Termination of transcription in vitro in the escherichia coli tryptophan operon leader region

J. Mol. Biol. (1976) 103, 383-393

Termination FRANK

of Transcription in vitro in the Escherichia coli Tryptophan Operon Leader Region

LEE, CATHERINE L. SQUIRES~, CRAIG SQUIRES~ AND CHARLES YANOFSKY Department of Biological Sciences Stanford University Stanford, Calif., 94305, U .S.d. (Received 23 May 1975, and in revised form 16 January

1976)

Transcription of the wild-type tryptophan (trp) operon of Escherichia coli was examined in vitro. Virtually all RNA polymerase molecules which initiate transcription at the by promoter cease transcription within the leader region of the operon after synthesizing about. 145 nucleotides of leader RNA, and thus rarely transcribe the structural genes of the operon. Transcription stops with approximately equal frequency at either of two adjacent nucleotide pairs within an A + T-rich region, giving rise to transcripts with U-rich 3’ termini. The site of transcription termination is in a segment of the leader region proposed on the basis of genetic and biochemical evidence to contain a new regulatory element, a transcription attenuator, which functions in cont.rolling the maximum level of expression of the operon.

1. Introduction “leader” region of the trp operant of Escherichia coli consists of approximately 160 base-pairsimmediately preceding the first structural gene, trpE, and is known to be transcribed in vivo into RNA contiguous with structural gene mRNA (Bronson et al., 1973; Squires et a,Z.,1976). In the accompanying papers and elsewhere(Squires et al., 1976; Bertrand et al., 1976; Korn & Yanofsky, 1976; Platt et al., 1976) detailed information is presented on the nature and possiblefunction of this region. In particular, several lines of evidence indicate the existence of a regulatory site, a transcription attenuator, near the end of the leader region, which participates in regulating the maximum level of transcription of the structural genesof the operon (Bertrand et al., 1975). Bacterial strains in which the attenuator is genetically deleted can exhibit elevated levels of both distal trp mRNA and enzymes (Jackson & Yanofsky, 1973). Quantitative measurements of mRNA production in vivo demonstrate that when the trp operon is intact, an eight to tenfold molar excessof leader RNA sequences is synthesized compared to distal structural gene sequences(Bertrand et al., 1976). This disproportional synthesis is postulated to result from transcription termination events occurring within the leader region of the operon, at the attenuator (Bertrand et al, 1976). Characterization of the 3’ termini of several RNA molecules made in vivo and in vitro has shown that sequencesrich in U residues are shared by all (Lebowitz The

t Present address: 03755, U.S.A.

Department,

$ Leader region of the operon shall denote the RNA transcript

of Biological

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shall refer to the of this region. 383

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College, above;

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et al., 1971; Pieczenik et al., 1972; Smith & Hedgpeth, 1975; Dahlberg & Blattner, 1973; Ikemura & Dahlberg, 1973). These results have led to the suggestion that such sequencescorrespond to at least part of a termination sequencerecognized by RNA polymerase. In trp leader RNA there is a U-rich sequencein the vicinity of position 140 which bears a striking similarity to these 3’ sequences (Squires et al., 1976). Furthermore, this sequencecorresponds to the segment of the leader region which, on the basis of findings with genetic deletions, is believed to contain the attenuator (Bertrand et al., 1976). Thus it was of interest to determine whether RNA polymerase terminates transcription in vitro in the attenuator region. In this paper we report that this is the case;when wild-type trp operon DNA is transcribed in vitro, transcription stops in the attenuator region, in the absenceof accessory factors.

2. Materials

and Methods

(a) Reagents and enzyme8 triphosphates (spec. act. 50 to 150 Ci/mmol)

