Tlhe EMBO Journal vol.3 no.12 pp.2857-2861, 1984

Mechanism of UTP-modulated attenuation at the pyrE gene of : an example of polarity control through the coupling of to

Fons Bonekamp, Kire Clemmesen1, Olle Karlstrom and (rpoBC) suggesting that the pyr genes are regulated by the Kaj Frank Jensen level of saturation of RNA polymerase with UTP (Jensen et University of Copenhagen, Institute of Biological Chemistry B, S0lvgade al., 1982). 83, DK-1307 Copenhagen K, and 'University of Copenhagen, Institute of We have previously shown that pyrE is the second gene of Microbiology, 0. Farimagsgade 2A, DK-1353 Copenhagen K, Denmark an operon preceded by an open reading frame (orJE) 238 Communicated by A.Munch-Petersen codons long, which directs the formation of considerable quantities of a polypeptide (mol. wt. 25 497) of unknown The pyrE gene of Escherichia coli is part of an operon where function (Poulsen et 1983, Transcription of it is preceded by an unknown gene (orfE) that ends 8 bp al., 1984). the operon is initiated at two in front of a before the start of the symmetry of promoters orfE at fre- the UTP-modulated pyrE quency independent of the cellular concentration attenuator. On a plasmid we have inserted this of UTP. attenuator However, a fraction of the mRNA region in a synthetic cloning site early in lacZ. The resulting only chains reach thepyrE gene due to a UTP a structure contains the lac promoter-operator, the first few regulated attenuation at transcription in the intercistronic space between orfE and pyrE codons of lacZ, 42 bp of DNA from the orfE end, the pyrE et and an (Poulsen al., 1984) (Figure 1). attenuator, in-frame fusion pyrE-IacZ+. The syn- The present experiments aim at elucidating the mechanism thetic cloning sites have been used to vary the length and behind this attenuation, in particular the role translation reading frame of the translation that begins at the lacZ start of of orfE in the control. In essence we have taken thepyrE and proceeds towards the attenuator. of attenu- The effects these ator out of its normal remove variations on pyrE attenuation were determined by monitor- context to it from other poss- ible regulations, e.g., on ing the synthesis of from the level of transcription initiation. ,B-galactosidase the pyrE-lacZ Thus we hybrid gene in grown have inserted fragment A (Figure 1) containing the cells with either low or high pools of end of orfE, the attenuator region, and the beginning ofpyrE UTP. Thus, a very low level of pyrE expression was observ- into the very first part of the lacZ gene on a plasmid. Since ed, regardless of UTP pool size, when the translation from the resulting pyrE-lacZ fusion is in-frame, the lacZ start ended 31 or 62 nucleotide residues upstream to ,B-galactosidase the pyrE activity could be measured to monitor pyrE expression. The attenuator symmetry, but a proper UTP controlled lacZ translation start and attenuation could be established if this translation ended only the cloning region following it were 8 essential for testing the effect of translation into the attenu- bp before the symmetry region of the attenuator (as the ator region. minor native orfE gene) or 10 bp after this structure. However, a By manipulations of the DNA in the clon- ing region we single 'leader read from could modify the length of a short artificial peptide' only frequently used codons peptide read from the lacZ translation start gave a high level of pyrE expression both at high and low towards the attenuator. The effects on UTP pools. These observations indicate that the coupling the UTP modulation of attenu- ation could then be measured. we between transcription and translation determines the degree From the results conclude of mRNA chain terminations at the pyrE attenuator. The level of saturation of the transcribing RNA polymerase with A UTP determines the tightness of this coupling, but the codon usage in the translated area also seems crucial. Key words: attenuation modulated by UTPAeader peptide variations/polarity control/pyrimidine nucleotide biosynthe- sis/translational coupling to transcription

