-life related to location of each region within the polycistronic messenger ? This question was answered by examining mutants with deletions entirely within the trp operon and operator-proximal to the B and A genes (see Figure 1). The deletions studied (Jackson & Yanofsky, 1972b) were all transduced into the A19 background (see Materials and Methods).

The decay of trp-mRNA in these deletion strains was measured after [3H]uridine pulses administered during the last minute of transcription as estimated from Figure 3 and the extent of the deletions. The curve obtained with each deletion strain indicated that transcription stopped at the expected time. Two of the half-life determinations are shown in Figure 5, and the results of all the measurements with deletion strains are summarized in Table 1.

- 0

Time after addltion of tryptophordmmi

Pm. 5. Degradation of pulse-labeled trp-mRNA in strains with internal deletions. A 226-ml. culture of strain A19 trpR+ (d4C8 or AEC20) wan grown exponentially in miniiel

medium +0*4% glucose and 40 pg tryptophan/ml. At O.D. = 100 the cultures were filtered and resuspended in the same medium without tryptophan. Thereafter the experiments were exactly as in Fig. 4(a). After 10 min derepression (fi-3-indolyl 8orylic acid 20 &ml.), tryptophan (100 pg/ml.) and [3H]uridine (8 Ci/m-mole) were added followed 1 min later by cold uridine (121 pg/ml.). The tqBA-mRNA was determined as before. tr~BA-mRNA in deletion A&C8 (-O-O--) and trpBA-mRNA in dlC20 (-+-+-) calculated as cts/min in 35 pg RNA.

It can be seen that when most of the region between the trp operator and the B and A genes is deleted, degradation of trpBA-mRNA (Figure 5) starts at the same time and shows the same kinetics, as degradation of trpE-mRNA in the control strain (Figure 4(a)). Furthermore, the data in Table 1 show that introduction of smaller deletions between the operator and the B and A genes generally results in trpBA- mRNA half-life values intermediate between the value observed with no deletion and the greatly reduced half-life value illustrated in Figure 5. Since trpBA-mRNA half- life is reduced in all the deletion strains, the nucleotide sequence of trpBA-mRNA,

HALF-LIVES OF trp-mRNA REGIONS 696

as such, does not determine the half-life of this mRNA segment. Rather, the physical half-life of an mRNA segment seems to be influenced by the fate of the mRNA cor- responding to the more operator-proximal regions of the operon or by location in the polycistronic messenger.

(d) Pohwity a& the half-f-life of trpBA-mRNA

Since internal deletions were selected by virtue of their ability to relieve the strong polarity caused by operator-proximal amber or ochre mutations (Jackson dz Yanofsky, 1972b), the deletion mutants obtained could be weakly polar or non-polar. None of the deletions studied terminate in trpB; therefore, if the gene fusions resulting from the deletions were out-of-phase, the generated chain termination codons (and un- translated mRNA region) would not be in the t@BA-mRNA region. Thus the trpBA-mRNA region, the region measured, should not be subject to detectable polarity degradation (Morse t Yanofsky, 1969a). To verify this conclusion trpBA- mRNA half-life was determined in an experiment with a moderately strong polar point mutant, trpD795. The alteration in this mutant was introduced into strain A19 tyR- and trpR+. In these strains there was the expected threefold reduction in tryptophan synthetase a and & activity compared to the trp+ non-polar control culture, whereas the physical half-life of trpBA-mRSA WM 130 seconds, a value slightly higher than that of the control culture.

The degree of polarity caused by the internal deletions was estimated in A19 tvR- strains containing the different deletions. All cultures were grown exponent- ially for 3 generations at 30°C in the presence of 40 pg tryptophan/ml. One half of each culture was then chilled, harvested and immediately extracted and the activity of tryptophan synthetase subunits was measured. The other half was divided into 4 parts and pulsed with [3H]uridine for 30 and 60 seconds and for 5 and 8 minutes in order to estimate the rate of mRNA synthesis and the amount of niRNA present during exponential growth (using the method applied to the data in Figure 2). In each experiment LS control culture of A19 trpR- was grown, labeled and extracted in parallel to correct for day to day variation. Variation was less than 10% with regard to the enzyme assay, but up to 50% with regard to the specific activity of the [3H]uridine, which was diluted and used for labeling on different days. However, the same isotope dilution was used for all cultures on a particular day, and the counts identified as trpBA-mRNA sre expressed as a percentage of the incorporation in the control culture. The results obtained are shown in Table 1, together with the half- lives of trpBA-mRNA determined (see section (c) above).