[cr-32P]ribonucleoside were obtained from New England Nuclear Corporation through the generousefforts of Dr Winston Salser. DNase A, T, RNase, RNase A and alkaline phosphatase enzymes were from Worthington Biochemical Corporation. The RNA polymerase holoenzyme employed was from the following sources: Dr B. Marrs, who used the purification procedure of Burgess (1969); Dr W. Mangel, who generously provided a sample of enzyme prepared by the method of Berg et al. (1971); or the authors, who used the method of Yarbrough & Hurwitz (1974). (b) Bacteriophage and DNA The DNAs obtained from 2 trp transducing phage were used as templates for transcription in vitro; $80trpEDCBl and #SOtrpEDCBA (pt 190). Each of these phage carries the trp operon promoter and operator (Rose et al., 1973; Zalkin et al., 1974). Template DNA was purified from phage as described previously (Rose et al., 1973). For isolation of trp-specific mRNA, the separated DNA strands of XtrpEDl, a phage which contains the operator-proximal portion of the operon, were used (Rose et al., 1973). DNA strand separations were performed by the method of Shapiro et al. (1969). (c) In vitro transcription and isoZation of leader RNA The method used for synthesizing trp RNA was modified slightly as follows from that described previously (Squires et al., 1975). A portion of the particular labeled nucleoside triphosphate (supplied in a solution of 50% ethanol) was dried in a small tube and then dissolved to give a final concentration of 10 PM t,o 74 PM, as appropriate. The following were added to the indicated final concentrations: 20 mM-Tris.HCl (pH 7*9), 52 mM-KCl, 1.1 mu-MgCl,, 0.11 m&x-EDTA, 83 PM-dithiothreitol, 25 pg RNA polymerase/ml, 25 pg template DNA/ml. This mixture was preincubated for 10 min at 37”C, after which transcription was initiated by the addition of a solution of the remaining 3 unlabeled nucleoside triphosphates, each at a final concentration of 75 PM. Termination of transcription, processing of the RNA samples and hybridizations uTere carried out as described by Squires et al. (1976). trp mRNA was isolated by a single hybridization t,o the Z strand of htrpED1 DNA. RNA samples were digested and fingerprinted as described in Squires et al. (1976). (d) Analysis of 3’ oZigonucZeotide.s Two unidentified oligonucleotides, X and Y (set Fig. 3), prosent on a T1 RNase fingerprint of [cr-32P]UTP-labeled trp RNA were eluted from the polyethyleneimine-cellulose thin-layer plate as described by Squires et al. (1976). Each oligonucleotide was digested with RNase A and the products separated by electrophoresis on paper (Whatman 3MM) in pyridine acetate, pH 3.5. In addition, samples treated with alkaline phosphatase were run side by side on DEAE-paper (Whatman DE-81, pH 3.5) with untreated samples, along with a series of oligonucleotides of known length wit#h the general sequence (UPKP (U&P-U&P).

TERMINATION

OF

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TRANHCHIP’J’IOS

3. Results (a) CharactekGx

of trp operon

tmnscription

in vitro

RKA

was synthesized in vitro using purified E. coli RNA polymerase and a @I phage template carrying the intact trp operon. The trp mRNA (labeled with [a- 32P]nucleoside triphosphates) produced was isolated by a singIt* hybridization to htrpED1 DNA. When the purified RNA was digested with T, RNase or RNaseA and fingerprinted, the oligonucleotide pattern obtained resembled only t,he 5’-proximal portion of the leader sequence. Figure l(a) shows a T, RNase fingerprint of uniformly labeled in vivo trp mRNA isolated from strain trpAED24. This fingerprint displays the oligonucleotide pattern of the ent,ire leader sequence ant1

trp transducing

FIG. 1. RNase T, fingerprints of trp leader RNA. (a) Uniformly in viva labeled [3ZP]Rh’A obtained from strain trpAED24 including the complete leader sequence as well as several oligonucleotides corresponding to the beginning of tTpE (those numbered 37.1, 39, 34 and 22). The spots labeled 34 and 22 are each composed of 2 oligonucleotides, one from trp leader RNA and thr other from trpE mRNA. (b) RNA synthesized in vitro from @OlrpEDCBI and labeled with [32P]GTP. Tao-dimensional fingerprints prepared as described bv Squires et al. (1976). electro. ljhoresis at. pH 3.5 from left to right and homochromatography from bottom to top. 25