Introduction ThepyrE gene of Escherichia coli encodes the enzyme orotate phosphoribosyltransferase that catalyses one of the reactions in the de novo synthesis of UMP - the precursor of all other pyrimidine nucleotides. The gene is located at 81 min on the "PUF" OPRTase E. coli chromosome and is transcribed in a counter-clockwise (Mr:25,497) (Mr: 23,326) direction towards dut (Bachman and Brooks Low, 1980; Fig. 1. Structure and expression of the orfE-pyrE operon. Symbols and Poulsen et al., 1983). Expression ofpyrE as well as ofpyrB is abbreviations: P2, P1, the two promoters of the operon; atn, attenuator. controlled by the UTP pool, being low when the concen- orfE: 238 triplet codons long open reading frame. T(5), T 8) blocks of five tration of UTP is high and vice versa (Schwartz and Neuhard, and eight thymidylate residues flanking the symmetry of t(he attenuator. 1975; Kelln et al., 1975; Pierard et al., 1976; Turnbough, The arrows and wavy lines underneath indicate the transcripts and gene 1983). So far, the identified mutants products of the operon, respectively. 'PUF': protein of unknown function. only regulatory that OPRTase: orotate phosphoribosyltransferase. A: 510-bp TaqI-TaqI show elevated expression ofpyrE and pyrB in the presence of fragment used for the construction of IacPO-pyrE4acZI gene fusion a high UTP pool are defective in their RNA polymerase herein (see text). ©C IRL Press Limited, Oxford, England. 2857 F.Bonekamp et al. pUC 9 HaeH+ XmaI P E HPABXB Av T atn T P-PROXIMAL P-DISTAL S1 PPP2 l-7-M I WIJ FUSION FUSION Ligation, L-]J ilil J pKCL4 orf E - - pyrE I Ligation pMLB1034 XmaI (pKCL 11) p HpA TaqI HT p ABamHI PKCL 10 l[Fj~4.~psniai~PJrP8NACC,L L HPi/P1L~latnT_ 101. Hindf J E /Ap I ISi P E HPAB Av p pQ i NpTatn T Li!-- J pKCL5 PKCL 13 (pKCL 10) NcoIPNNS1~~~~Ll9@t,OnS....l JL1 S1 _PKCL104- AAGCTT HindX p1 Ligation + Klenow pot. p F p F p1T atn AAGCTAGCTT igation pKCL 15 - L~~~~=~~~4~IcTL1CLO0

Av P E PAB Av P P p pT atn T, Rt LiP- IL- J pKCL6 pKCL 6 L_ _J.w- i b L PJ I L1t-,-PK~CL 106 am am am:

P Pp P pT atn T Fig. 2. Construction of cloning vehicles. A lacZ-translational fusion vehicle Pi I PKCL 107 pKCL4 was constructed by cloning the 254-bp HaeII-XmaI fragment of pCN 102 pCN1O21 --- 7 * .... .6mzj..i.PIf -1i_ ==X pUCG8 I pUC9 into pMLB1034 opened with XmaI. pKCL4 contains the EcoRl site I of pMLB1034 and the lac promoter and operator with a cloning region CLONING REGION I \ within the start of the lacZ gene. This cloning region is in-frame with the I,TI rest of lacZ allowing formation of active fl-galactosidase. A derivative of this plasmid (pKCL5), in which the lacZ gene is brought out-of-frame, was constructed by deleting the small BamHl fragment of the cloning region. Since the further cloning strategy required as few Accl sites as possible (see Figure 3), the 3320-bp EcoRI-AvaI fragments of pKCL4 and 5, where the two AvaI sites in lacZ were left uncut, were in turn ligated to the ,3- lactamase encoding AvaI-EcoRI fragment of pBR327. The resulting plasmids are called pKCLI I and 10, respectively. The fact that the two ends of lacZ in pKCL5 are out-of-frame by - 1 bp inside the cloning region could be used to construct an amber codon in the start of this region by opening the HindIII site, filling it up with Klenow polymerase, and religating. While the cloning region was thus brought back in-frame Fig. 3. Construction of lacPO-pyrE-lacZ+ gene fusions. The 510-bp Taql- with the rest of lacZ, an amber codon was generated within the former TaqI fragment of pPP2 (fragment A, Figure 1) was cloned behind amino HindIII site. The proper clones were selected by testing for f-galactosidase acid codon no. 8 of the lacZ gene in partially Accl digested plasmid activity after cross-streaking with 080pSU3 +, which carries an amber pKCL1O (Figure 2), where the AccI site in lacZ was left uncut. This suppressor and which made those colonies that contained the amber codon resulted in a 1acPO-pyrE-lacZ+ fusion with ,B-galactosidase activity turn blue on XG plates after infection. The sites for the restriction (plasmid pKCLI01). This plasmid also contains a coding frame for a endonucleases are indicated as follows: A, site for the endonuclease AccI; 'leader peptide' originating at the lacZ start (see Figure 4). To be able to Av, AvaI; B, BamHI; E, EcoRl; H, HindIII; P, PstI; X, XmaI. P (in change the nature (length and reading frame) of the leader-translation, we