Regulated in Vitro Synthesis of Escherichia coli Tryptophan Operon Messenger Ribonucleic Acid and Enzymes*

SUMMARY DNA-dependent synthesis of tryptophan (trp) operon mRNA and enzymes was studied in vitro. With 480 trpEDCBA template DNA trp operon transcription was initiated primarily at a phage promoter estimated to be approximately ‘7400 to 8000 nucleotides from trpE. Sequential transcription of trp mRNA in the correct direction proceeded at a rate of about 19 nucleotides per s. Trp mRNA resulting from read-through transcription from the phage promoter was larger than 23 S. Coupled sequential synthesis of trp operon enzymes occurred at a rate of approximately 4 amino acids per s. Experiments were conducted to determine if ribosomes discharge from polycistronic trp mRNA following translation termination at natural chain termination codons. Kasugamycin was used to inhibit reinitiation of translation. Kasugamycin shut down of steady state translation required 6.5 min for trpE enzyme, 7 min for trpD enzyme, and 3 min for trpA enzyme. These results indicate that ribosomes which translate trpE mRNA must discharge and reattach in order to translate trpD mRNA, and that ribosomes that translate trpD mRNA must discharge and reattach in order to translate trpA

Kasugamycin was used to inhibit reinitiation of translation. Kasugamycin shut down of steady state translation required 6.5 min for trpE enzyme, 7 min for trpD enzyme, and 3 min for trpA enzyme.
These results indicate that ribosomes which translate trpE mRNA must discharge and reattach in order to translate trpD mRNA, and that ribosomes that translate trpD mRNA must discharge and reattach in order to translate trpA mRNA.
Kasugamycin inhibition of trpD enzyme and trpA enzyme synthesis under appropriate conditions of sequential transcription and coupled translation provided further evidence for obligatory discharge and reattachment of ribosomes.
Enzyme synthesis coupled to trp operon-promoted transcription from Ah80 trp template DNA was repressible by tryptophan in reaction mixtures containing S-30 extract from trpR+ cells.
In contrast, enzyme synthesis coupled to read-through transcription from 480 trp template DNA was essentially irrepressible. It is concluded that read-through transcription is not repressible in vitro. Trp operon mRNA transcribed from a Ah80 trpEDCBA DNA template was smaller than 23 S.
The polar mutation trpC6 was expressed in uivo in phage Ah80 trpEDCBA 190-9 but not in phage 480 trpEDCBA 190. Read-through transcription of the trp operon occurs in the latter phage but not in the former. Polarity was not expressed in vitro using template DNA from either phage. Trp operon mRNA was extremely stable in vitro; the halflife varied between 30 min and more than 2 hours.
Recent work on DNA-dependent in vitro mRNA and enzyme synthesis catalyzed by preparations from Escherichia coli has helped to clarify mechanisms for positive and negative regulation of gene expression for the lac operon (l-4), gal operon (5-7), am operon (8-lo), trp operon (ll- 13), and arg regulon (14). With regard to regulation of tryptophan biosynthesis, Zubay et al. (11) isolated and partially purified the protein product of trpR, the repressor gene for the tryptophan operon.
Partially purified trpR protein functioned in vitro to repress @galactosidase synthesis directed by a DNA template in which lac operon genes were fused to a portion of trpE and contiguous trp operon regulatory elements.
Synthesis of trpED1 and trpBA enzymes in vitro was reported by I'ouwels and Van Rotterdam (15), but repression was not observed.
More recent experiments (12, 13), have shown that tryptophan and partially purified trpR protein repress trp operon transcription in a partially purified system. Repression was dependent upon a functional operator and showed no requirement for tryptophanyl-tRNA or trypt,ophanyl-tRNA synthetase.
The present work was initiated with the objective of developing an in vitro system in which trp operon transcription coupled to enzyme synthesis could be studied biochemically, thereby providing the opportunity to examine various aspects of gene expression.

EXPERIMENTAL PROCEDURE
Enzyme Synthesis-S-30 extracts were prepared from E. coli strains derived from strain A19 (16). A tonBtrpAE deletion was transduced into isogenic trpR+ and trpR-derivatives of strain A19 used previously (17). The resulting genotypes were rns-X-AtonBtrpAEl trpR-and rns-X-AtonBtrpAEl trpR+ where rns is RNase 1 and X stands for an unknown growth requirement satisfied by 0.05% acid hydrolyzed casein. Bacteria were grown at 30" with vigorous shaking in 2-liter flasks containing 1 liter of medium substantially according to the procedure of Zubay et al. (2). The growth medium was modified to include 20 pg per ml of tryptophan, 0.2% acid-hydrolyzed casein, and 10 pM FeC13. S-30 extracts were prepared from freshly harvested and washed (2) log phase cells. Extracts were treated at 37" for 80 min (18) and then dialyzed as described by Wetekam et al. (6). S-30 extracts stored in liquid N2 retained activity for three to four freeze-thaw cycles.