386

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includes several spots corresponding to the initial segment of trpE mRNA (spots numbered 37.1, 39, 34 and 22). Figure l(b) presents a T, fingerprint of trp mRNA synthesized in vitro and labeled with [c+~~P]GTP. Comparison of the two flngerprints in Figure 1 and reference to Figure 2 shows that the 5’ portions of the in vivo and in vitro transcripts are indistinguishable. It is also clear from examination of the fingerprints that several oligonucleotides are absent from the material synthesized in vitro, in particular, oligonucleotides t48, t37.2, t37.1, t39, t34.2 and t22.2. The missing oligonucleotides all come from the operator-distal (3’) segment of the leader sequence or the sequences corresponding to the beginning of trpE, i.e. only the 5’-proximal 140 nucleotides are present in the GTP-labeled in vitro transcript. This result is reproducible under a variety of experimental conditions (see below) and is independent of which nucleosidetriphosphate is labeled. Following the initial observation that transcription apparently stops near the end of the leader region of the operon, we examined the kinetics of synthesis of trp mRNA. The fingerprint shown in Figure l(b), representing 140 nucleotides of the leader sequences,was obtained with mRNA isolated following a labeling period of three minutes. A seriesof syntheseswere performed, ranging from 15 secondsto 10 minutes in duration, and the trp mRNA was isolated and fingerprinted in each case. The results showed that as the labeling period was increased, leader olignoucleotides appeared more or less sequentially in the 5’ to 3’ direction, and that by 60 seconds the pattern representing the first 140 nucleotides of the leader sequencewas complete. The time required to transcribe a region of this length is consistent with the previous estimate for the rate of RNA chain elongation in this system of approximately three nucleotides per second (Rose et al., 1973). Increasing the time of incubation up to ten minutes results in additional transcription of the leader segment, but oligonucleotides corresponding to the region beyond nucleotide pair 140 are never present in significant amounts. The standard conditions we employ in synthesizing RNA in vitro involve the use of a single [u-32P]nucleosidetriphosphate at a concentration of 10 to 15 pM and three unlabeled nucleoside triphosphates, each at a concentration of 75 pM. To examine the possibility that the observed transcription termination in the leader region is a consequence of the low concentration of one of the triphosphates, we performed synthesesin which all four substrate triphosphates were present at a high concentration (75 PM). The results were unchanged; synthesis did not proceed beyond nucleotide 140. Cessationof transcription near the end of the leader region is not unique to a single preparation of RNA polymerase. With two enzyme preparations purified in this laboratory and one kindly provided by Dr W. Mangel, each prepared according to a different procedure, essentially identical results were obtained. In other experiments the concentration of RNA polymerase, normally kept 60-fold higher than the DNA concentration, on a weight basis, was increased to 300 times the template concentration with no effect on termination of transcription. Two different 480 trp phage templates were also tested in these studies, and the short leader transcript was made with each of them. In addition, preparations of each template DNA were treated extensively with Pronase prior to useto investigate the possibility that small amounts of protein associated with the DNA were responsiblefor the observed transcription termination. Hybridization assays of [3H]UTP-labeled mRNA transcribed from such protein-free DNA templates indicate that this possibility is unlikely.

5’ t.4 _-

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FIG. 2. The relative positions of the unique unlabeled regions represent small oligonucleotides

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t5

t-13

digestion products in some cases occur

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of trp several

t24

t2.5

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actual

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see Squires

t37.1

1976).

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Short

t22 . 2

388

FIG. different

F.

3. RN&se lengths

LEE

T1 fingerprints of [32P]GTP-labeled of time. (a) 60 8; (b) 10 min.

h’T

AL.

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(b) Identity of the terrnimtion

RNA

synthesized

in

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for

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On a T, RNase fingerprint of [a-32P]GTP-labeled RNA such as that shown in Fig. l(b), oligonucleotide t48 (CUsG) and all more distal oligonucleotides are absent (see Fig. 2). On an RNase A fingerprint of UTP-labeled material oligonucleotide ~24, G-G-G-C(U), is present. Transcription termination must therefore occur within the region corresponding to the C residue of p24 and the G residue of t48 (i.e. between nucleotide pairs 137 and 146 in the leader region). We determined the approximate ceases from the following information. position at which transcription On the T, RNase fingerprint of [a-32P]UTP-labeled leader RNA two oligonucleotides appear in approximately equimolar yield which are not detected following

Fm. 4. The nucleotide sequence of the RNA transcript in the region of the transcription termination site and deduced DNA sequence (Squires et al., 1976). Pertinent RNase A (1)) and T, RNase (t) oligonucleotides are also indicated on the RNA sequence. The arrows denote t’hr estimated location of the 2 transcription termination sites. The exact number of U residues in t48 and at the 3’ terminus of the terminated transcripts is uncertain. Lines on the DNA sequenre show the regions of “-fold symmetry.