It is clear from the data in columns 3 and 4 of Table 1 that, with the exception of deletion strains trpAEC8, trpAE41 and tqADC71, the tryptophan synthetase a and pS specific activities are reduced more than twofold, i.e. more than the extent expected if the physical half-life difference were responsible for the reduction. Thus, in the latter group there would appear to be a polar effect on enzyme production. This conclusion is supported by the mRNA synthesis data in column 6, which show that less than the control level of &pBA-mRNA can be detected in all strains but trpAEC8 and possibly trpADC71. Despite these indications of polarity, it is evident that the extent of the reduction of tryptophan synthetase a and pa synthesis (columns 3 and 4) does not correlate with the change in half-life (column 2). This is most obvious in the three strains trpAEC8, trpAECl7 and trpAEC20 with large deletions covering approximately the same segment of the operon. These strains give identical trpBA-

696 J. FORCHHAMMER, E. N. JACKSON AND C. YANOFSKY

TABLE 1 Typtop?un “yntiaetase spec@c activities and trpBA-mRNA half-life in strains

with trp intern.& deletions

strains Half-life of Tryptophan synthe- trpBA-mRNA labeled dur- Ratio of tryp-

tquBA-mRNA tase spec. activity as ing exponential growth as y0 tophan synthe- in secondst O/e of trp+ control of control tam to amounts

Ba CL 30 and 60 set 5 and 8 ruin of trpBA- pulse Pulse mRNA$

1 2 3 4 5 6 7

trp + control 101-114 100 100 100 100 1.00 trpAEC8 66 70 78 113 76 1.01 trpAECl7 62 10 8 40 22 0.46 trpAEC20 69 17 16 32 24 0.69 trpAED11 86 20 18 32 24 0.79 trpAEl1 86 48 49 66 66 0.88 trpADC71 67 66 63 82 61 0.97

The strains used are described in Materials and Methods. All have a doubling time of 96 min f 8 min at 30°C in minimal medium with glucose end tryptophan added.

The assay for tryptophan synthetaae is described in Materials and Methods. Extracts were from trplt- strains otherwise isogenio to those used in col- 2, grown exponentially for 3 generations in the presence of 40 pg n-tryptophan/ml. Half of this culture was used for enzyme assays (columns 3 and 4), and the other half was used for trp-mRNA determina tions (columns 6 and 8) as illustrated in Fig. 2.

t Measured in trpR+ strains as in Figs 4 and 6. $ Values from oolumns 3 and 4 divided by values in column 6. 8 This strain is ttyA- (A9761) and the A mutation may be slightly antipolar.

mRNA half-lives within the limits of the methods used, yet they exhibit a sevenfold difference in enzyme-specific activities. It can be argued that trpAEC8 is not polar at all and that the reduced amount of tryptophan synthetase 0: and /la reflects the reduced physical half-life of trpBA-mRNA in this strain.

The conclusion can be drawn therefore that the observed decrease in the half-life of tqBA-mRNA (Table 1) in mutants with internal deletions is influenced by the size of the deletion and is not attributable to polarity.

4. Discussion

The five contiguous structural genes of the tryptophan operon in E. c&i are transcribed into a single polycistronic mRNA (Imamoto & Yanofsky, 1967). The results presented here show that the mRNA complementary to each of three adjacent trp operon regions of equal length is degraded at a different rate. Direct measurements of the rate of trp-mRNA decay at 30°C indicate the following physical half-lives: trpE-mRNA, 60 seconds; trpDC-mRNA, 76 seconds; and the half-life of trpBA-mRNA, 95 to 115 seconds. The 20% difference between the half-lives of trpE-mRNA and trpDC-mRNA was not detected by Mosteller et al. (1970). However, we consider this small difference observed here in our more detailed studies to be significant. It was noted in several independent direct determinations of degradation rates, and the indireot estimates obtained from the experiment summarized in Figure 2 also indicate that trpDC-mRNA half-life is 20% greater than that of trpE-mRNA. No further

HALF-LIVES OF tsp-mRNA REGIONS 697

attempts were made to confirm this small difference, and in the remaining experi- ments we exploited the larger difference between the rates of degradation of trpE- mRNA and tqBA-mRNA.