The composition of the reaction mixture was essentially as &scribed by Zubay et al. (2). The reaction mixture was modified by decreasing the tryptophan concentrat,ion to 0.1 nlM, omitting adenosine 3':5'-monophosphatc, substituting calcium acetate for calcium chloride and $80 trp DNA or Xh80 trp DNA for lac DNA. The optimal Mg2+ concentration was 12.8 mM and did not varl for various l>h'A or S-30 preparations.
Contrary to result's of earlier work (15), omission of other ingredients resulted in decreased activity.
Reactions for enzyme synthesis were usually conducted in a volume of 0.1 ml, and incubations were for 30 to 60 min at 34" unless specified otherwise.
Reactions were stopped by rapid chilling and 2. to 25.fold dilution into cold enzyme assay mistures.
In some experiments 50 pg per ml of chloramphenicol were used to t,erminate enzyme synthesis.
The two methods gave comparable results.
Unless specifically noted otherwise, S-30 extract was from the trpn-strain. by infection of sensitive host strains.
Phages were precipitated from lysates with 140 g per liter of polyethylene glycol 6000 plus 40 g per liter of NaCl and were then purified by twice banding to equilibrium in ccsium chloride density gradients. Template DNA was prepared by extracting purified phage in 0.02 M Tris-SO4 (pH 8.8)-0.01 M MgClz with redistilled phenol.
DNA was dialyzed against 50 to 100 volumes of 0.02 M Tris-acetate (pH 7.8).0.1 mM EDTA for 2 days with at least three changes of the buffer solution.
DNA for detection of 311-labeled RNA by filter hybridization was extracted with phenol and dialyzed against 0.15 M NaCl plus 0.15 M sodium citrate, pH 7.0. Separated DNA strands from X trp phage used for detection of 3H-labeled trp mRNA by filter hybridization were prepared by a modification (21) of the method of Hradecna and Szybalski (22).
DNA preparations used to direct in vitro synthesis or for detection of 3H-labeled trp RNA by filter hybridization are described in Fig. 1 were constructed in this laboratory and will be described elsewhere.
Phuge Infection-E. coli strain trpE9829 trpR-was infected at a multiplicity of two to four phages per bacterium according to the procedure of Franklin (23). Infected cells were incubated with vigorous shaking for 30 min at 37" in minimal medium supplemented with 0.05% acid-hydrolyzed casein, 0.2 mg per ml of 5-fluorouracil, 0.2 mg per ml of uridine, and 50 pg per ml of tryptophan.
Infected cells were then harvested and disrupted by sonic oscillation.
Following centrifugation at 27,000 x g for 30 min, extracts were assayed for enzymes of tryptophan biosynthesis.

Properties of in Vitro Enzyme
Synthesis-E. coli trpED enzyme is an oligomer apparently (26) containing two subunits of trpE protein and two subunits of trpD protein; each subunit has a molecular weight of approximately 60,000 to 65,000. TrpE enzyme catalyzes NHt-dependent anthranilate formation and carries the feedback site for tryptophan. TrpD enzyme confers glutamine reactivity to trpE enzyme and also catalyzes conversion of anthranilate plus 5-phosphoribosyl-1-pyrophosphate to phosphoribosyl anthranilate.
TrpED enzyme synthesized in vitro catalyzes chorismate-and glutamine-dependent anthranilate formation which is subject to feedback inhibition by tryptophan ( Table I). Synthesis of trpED enzyme is dependent on template DNA containing trpED genes (Tables I and II). As shown in Table II In vivo In vitro nmolcs/mi*//ng 2.58 0.083 trpE enzyme is synthesized, these data show that the assay for trpBD enzyme is absolutely specific for glutamine-dependent anthranilate synthetase and therefore can be used as an assay for trpD enzyme. TrpA enzyme is also synthesized in vitro, and its synthesis is not obtained with DNA templates lacking trpA. Variations in amount of enzyme synthesis with different DNA preparations were routinely observed. Generally, template DNA from Xh80 trp phage was inferior to that from @SO trp phage.