FIG. 5. RNase T, fingerprint of [32P]UTP-labeled two 3’ oligonucleotides resulting from transcription Note that on a fingerprint of ill viva leader RNA Fig. l(a)).

trp leader RNA synthesized in vitro. The termination are labeled X and Y (see Results). these 2 oligonucleotides are not present (see

labeling with any of the other three nucleoside triphosphates (Fig. 5, spots X and I’). From their positions on the fingerprint these oligonucleotides are potentially rich in U residues and thus could be fragments of oligonucleotide t48. Such fragments would have the sequence CU,-OH, with both oligonucleotides lacking at least the S-terminal G residue of t48, since neither is labeled with [a-32P]GTP. When each oligonucleotide was subjected to digestion with RNase A, the products exhibited the labeling pattern expected for termination fragments of t48, that is, only labeled Up and Cp were produced.

390

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E!Z’ AL.

The 3’ terminus of an RNA molecule generated by termination of transcription characteristically possessesa 3’-hydroxyl group. That this was the case for both oligonucleotides X and Y was demonstrated by treating these oligonucleotides with bacterial alkaline phosphatase. This enzyme hydrolyzes terminal phosphate residues at either the 5’ or the 3’ end of an oligonucleotide. The removal of a phosphate from an oligonucleotide increasesits electrophoretic mobility on DEAE-paper at pH 3.5 relative to the intact molecule; however, both treated and untreated samplesof spots X and Y had identical mobilities (seeFig. 6).

0

0

0 0

-+-+-+-+-+-+ U,G U,G

U,G

00-a U,G

X

Y

FIG. 6. Tracing of electrophoretic comparison of oligonucleotides X and Y with nucleotides (DEAE, pH 3.5) with and without treatment by alkaline phosphatase. nucleotide was spotted adjacent to an equal portion digested with phosphatase. marker oligonucleotides had the general structure (Up),Gp and were identified fingerprint (see Squires et al., 1976) of in viva labeled total RNA.

marker oligoEach oligoThe series of from a PEI

With the knowledge that each oligonucleotide ended in a 3’-hydroxyl we were able to estimate the number of U residuescontained in each. The electrophoretic mobilities of X and Y were compared to those of a seriesof oligonucleotides of known length with the general formula (Up),Gp (seeFig. 6). From their mobilities relative to those of the markers, we estimate that spots X and Y have seven and eight U residues,and

TERMINATION

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trp

TRANSCRIPTION

391

thus the sequences C-U-U-U-U-U-U-U,, and C-U-U-U-U-U-U-U-U,,, respectively. Due to the high U : C ratio, we were unable to obtain a reproducible estimate of the relative number of U residues by counting the alkaline digestion products of X and Y labeled with UTP.

4. Discussion Information presented in the accompanying papers and discussed elsewhere (Bertrand et al., 1975) provides in vivo evidence for the existence of a transcription termination site or attenuator within the leader region of the trp operon. Measurements of mRNA corresponding to defined regions of the operon establish that under certain conditions an eight to tenfold excess of leader RNA over structural gene RNA is synthesized (Bertrand et al., 1976). Internal deletion mutations with one endpoint within the leader region define a site within about 30 base-pairs of the sequencecorresponding to the translation initiation codon for trpE (see Fig. 2) whose presenceis necessary for disproportional RNA synthesis (Bertrand et al., 1976). The intriguing observation has also been made that unlinked mutations which relieve polarity imposed by nonsensemutations increase the level of expression of the wildt.vpe trp operon, and this effect involves a site in the operon at or near the attenuat’or (Korn & Yanofsky, 1976). Furthermore, these mutations appear to affect the transcription termination protein, rho, implying a function for rho at the attenuator (Korn, unpublished results). The attenuator thus is a regulatory site which functions to control the maximum level of expression of the operon, with control being accomplished by influencing termination of transcription within the leader region. The frequency of transcription termination in vivo is responsive to the intracellular concentration of tr.vptophan (Bertrand & Yanofsky, 1976) but the identity of all the molecule+ acting at the attenuator to cause termination or readthrough is uncertain. There is, however, evidence suggesting the involvement of tryptophanyl-tRNA and/or its cognate synthetase (Bertrand & Yanofsky, 1976; Morse & Morse, 1976) in regulation at this site. An analogousregulatory site involving termination by rho factor and antitermination has previously been demonstrated in the N-operon of h bacteriophage (Roberts, 1969). More recent studies suggest that antitermination is used more generally as a regulatory mechanism in h (Roberts, 1975). That transcription does stop at this site in vitro is demonstrated by the results presented here. RNA polymerase molecules, in the absence of any added fact,ors. appear to terminate transcription within the leader region, 20 nucleotide pairs before t,he beginning of trpE. Termination occurs in an A + T-rich region with approximately equal efficiency at either of two adjacent base-pairs, generating transcripts which end in a run of U residues. The relationship between the observed termination site in vitro and the putative termination site in vivo is uncertain; however, termination in vivo is believed to occur within the same region (Bertrand et al.. 1976). The U-rich 3’ termini determined here for the trp leader transcript are strikingly similar to the 3’ sequencesof several RNA molecules isolated from both in viva and in vitro sources. Two small RNAs transcribed from h DNA in vitro, designated 2S and 6s: end in U,A-OH (Lebowitz et al., 1971; Dahlberg & Blattner, 1973). A low molecular weight RNA isolated from $80 phage-infected E. coli and thought to bc