bold type) indicates the position of the lac promoter; the arrow indicates deleted the smaller PstI-PstI fragment, which contains the lacZ start and the direction of transcription from this promoter; am, amber codon. the linker, from pKCLIOI. The larger PstI-PstI fragment, containing the pyrE attenuator and the hybrid pyrE-lacZ+ gene was used for the cloning that the attenuation frequency is controlled through UTP- of similar small PstI-Pstl fragments, manipulated in the restriction sites of the linker region. This was done in the following way. Plasmid pKCLIO induced variations in the coupling between translation and was cut with HindIlI, treated with SI nuclease, and religated. In this way transcription in the end of orfE. four nucleotides were deleted, giving rise to a new plasmid (pKCL13). A unique NcoI site generated in this construction (Figure 4) was used to Results delete, again by nuclease S1, four more base pairs from the cloning region. In the resulting plasmid (pKCL15) the lacZ gene after the linker is read in To investigate if translation plays a role in the intercistronic its proper frame. We then inserted the small PstI-Pstl fragments of attenuation in front of pyrE we first constructed a vector plasmids pKCLI3, pKCLl5 and pKCL6 (Figure 2) in pKCLI01 in place of (pKCL10) with the cloning sites of pUC9 in a full-length lacZ its original PstI-PstI fragment. In this manner, a set of lacPO-pyrE-lacZ+ A fusions were made (pKCL104, pKCL105 and pKCL106) differing in the gene (Figure 2). By insertion of fragment (Figure 1) into frame of translation after the Pstl site. The resulting reading frames for the this gene, plasmid pKCL101 and subsequently its derivatives artificial leader peptides are shown in Figure 4. Plasmid pKCL107 was pKCL104-107, were made (Figure 3). Each of these plas- constructed by cloning the smaller PstI-PstI fragment of pCN102 (kindly mids contains a pyrE-lacZ gene fusion encoding a fusion pro- given to us by Carsten Petersen) into pKCLIOI as described above. The tein with stable ,B-galactosidase activity, which could be used PstI fragment of pCN102 is analogous to that of pKCL13, but contains the cloning region of pUC8. Sequence determinations of the DNA from to monitor pyrE expression. The length and nature of the the lacZ start and beyond the pyrE attenuator in plasmids pKCL10I - 107 'leader' translation from the lacZ start towards the pyrE confi'rmed the outcome of the described manipulations. The cleavage sites attenuator is indicated for each plasmid by filled bars in for the restriction endonucleases and other abbreviations are as in the Figure 3, and by brackets in Figure 4. legend to Figure 3. Additionally we use: F, FnuDII; N, Ncol; T, Taql; Derivatives of the strain Atn, the pyrE attenuator. Dotted areas represent DNA derived from the uracil-requiring S41256 (pyrB) orfE gene; white areas, lacZ-derived DNA; streaky areas, pyrE DNA. The containing F'lacJq1Z::Tn5 and one of the five plasmids de- black bars below the vectors denote the length of translation of the scribed in Figures 3 and 4, or the control plasmid pKCLl1 artificial 'leader peptides'; (+ +), pUC8 cloning region. (Figure 2), which contains an intact lacZ gene, were grown with UMP as a growth-limiting pyrimidine source or in the From the results (Figure 6 and Table I) it is seen that the presence of excess uracil as exemplified in Figure 5. During control plasmid pKCL1 1 (lacZ+), which does not carry any induction with isopropyl-f-D-thiogalactoside (IPTG), ,3- pyrE DNA, gives the same rate of fl-galactosidase formation galactosidase activity was followed. with the two pyrimidine sources. Virtually no ,B-galactosidase 2858 Mechanism of UTP-modulated attenuation at the E. coli pyrE gene