The slower decay of t+yBA-mRNA is clearly attributable to the location of the trpBA region in the operon. When the trpBA region is located at a position equivalent to that of tqvE in the wild-type trp operon, aa in strains trpAEC8, trpAECl7 and trpAEC20, the half-life of trpBA-mRNA is the same as the half-life of trpE-mRNA in the wild-type strain. When the trpBA region replaces trpD and trpC as in strains trpAEDI1, trpAE41 and trpADC71, the half-life of trpBA-mRNA is similar to the half-life of trpDC-mRNA in the wild-type strain. The slight discrepancy from the exact values expected for each deletion strain may reflect inaccuracies in half-life determinations as we perform them. Since the half-life of &pBA-mRNA is decreased in all six deletion strains studied, the greater stability of tqBA-mRNA relative to trpE-mRNA in the wild type cannot be due to the primary nucleotide sequence of the trpBA region. Rather, stability must be a function of the location of the trpBA region within the polycistronic messenger.

An alternative interpretation of these data is that some specific short nucleotide sequence between &pE and trpB is a barrier to messenger degradation, and this barrier prolongs the lifetime of trpBA-mRNA. The properties of strains trpAE41 and trpADC71 are inconsistent with this hypothesis. The deletions in these strains remove different non-overlapping segments of the initial half of the operon. Thus, if the deletion in strain trpADC71 removed the region generating the postulated barrier to messenger degradation, the barrier region would be retained in strain trpAE41 and vice versa. Yet trpBA-mRNA half-life is reduced in both strains.

Since the trpBA-mRNA half-life varies as a function of the position of the trpB and trpA genes in the operon, we have asked whether there is a parallel variation in the steady-state levels of the proteins specified by these genes. Unfortunately, it is not possible to answer this question by comparing tryptophan synthetase enzyme levels in the deletion strains with those from the wild-type control, since a reduced enzyme level could result from polarity as well as from a shift of the respective region within the operon and a concomitant increase in the rate of mRNA break- down. In fact, the data in Table 1 (column 5) do indicate that in all the deletion mutants except trpAEC8 there is a polar effect on trpBA-mRNA production. However, since the polarity of the internal deletions does not influence trpBA-mRNA half-life as measured by the procedure described in the legend to Figures 4 and 5, it should be valid to compare steady-state t?BA-mRNA levels and tryptophan synthetase a and /la levels for each deletion strain (see column 7 of Table 1). Polarity might reduce both steady+tate trpBA-mRNA levels (column 6) and tryptophan synthetase levels (columns 3 and 4) equally, so the effect of polarity may cancel out in the ratio cal- culated in column 7. If the steady-state level of tryptophan synthetase subunits is reduced in proportion to trpBA-mRNA halflife in the deletion strains, the ratio in Table 1, column 7, should be equal to the wild-type ratio of 1. If the steady-state level of tryptophan synthetase subunits is not reduced when trpBA-mRNA half-life is reduced, the ratio for the deletion strains should be greater than 1. All the deletion mutants but one have ratios of tryptophan synthetaae a and /32 activity to steady- state trpBA-mRNA levels which vary from O-7 to l-0, and none is greater than 1.0. The low value of 0.46 found for trpAECl7 is probably due to the antipolar effect (Yanofsky, Horn, Banner & Staaiowski, 1971) of the trpA nonsense mutation in this

698 J. FORCHHAMMER, E. N. JACKSON AND C. YANOFSKY

strain on trpB polypeptide synthesis. These results suggest therefore that the level of polypeptide product of trpBA-mRNA does vary in relation to the half-life of trpBA-mRNA.