392

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ET

AL.

analogous to the X 6s transcript bears the terminal sequence CU,-OH (Pieczenik et al., 1972; Roberts, 1975). In addition, a small RNA isolated from uninfected E. co.5cells ends in U,A-OH (Ikemura & Dahlberg, 1973). These results, together with those presented here for trp transcription, strongly suggest t’hat RNA polymerase can recognize particular nucleotide sequencesas termination signals both in vivo and in vitro, and that these signalsare rich in A-T base-pairs.The trp leader transcript and the two small /\ transcripts were detected in a purified transcription system employing RNA polymerase and no additional components. In the casesof the h 4S RNA and the short typ leader transcript, similar or identical RNA species have also been detected in vivo (Smith 6 Hedgpeth, 1975; Bertrand et al., 1976). Several groups have reported that RNA polymerase transcribing certain templates in vitro pausestransiently at particular sites on the DNA, resulting in a non-uniform rate of chain elongation, and thereby generating discrete size classesof RNAs detectable as bands on polyacrylamide gels (Dahlberg & Blattner, 1973; Maizels, 1973). At least in some cases such pausesin transit are apparently related to the use of low concentrations of substrate nucleoside triphosphates (Maizels, 1973; Darlix & Fromageot, 1972). Although we attempted in several ways to overcome the transcription block within the leader region, for example, by varying the substrate concentration, we were unable in any case to observe synthesis of distal sequences. Termination of transcription within the leader region may involve simply the cessation of chain elongation by the enzyme-DNA complex (this would resemble a polymerase molecule “pausing”) or it may be accompanied by actual dissociation of RNA polymerasefrom the template with the concomitant releaseof the RNA chain. Preliminary experiments suggest that the termination event in vitro does not include release of the transcript from the template. It is difficult to estimate accurately the fraction of polymerase molecules, if any, which do not terminate within the leader region in vitro but instead continue to transcribe into the structural genes.From the fingerprint data me would estimate that this fraction is at most a few per cent of the total. This estimate is based on the absenceof traces of readily identifiable distal oligonucleotides. Termination, therefore, occurs with a very high frequency. This observation is in apparent contrast to the results of others, who, in examining trp transcription in vitro, do detect transcription corresponding to the structural genes (Rose et al., 1973; Pannekoek et al., 1974; Shimizu & Hayashi, 1974). However, Pannekoek et al. (1975) in recent experiments have also detected disproportional transcription of the operon with the relative molar amount of structural genetranscripts perhaps aslow as 10% of transcripts terminating within the leader region. In the other studies, an appreciable excessof leader relative to distal RNA may have gone undetected if insufficient levels of DNA were used in the hybridization assays. As detailed elsewhere (Squires et aZ., 1976) examination of the DNA nucleotide sequencesurrounding the transcription termination site reveals two regions of 2-fold symmetry, one ten base-pairs in length, and the other extending 12 base-pairs (see Fig. 4). Such unusual sequenceshave been demonstrated in several DNA regions at which regulatory proteins interact, such as the lac operon and X N-operon regulatory regions (Dickson et al., 1975; Gilbert et al., 1973; Maniatis et al., 1974). The presence of such a unique structure in this region of the trp operon allows one to at least speculate that it functions in vivo as the recognition site for a protein or proteins which influence the frequency of transcription termination.