ind E Pa I req I pKCL 101 (tAACC Arc ATl ACC CCA ACF TiG GCMTEMFcA MT ATC CAT IGT AGC GAC GCA GAA GGC GGC GCC GGC AAA C1G AT 1AAGITT GCG ACT GCT GAG TCG CCT iTT TTT 30 TGI CTC TAG] AAAAGTAAGATGAGGAGCGAAGGC A1C PyrE McoI Pst I rT pKCL104 (AjlaACC AIG AlT ACC CCA TG GTG A ICC ATI GTA GCG ACG CAG AAG GCG GCG CTC GCA AAC TGA] ITITAAGGCGACTGA.IGAGCGCCT!TTTTTTGTCTGTAGAAAAGT 20 AACAICACCAGCGAAGGC AIG PyrE FnuDI PstI TeqI PKCL 105 [AjTACC ATG AT! ACG CGC TGC AGG TCG AAT CCA TTG TAG]CGACGCAGAAGGCCCCGCCT GGCAAACI GAT T I TlAGGCGACIGAIGAGICGCC'T ITTTGCTCTGTAGAAAAGTAAGATGAGGAGCGA 10 AGGC ATG PyrE PatI TaqI pKCL106 (A>j~ACC AIG AlT ACG CCA AGC IA CITGGCCTCCACGTCGAATCCATTGTAGCGACCCACAAGG CGGCGCTGGCAAACTGATTITT9AGGCGACTGATGAGTCGCCTTTTTTGTTCTGTAGAAAACTAACATCA GGAGCGAAGGC ATG PyrE 30 Eco RI XmaI Bm H Sill Pstl TaqI pKCL 107 [AJ,jACC ATG AT! ACC AAT TC( ICCICGGA TCC GTC GAC CTG CAG GTC GMA TCC ATT GTA GCG ACG CAG AAG GCG GCG CTG GCA AAC TGAITTTTIIAGGCGACTGATGAGTCGCCTT 20 TTTTTTGICTGTAGAAAAGTAAGATGAGGAGCGAAGGC ATG PyrE E Fig. 4. 'Leader peptide' sequences in plasmids pKCLI0I - 107. The small arrows mark the translation start points of the artificial leader peptides and .E 10 of the pyrE-lacZ+ gene fusion. The part of the DNA sequence within brackets denotes the leader in each plasmid. Opposite arrows mark the position of the attenuator. The codons in boxes are poor codons. en CUco ~0 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140 ° 30 0 0.9 09 0.8 0.8 0,7 0.7 0,6 0,6 "-20 0,5 0,5 (D 04 04 0 0Q3 -IPTTGIPTG 7LI 0,3 10

0,2 0.2

| " j fl=/ ~~URACIL 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140 TIME AFTER INDUCTION (min.) 30

Flg. 5. Growth curves for 541256 (pyrB,A(ac)U169) transformed with pKCL107. Growth conditions and concentrations of nutritional 20 supplements as described in Materials and methods. Symbols: (0) growth on 5' UMP (100 ,g/ml); (@) growth on uracil (25 ug/ml); (A) growth on 5' UMP (100 pg/ml) together with a limited amount (1.3 pg/ml) or uracil; 10 (U) addition of excess uracil (25 Ag/ml) to a UMP grown culture (100 pg/ml). When grown with UMP as pyrimidine source the UTP pool was 6-fold lower while the level of orotate phosphoribosyltransferase was 12-fold higher than when uracil served as source of pyrimidine (not shown). OD436 is made from any of the plasmids in the absence of IPTG, Fig. 6. Expression and control of the hybrid pyrE-lacZ+ gene. Growth, showing that the pyrE-lacZ+ gene is indeed expressed from induction, sampling and assay procedures are described in Materials and the lac promoter. methods. In A, C, D, E and G, the open symbols mark growth with 5' UMP, while the closed symbols mark growth with uracil. B, D and H Plasmid pKCL101, which contains fragment A inserted show the results of shift experiments with plasmids pKCLIOI, pKCL104 into lacZ (Figure 3), gives a much lower rate of ,B- and pKCL107, respectively. Open symbols here represent experiments galactosidase formation during growth with uracil than the where cultures were grown in the presence of excess UMP together with a control plasmid pKCL1 1; when the are grown with limited amount (1.3 pg/ml) of uracil, while the closed symbols represent experiments in which uracil was added to a UMP-grown culture. The data- UMP as pyrimidine source instead of uracil, however, the points shown are means of duplicate assays. The differential rate of rate of enzyme formation is considerably increased (-8- synthesis of ,B-galactosidase derived from these plots is listed in Table I. fold). Moreover, if uracil is added to a UMP-grown culture, The chromosomal pyrE activity was in all cases 12-fold higher for the rate of formation of ,B-galactosidase activity is promptly cultures grown with UMP than for cultures grown on uracil (not shown). reduced. Conversely, the rate of formation of f,-galactosidase activity abruptly increases when UMP utilization begins after Table I. Differential rate of ,B-galactosidase synthesis (units/ml. OD436 = 1) exhausting a limited uracil supply (Figure 6B). Similar experiments were carried out using plasmids Pyrimidine Plasmid pKCL 105 and pKCL 106, which contain the same pyrE-lacZ+ supply pKCL1I pKCL101 pKCL104 pKCLI05 pKCLI06 pKCL107 fusion as in pKCL1I1, but deviate from this plasmid the Uracil 190 9 36 7 7 11 leader-translation. Thus, while this translation in pKCLlQ1 5' UMP 190 67 67 7 7 129 can continue across the region of dyad symmetry of the tran-