The dependence on DNA for synthesis of trpED and trpA enzymes is shown in Fig. 2. Nonlinearity of trpED enzyme synthesis at low DNA concentrations may reflect inefficient subunit aggregation at low subunit concentrations.
At higher subunit concentrations aggregation occurred rapidly since the activity obtained immediately after synthesis was not further increased by incubation at 0" for several hours.
An estimate of the amount of enzyme synthesis obtained in vitro is shown in Table III.
The specific activities of trpED and trpA enzymes resulting from in vitro synthesis were approximately 1.7 to 3.2 y. of those obtained from trpR-cells. This comparison does not take into account differences in DNA concentration between in vivo and in vitro syntheses and only serves to indicate that significant enzyme synthesis is obtained in vitro. The specific activity of trpED enzyme relative to trpA enzyme from in vitro synthesis is within 2-fold of that obtained by in viva synthesis (see also Sequential Transcription and Enzyme Synthesis-Zn viva experi-ment@ (23) have shown that in trp transducing phage transcription of the trp operon is initiated at either or both of the following sites, the trp operon promoter and the phage N operon promoter. Rates of read-through transcription from the phage promoter into the trp opcron frequently exceed rates of trp-operon promoted transcription.
Sequential transcription and translation of trp genes in the order trpE to trpA should result from initiation at either the phage or trp promoter. Data in Fig. 3 show the time course for transcription and enzyme synthesis from @'O trpEDCBA 190 template DNA.
Transcription under the conditions employed gave greater than 90% correct 1 strand trp mRNA.
Transcription and enzyme synthesis were assayed in similar reaction mixtures which differed only in the concentration of UTP.
Extrapolation from the initial linear rates indicates that trpED mRNA begins to appear at 8 min while trpCB mRNA first appears at 11 min. In another experiment, initial appearance occurred at 7 and 10 min for trpED and trpCB mRNA, respectively.
Transcription of trpED (approximately 3460 nucleotides, Fig. 1 190. Rifampicin (10 pg per ml) was added at each point shown, and incubation was continued for a total of 60 min. Samples'were removed for determination of trwED mRNA.
The 100% value is 5120 cnm of trvED mRNA. Dita are corrected for 50'to 140 cpm of nonspecific hybridization of 3H-labeled RNA to X DNA.
trp operon promoter are relatively infrequent.
TrpE and trpA enzymes routinely appeared following a longer lag. For the experiment shown in Fig. 3, the extrapolated initial appearance was at 17 min for trpE enzyme and 24 min for trpA enzyme. For an estimated operon segment of 5030 nucleotides for trpD through trpA, a translation rate of approximately four amino acids (or 12 nucleotides) per s is calculated from the 7-min delay between the appearance of trpE enzyme activity and trpA enzyme.
The initial lag of approximately 7 to 8 min in appearance of trpED mRNA appears to reflect mainly the time for read-through transcription from the phage N operon promoter. This is shown by the data in Fig. 4. The capacity for synthesis of trpED mRNA was rifampicin-sensitive within 1 min, yet as shown in Fig. 3 trpED mRNA did not appear for 7 to 8 min. This indicates that the site of transcription initiation for synthesis of trpED mRNA must precede trpE in the phage template by a relatively large region. The linear initial increase of trpED mRNA appearance is consistent with this interpretation as is the observation (see "Repression of Enzyme Synthesis") that readthrough transcription is not subject to inhibition by the repressor of the trp operon.
RNA Polymerase "Run-o$)'-An alternative method was employed to estimate the location of the major trp operon transcription initiation site of 480 trpEDCBA template DNA. During steady state transcription, rifampicin was added to block initiation, and the time course for shut down of trpED mRNA synthesis was measured.
Since transcription of an operon region ceases following run-off of all RNA polymerase molecules, the run-off time is a measure of the distance from the promoter to the end of the operon region being assayed. Such an experiment using 480 trpEDCBA 190 template DNA is shown in Fig. 5. Following addition of rifampicin at 15 min, synthesis of trpED mRNA proceeds similarly to the untreated control for several minutes and then stops. Ext,rapolation yields an RNA polymerase run-off time for trpED transcription of approximately 9.5 min. At a t'ranscription elongation rate of 19  Although not shown in Fig. 5, the translation inhibitors kasugamycin and chloramphenicol were entirely without effect on transcription under these conditions. Riboso,tte DischargePEsperiments were conducted to determine if ribosomes discharge from polycistronic trp mRh-A following translation termination at natural chain termination codons. Aurintricarbosylate (28)(29)(30) and kasugamycin (31)(32)(33) have been reported to act as translation inhibitors that specifically inhibit ribosome initiation.