However, in a recently completed study (Jackson t Yanofsky, 1972b), in which a large number of similar trp deletion mutants were examined, there was no reduction in tryptophan synthetase z and pa levels (trpBA-mRNA levels were not determined) in deletion strains in which trpB and trpA replaced trpE in the operon. Obviously, additional experiments are required before we can state unequivocally whether there is a reduction in enzyme yield paralleling the reduced half-life of trpBA-mRNA when trpB and trpA replace trpE in the operon.

Our results can be used to examine various models which have been proposed for the normal mechanism of mRNA degradation (Lennette, Gorelio & Apirion, 1971; Blundell et al., 1972; Mosteller et al., 1970). Previous investigations have shown that trp-mRNA is degraded in the 5’ to 3’ direction (Morikawa & Imamoto, 1969; Morse et al., 1969), disappearance of trp-mRNA is exponential (Mosteller et al., 1970) and onset of degradation of a trp-mRNA molecule is random with respect to the time of initiation of transcription (Mosteller et al., 1970). These findings and the results reported here that trpBA-mRNA is degraded more slowly than trpE-mRNA as a consequence of the location of the trpBA region in the operon are consistent with a model in which trp-mRNA molecules are degraded sequentially from the 5’ terminus by a 5’ to 3’ exonuclease which attacks trp-mRNA molecules randomly with respect to time of initiation of transcription. To explain the half-life gradient along the polycistronic messenger, it can be assumed that degradation is slowed or interrupted, either randomly with a frequency of 10m4 per nucleotide, or at each intergenic junction with a frequency of about O-2. This model is modified only slightly from that proposed by Mosteller et al. (1970).

Quite different models for normal mRNA degradation have been considered by others. Lennette et al. (1971) found a change in the functional lifetime of lactose operon mRNA in a mutant strain of E. coli having an RNase hyperactive in the assay system usually employed to measure RNase II activity. Since RNase II is a 3’ to 5’ exonuclease, they propose that an endonuclease cleaves mRNA near the 5’ end as soon as a gap in translating ribosomes occurs. Thereafter, endonucleolytic attacks proceed generally in a 5’ to 3’ direction following translating ribosomes, and further degradation occurs via RNase II in a 3’ to 5’ direction. Our data could be accommodatedin this modelwith the modificationthat the frequencyofendonucleolytic attacks is reduced in operator-distal regions relative to more operator-proximal regions of the mRNA. However, evidence for the model of Lennette et al. (1971) rests on the identification of the mutant RNase 8s a 3’ to 5’ exonuclease, and this has not been rigorously demonstrated as yet. In addition, further assumptions are necessary to explain why increased 3’ to 5’ exonuclease activity should increase the rate of mRNA breakdown.

On the basis of their studies of functional inactivation of Zuc-mRNA, Blundell et al. (1972) have proposed that the half-life of each region within a polycistronic mRNA is determined by the relative vulnerability of a specific number of susceptible sites in the mRNA molecule. According to this model, the primary nucleotide sequence of each segment of the mRNA would determine its rate of degradation. Our results with trp-mRNA clearly conflict with this model.

Since both of the latter models postulate that endonucleolytic attack determines

HALF-LIVES OF trp-mRNA REGIONS 699

the rate of mRNA degradation, we investigated whether endonuclease A (Kuwano, Schlessinger $ Morse, 1971) is involved in normal degradation of trp-mRNA. Endo- nuclease A is thought to be involved in the rapid degradation of untranslated mRNA on the operator-distal side of a mutationally introduced nonsense codon (Morse & Primakoff, 1970). We transduced an endonuclease A mutant allele, WA, into the same background used in the experiments reported here, and determined the half- life of trpBA-mRNA as shown in Figure 4(b). The SUA mutation did not change the half-life of trpBA-mRNA (tt = 110 set). Thus endonuclease A is probably not involved in normal degradation of tqn-mRNA.

This project was supported by grants from the National Science Foundation (GB 6790), the U.S. Public Health Service (GM-69738) and the American Heart Association; one of us (J. F.) held a long-term fellowship from EMBO (European Molecular Biology Organiza- tion). One of us (E. J.) is a Predoctoral Fellow of the U.S. Public Health Service. The other author (C. Y.) is a Career Investigator of the American Heart Association.

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