2859 F.Bonekamp et al. scription terminator, it stops at some distance upstream to al., 1982). This idea is supported by the finding that the puri- this structure in pKCL105 and pKCL106 (Figures 3 and 4). fied RNA polymerase from the above mentioned rpoBC mu- As seen from Figures 6C and D the rate of appearance of f- tant has an increased Km for UTP, and by the observation galactosidase activity from these plasmids after induction is that RNA polymerase in wild-type cells is subsaturated with low both with uracil-grown and 5' UMP-grown cells. Appar- UTP (K.F.Jensen, unpublished observations). Hence, the ently thepyrE attenuator functions as a permanent transcrip- UTP pool appears to control attenuation by affecting the rate tion-stop, when no translation approaches from the promoter of mRNA chain elongation in such a way that transcription proximal side. Thus, such a translation is needed to suppress more frequently proceeds past the attenuator when the RNA attenuation when the cellular UTP pool is low. In the intact chain growth rate is reduced due to shortage of UTP, than pyrE operon, translation of the end of the orfE message may when chain growth rate is normal (Poulsen et al., 1984). serve this purpose. The experiments presented here were aimed at identifying a To test this possibility we used plasmid pKCL 104, where possible regulatory role of translation of the orfE gene that the first triplet codons of lacZ were brought in:-phase with the precedes the attenuator (Figure 1). Clearly, translation last 14 codons of orfE. Thus the leader peptide ends like orJE towards the attenuator is needed for mRNA chain termi- just in front of the block of five thymidylate residues up- nation to be suppressed when the UTP pool is low. In plas- stream to the symmetry region of the pyrE attenuator (Fig- mids pKCL105 and pKCL106, where such a translation is ures 1 and 4). Upon induction with IPTG of cells containing prevented, at most 507o of the mRNA chains escape termi- pKCL104, a high level ofpyrE-lacZ+ expression was indeed nation and continue into pyrE, regardless of pyrimidine seen during grown on UMP, but surprisingly this level was supply. In all cases where translation continues close to, or decreased only by a factor of two when uracil was used as past, the attenuator symmetry (pKCL1I1, pKCLI04 and pyrimidine source instead of UMP (Figure 6E and F). It pKCL107), the mRNA chain termination is suppressed when seems therefore as if translation at the end (the last 14 codons) the UTP supply is poor. This condition of slow mRNA chain of orfE is able to suppress mRNA chain termination at the elongation can be expected to result in a very tight coupling pyrE attenuator, but that it does so in plasmid pKCL104 even between translation and transcription and thereby the leading at high UTP pools, thereby resulting in a very poor UTP may prevent the formation of a termination- regulation. On the other hand, a translation of the same part inducing RNA structure. Apparently, it is not necessary for of orfE, but in a frame-shifted manner, as in pKCL1I1, does the coding frame for the leader peptide to include the region result in a substantial pyrimidine regulation. This indicates of dyad symmetry of the attenuator in order for translation to that the nature of the translation, perhaps the codon usage, is prevent termination, as was suggested for the putative leader of importance in obtaining attenuation which is normally of the pyrB gene (Turnbough et al., 1983). controlled by the UTP pool. Therefore we constructed The behaviour of thepyrE attenuator in plasmid pKCL104 another plasmid (pKCL107, Figures 3 and 4) with the capaci- relative to pKCL 107 is particularly interesting. During growth ty to encode a new leader peptide partially identical to the with a poor pyrimidine source (UMP) very little mRNA chain leader peptide of pKCL104 (the first five and the last 16 termination occurs in either of the plasmids. When the UTP amino acid residues), but different and longer in the middle. pool is high, however, the attenuator causes a high frequency This alteration had a dramatic effect. ThepyrE-lacZ+ fusion of termination in pKCL 107, but only little termination in is now fully pyrimidine regulated, i.e., the synthesis of ,B- pKCL104. The leader peptides in these two plasmids are iden- galactosidase is 12-fold higher during growth on UMP than tical in their first five and last 16 codons but differ in the when the cells are grown with uracil (Figure 6G and Table I). middle, pKCL107 being five triplets longer than pKCL104 Furthermore, the synthesis of the enzyme promptly adjusts