Because of the recently reported greater selectivity of kasugamycin (33) and our finding of partial inhibition of transcription by 70 PM aurintricarbosylate, kasugamycin was used to block translation initiation. Data in Fig. 6 show that approximately 0.2 mM kasugamycin was required for 90% inhibition of h-p.& enzyme synthesis. The data in Fig. 7 show the kinetics of synthesis of trpl3 and trpA enzymes following addition of kasugamycin.
Note that enzyme synthesis is un-3A length of 10,800 nucleotides minus 3460 nucleotides (the approximate length of trpED). affected for several minutes following addition of the drug. It appears from this and other similar experiments that the rate of polypeptide elongation is not detectably changed by kasugamycin.
In vitro polypeptide elongation on E. coli polysomes was also unaffected by kasugamycin (33). At the concentrations used in these experiments, kasugamycin was without effect on transcription of trp mRNA. The abrupt inhibition of enzyme synthesis shown in Fig. 7 is attributed to inability of ribosomes to reinitiate translation following discharge from mRNA at putative intercistronic termination sites. The secondary slope of enzyme appearance presumably reflects kasugamycin-resistant synthesis (see Fig. 6).
The times for shut down of translation due to discharge of ribosomes from segments of trp mRNA were approximately 7 and 3 min for synthesis of trpE and trpA enzymes, respectively, in the experiment shown in Fig. 7. From the average of three experiments, ribosome run-off times of 6.5 and 3.5 min for trpE and trpA enzyme synthesis, respectively, were calculated. A ribosome rurl-off time for trpA enzyme synthesis less than or equal to that for trpE enzyme synthesis indicates that ribosomes which translate trpE mRNA must discharge before reinitiation of trpA enzyme synthesis. According to this model for ribosome translation, it should be possible to add kasugamycin following synthesis of trpE mRNA but prior to synthesis of appreciable trpA mRNA, and thereby prevent trpA enzyme synthesis.
Au experiment consistent with this expectation is shown in Table 1V. Kasugamycin added from 0 to 8 min, before initiation of significant synthesis of trpE mRNA and enzyme, allowed the expected low level of drug-resistant syuthesis of trpE and trpA enzymes. At various times 0.2 mM kasugamycin was added. Synthesis was for a total of 50 min. Addition of kasugamycin at 50 min therefore provides a drug-free control reaction. Samples (0.02 ml) were removed for assay of trpE and trpA enzymes. 'I'llis amount is less than 10yG of the control as shoal by comparison of the first three lines in Table 1V with the last line. 1Iore importantly, addition of liasugamycin at, 12 min allows completion of 1.19 units of drug-sensitive trp/E enzyme, but essentially no drug-sensitive trprl enzyme.
Addition of kasuga-my& at 16 and 20 min also shows strong selective inhibition of trp-1 cnzymc synthesis.
Tlic preceding experiments indicate that ribosonies discharge at a site following trpB mRNA and reattach at a site preceding trpll mRl\jA.
Similar experiments were conducted to determine if ribosomes discharge at the end of trpfi mRNii, or continue moving on the messenger to translate trpD mRh'A.
Following addition of kasugamycin, translation shut down times of approsimntely G min for trpE enzyme synthesis ad 7 min for trpED enzyme synthesis were obtained (Fig. SA). The apparently rctluced rate of trpEL> enzyme synthesis prior to kasugxmycindcpeudeiit shut down may result from slow association of trpD subunits with frpE subunits.
Since the shut down t)ime for synthesis of trpED enzyme (frpD enzyme) is not appreciably longc,r than the time for shut down of frpl!: enzyme synthesis, it appears that ribosomes discharge folloGig translation of frpi!: mRNA and must reattach to initiate translation of frpD mRKA. Further evitlrnce for this conclusion is shown in Fig. 8B and Table  V. The data in Fig. 8B show the kinetics for kasugamycin shut down of frpE enzyme synthesis using X&O frpE template DNA. Shut, down of trpE enzyme synthesis occurred in approximately 6 min in the absence of any possible complicating effects due to synthesis of trpD enzyme.
The data in Table V show kasugamycin inhibition of trpD enzyme synthesis. In this experiment rifampicin was added together with kasugamycin, to inhibit new rounds of mRNA synthesis.