TEHMINATION

OF

t,p

THANS(‘HII’TlON

3!13

\ve thank Terry Platt, Kevin Bertrand and Laurence Korn for helpful discussions un(l Miriam Bonner, Virginia Horn and Joan Hanlon for expert assistance. Support was provided by grants from the United States Public Health Service (GM 09738) and the National Science Foundation (GB 36967). One of the authors (F.L.) is a predo&oral t,raincr of t)hr U.S. Public Health Service, and another a,uthor (C.L.S.) was a postdoctoral follow of the Helen Hay Whitney Foundat.ion. Onv of thv altt.hors (C.Y.) is A Carcc,r I nvvst igator of thv Amcricwn Heart Association. REFERENCES Berg, D., Barret.t, K. & Chamberlin, M. (1971). Meth. Entymol. 21, 500-519. Rertrand, K. & Yanofsky, C. (1976). J. Mol. BioZ. 103, 339-349. B&rand, K., Korn, L., Loe, F., Platt, T., Squires, C. L., Squires, C. & Y~motsli~.. ( ‘_ (1975). Science, 189, 22 -26. B:lrtrand, K., Squires, C. & Yanofsky, C. (1976). J. iI1o.Z. Bid. 103, 319.-337. Bronson, M. J. & Yanofsky, C. (1974). J. Mol. BioZ. 88, 913--916. Bronson, M. J., Squires, C. & Yanofsky, C. (1973). I’TOC. Nat. Acad. Sri., IT.N.A. 70, 2335-2339. Burgess, R. (1969). J. BioZ. Chem. 244, 6160-6167. Dahlberg, J. E. & Blattner, F. R. (1973). In 1%us Ileueavclc, pp. 533-643, Aratltln>ic Press, New York. Darlix, J. L. & Fromageot, P. (1972). Biochimie, 54, 47. 54. Dickson, R. C., Abelson, J., Barnes, W. M. & Reznikoff, W. H. (1975). Science, 187. 27 35. Gilbert., W., Maizels, N. & Maxam, A. (1973). CoZd spring Harbor Symp. @rant. HioZ. 38, 845 -855. lkemura, T. 8: Dahlberg, J. E. (1973). J. BioZ. C/Len&. 248, 5024 5032. J-ackson, E. N. & Yanofsky, C. (1973). J. MOE. BioZ. 76, X9--101. Korn, L. & Yanofsky, C. (1976). J. Mol. BioZ. 103, 395-409. Tdehowitjz, P., Weissman, S. M. & Radding, C. M. (1971). J. BioZ. Chenc. 246, 51%l 5139. Maizels, N. (1973). Proc. Nat. Acd Sci., U.S.A. 70 3585-3589. Man&is, T., Ptashne, M., Barrel& B. G. & Don&on, J. (1974). Nnture (Lo,ctlo,r), 250. 394-397. Morse, D. E. & Morse, A. N. C. (1976). J. Mol. BioZ. 103, 209-226. Pannekoek, H., Perbal, B. & Pouwels, P. (1974). Mol. Gen. Genet. 132, 291-305. Pannekoek, H., Brammar, W. & Pouwels, P. (1975). l%!oZ. Gen. Gertet. 136, 199--214. Pi:aczenik, B., Barrell, B. G. & Gefter, M. L. (1972). Arch. Biochem. Biophys. 152, 152 1 (i.i. Plat,t, T., Squires, C. & Yanofsky, C. (1976). J. Mol. BioZ. 103, 411 420. Roberts, .J. W. (1969). Nature (London), 224, 1168.-1174. Rohcrts, .J. W. (1975). Proc. Nut. Acad. Sci., U.S.A. 72, 3300-3304. Rasp, ,J. K., Squires, C. L., Yanofsky, C., Yang, H.-L. & Zubay, (:. (1973). Sntzcr~ Svrv BioZ. 245, 133--137. Shapiro, J-., MacHattie, L., Eron, L., Ihler, G., Ippen, K. & Bcckait.h, J. (1969). dVrrtwe (London), 224, 768-774. Shin&u, N. & Hayashi, M. (1974). J. Mol. BioZ. 84, 315--335. Smith. G. R. & Hedgpeth, J. (1975). J. BioZ. Chem. 240, 4818--4821. Squires, C., Lee, F., Bertrand, K., Squires, C. L., Bronson, M. J. R- Yanofsky, C’. (197(i). J. MOE. BioZ. 103, 351-381. Squires, C. L., Lee, F. D. & Yanofsky, C. (1975). J. Mol. BioZ. 92, 93~~1 Il. Yarbrough, L. & Hurwitz, J. (1974). J. BioZ. Chem. 249, 5394-5399. %alkin, H., Yanofsky, C. & Squires, C. L. (1974). ,7. BioZ. Chem. 249, 465-475.