Under these conditions there was no kasugamycin-resistant enzyme synthesis. These data show clearly that ribosomes which translate frpF: mRNA do not translate trpD mRNA in the presence of kasugamycin. r:ncoupled Translation of Trp mRNA--ln order to study translation of trp mRiYA independent of transcription, translation was uncoupled from mRNA synthesis.
This was accomplished by interrupting transcription with rifampicin or streptolydigan. As shown in Fig. 9, the two drugs were added at times chosen so that approximately equal amounts of trpED mRNA were sy~ TAHLIC V Kasugamycin inhibition of trpD mRNA lranslalion Template I)NA was from phage 480 IrpEDCB 1. Kasugamycin (0.5 mM) and rifampicin (10 pg per ml) were added simultaneously. Synthesis was for a total of 40 min. Enzyme synthesis was terminated with 50 pg per ml of chloramphenicol and the tubes were then incubated for 3 hours at 37" to insure subunit aggregation (15). Samples of 0.05 ml were used for assay of trpE and trpED enzymes at 34" for 30 min. thesized in reactions containing either of the two drugs. Rifampicin allowed completion of transcription in progress as shown by the appearance of trpCB mRNA, while streptolydigan blocked transcription in progress as indicated by the absence of trpCB mRNA.
Since streptolydigan blocks all transcription instantaneously, some mRNA chains will contain incomplete gene transcripts and therefore will not yield functional enzyme. The fraction of such incomplete chains will decrease with time, as the period allowed for frp mRNA synthesis increases before addition of streptolydigan.
In Fig. 9 it is seen that although approximately the    20  39  25  13  30  43  25  13  40  45  27  0  50  43  27  0  60  42  30  0 same amount of trpED mRNA is present in both drug-treated mixtures, at early times the trpE' enzyme yield in the streptolydigan-treated mixture is only about half that of the rifampicintreated mixture.
The rates of coupled and uncoupled translation calculated from the data in Fig. 9 are presented in Table VI.
In both the control and rifampicin-treated mixtures the rate of translation was proportional to the amount of trpED mRNA. Enzyme synthesis in the rifampicin uncoupled mixture was approximately 647, as efficient as coupled translation (control). Translation efficiency in the streptolydigan-treated mixture was lower, as espccted, and trpE enzyme production was not maintained beyond 30 to 40 min. The data in Fig. 9 demonstrate that trp mRNA is quite stable in the drug-treated mixtures.

Repression of Enzyme
Synthesis-Repression of enzyme synthesis directed by fused trp operon-lac operon DNA has previously been detected by comparing enzyme synthesis in S-30 extracts prepared from isogenic trpR+ and trpR-strains (11). Similar comparisons of trp enzyme synthesis with several DNA templates are shown in Table VII. A marginal and variable repression of enzyme synthesis was obtained with @I trpEDCBA On the other hand, 80 to 90% repression of trpE enzyme synthesis was routinely obtained when the DNA template was from certain trp transducing phages (Xh80 trpED 9, 37, and 50; and Xh80 trpEDCBA 190-9) which show little or no read-through from an upstream phage promoter (23). When trpCB and -4 were incorporated into Xh80 trpED 9 by recombination within the trp operons of two phage, trpA enzyme synthesis was found to be repressible in vitro to the same extent as trpE enzyme. Franklin (23) has found little or no read-through trp transcription in vivo with the Xh80 trp phages used here as sources of template DNA. Titration of trpR protein in S-30 extracts was performed by varying the proportion of estract from the trpR-strain. Data in Fig. 10 show 90% repression of trpE enzyme synthesis, with Xh80 trp 50 template DNA.
Enzyme synthesis from template DNA with an operator constitutive mutation serves as a control and shows that decreased enzyme synthesis cannot be entirely due to low synthetic capacity of the trpR+ S-30 extract.
Repressor activity was also detected with S-100 extracts from    Table VIII show repression of Xh8O trpED $7 DNA-directed trpE enzyme synthesis when S-100 protein from trpR+ strains was added to S-30 extract from a trpR-strain.
It appears that S-100 extract from a strain diploid for trpR contains twice as much trp repressor as a haploid strain. Little or no repression of enzyme synthesis was obtained with 480 trpEDCBA 190 and 480 trpEDCB 1 DNA templates. S-100 prepared from a trpR-strain has little or no repressor activity.
A comparison of repression of transcription and enzyme synthesis is presented in Table IX. These data show somewhat greater repression of trpE enzyme synthesis than trp mRNA transcription but more extensive studies are required to determine the significance of this difference.
Repression of Transcription-The kinetics of transcription of trp mRNA catalyzed by trpR+ S-30 extracts in the presence and absence of tryptophan are shown in Fig. 11. Transcription from 480 trpEDCBA 190 template DNA exhibited biphasic kinetics (Fig. 11A).
A slow initial rate of trpCB mRNA synthesis (33 cpm per mm) was detected prior to the expected read-through transcription, which was at a rate of 430 cpm per min. Presumably the trpCB mRNA observed early was initiated at the trp operon promoter.
Trp promoted transcription was repressed by tryptophan whereas read-through transcription was not. The kinetics of transcription of trp mRNA from repressible Xh80 trp DNA templates was not obtained due to difficulties with the detection of the small amounts of trp mRNA transcribed from Xh80 trp hybrid phage. However, transcription kinetics were determined with template DNA from phage 480 AtrpEDlOZ trpCB (Fig. 11B). The extrapolated time of appearance of trpCB mRNA at about 4 min suggests that the initial transcription was initiated at the trp operon promoter.
The trp promoted transcription by trpR+ S-30 extract was 94% repressed by tryptophan.
Read-through transcription of trpCB mRNA appeared at approximately 15 min and accounts for the decreased repression. Under these conditions the trp promoted transcription on 480 AtrpEDlOZ trpCB DNA was at a rate approximately 13 times that on 480 trpEDCBA 190 DNA. E. coli strain trpAED 102 has a 4-to &fold increased rate of in viva trpCB mRNA synthesis compared to control strains (34).
Size of Trp mRNA Synthesized in Vitro-Since trp operon transcription is initiated at a phage promoter approximately 7400 to 8000 nucleotides from the start of trpE with 480 trpEDCBA 190 DNA as template, trp operon mRNA from this template should be approximately twice the size of that initiated at the trp promoter, provided that the transcription termination site at the end of the trp operon is functioning.
Profiles of trp mRNA made in vitro from 480 trpEDCBA 190 and Xh80 trpEDCBA 190-9 templates are shown in Fig. 12. TrpEDCBA mRNA transcribed from the 480 trp template DNA was larger than 23 S whereas trpEDCBA mRNA transcribed from the Xh80 trp template DNA was smaller than 23 S. Franklin has deduced that essentially all in vivo trp transcription is initiated at the trp promoter in phage hh80 trpEDCBA 190-9.4 These results demonstrate the relatively large size of trp mRNA resulting from read-through transcription. Polarity--In order to examine polarit,y in vitro the polar chain termination mutation trpC6 (35) was crossed into 480 trp and Xh80 trp phage. The data in Table X show the polar effect of trpC6 in vivo. A 'I-fold reduction of trpA enzyme activity was obtained for trpC6 relative to the parental bacterial strain.
In vivo mutation trpC6 exhibited polarity in the Xh80 trp phage but not in the 480 trp phage. Polarity was not obtained in vitro using template DNA from either the Xh80 trp or 480 trp phages (Table XI). DISCUSSION Enzymes of the trp operon have been synthesized by in vitro DNA-directed transcription and coupled translation. We have detected and measured synthesis of mRNA corresponding to the first four genes, and enzyme subunits corresponding to the first, second and fifth genes, of the E. coli trp operon.
We have studied expression of the trp operon in vitro using two types of DNA templates: (a) templates from 480 trp phage exhibiting mostly read-through expression in vivo and in vitro; (6)  190 DNA template more than 90% of trp transcription, as estimated from comparison of maximal and initial rates ( Fig. 1 lA), is initiated at a phage promoter approximately 7400 to 8000 nucleotides from trpE.
As a result of read-through, sequential transcription of the trp operon occurs in the correct   (Fig. 3). From the time required for completion of synthesis of trpED mRNA (Fig. 3) and the approximate length of this operon segment, the mRNA chain elongation rate was estimated as 19 nucleotides per s at 34". This compares with estimates of 25 to 28 nucleotides per s at 30" and 37 to 45 nucleotides per s at 37" for in vivo trp operon transcription (36). Information is not available to indicate whether or not i n vitro transcription terminates at the normal site following trpA. The size of the polycistronic mRNA resulting from phage-promoted read-through transcription into the trp operon is estimated to be approximately 14,400 nucleotides in length (approximately 7,700 nucleotides transcribed from phage genes trp operon is initiated at the phage promoter PL in the presence of the N protein of X.