to (Figure 4). Although this difference in length per se might its new rate following shifts in growth conditions (Figure 6H). allow the RNA polymerase to escape from the leading ribo- some, and hence to decouple and cause mRNA chain termi- nation, when the UTP pool is high, it seems more likely that Discussion the different behaviour is due to differenes in the codon usage The results in this paper show that the pyrimidine control of in the two leader peptides. Thus only efficiently translated, pyrE expression in E. coli is exerted exclusively within the good codons (Grosjean and Fiers, 1982) are used in the pep- intercistronic space of the orfE-pyrE operon, as previously tide of pKCL104, while two inefficiently translated poor suggested on the basis of a different experimental approach codons have been introduced in pKCL107 (Figure 4). Like- (Poulsen et al., 1984). Thus, expression of the pyrE-lacZ+ wise, pKCL1I1, which is frame shifted in its leader trans- hybrid gene of plasmid pKCL107, transcribed from the lac lation compared with pKCL104, and does show a pyrimidine- promoter, is pyrimidine-regulated to the same extent as the regulated attenuation, contains a poor codon (Figure 4). This pyrE gene of the intact operon transcribed from its own pro- indiates that the occurrence of poor codons in the 'leader pep- moters. We do not know whether the previously described tide' is needed for the transcribing RNA polymerase to de- inhibitory effects by guanine nucleotides on pyrE expression couple from the at high UTP pools. Hence the (Jensen, 1979) or the activation by the (unknown) product of poor codons may be needed to obtain attenuation properly the pyrS gene (Nowlan and Kantrowitz, 1983) affect the at- modulated by UTP. tenuation or the promoter activity. The intact orfE gene of the pyrE operon contains 15 The activity of thepyrE gene is high when the cellular UTP codons for arginine. All are good codons (Grosjean and pool is low and vice versa (Kelln et al., 1975; Pierard et al., Fiers, 1982) except for the very last one (CGA) which is found 1976; Schwartz and Neuhard, 1975; Turnbough, 1983). In an 16 codons from the carboxy-terminal end and which is rpoBC mutant, however, pyrE expression is 25- to 30-fold followed by two relatively rare glycine codons (Poulsen et al., increased at high UTP pools. This observation has led to the 1984). These poor codons are not present in pKCL 104, which proposal that the control of pyr gene expression involves the includes only the last 14 triplets of orfE and which gives a degree of saturation of RNA polymerase with UTP (Jensen et high level of pyrE expression even at high UTP pools. It is 2860 Mechanism of UTP-modulated attenuation at the E. coli pyrE gene therefore tempting to speculate that RNA polymerase and the Preparation of cell extracts for orotate phosphoribosyltransferase assay leading ribosome move in a very tightly coupled fashion 25 ml samples were withdrawn from the cultures at OD4N = 1, harvested, ex- through orfE until they reach the cluster of poor codons, tracted, and assayed for specific orotate phosphoribosyltransferase activity as described by Poulsen et al. (1983). One unit is defined as the amount of en- which may delay the ribosomes and decouple translation I substrate into min. from transcription if the UTP pool is high, while a tight zyme that converts pmol (orotate) product (OMP) per coupling may be re-established if RNA polymerase lacks Acknowledgements UTP and therefore elongates the mRNA chains more slowly. This work was supported by grants from the Danish Natural Sciences In this context it should be mentioned that the leader of the Research Council, the NOVO Foundation and from the Carlsberg Found- pyrB gene also contains poor codons (Roof et al., 1982; ation by a scholar stipendium to F.B. Turnbough et al., 1983). References Attenuator-like structures are found in the intercistronic Bachman,B.J. and Brooks Low,K. (1980) Microbiol. Rev., 44, 1-56. spaces of many . Hence, the UTP-modulated attenu- Clark,D.J. and Maaloe,O. (1967) J. Mol. Biol., 23, 99-112. ation in thepyrE operon might well be a perhaps extreme case Grosjean,H. and Fiers,W. (1982) Gene, 18, 199-209. of operon polarity control through the coupling between Jensen,K.F. (1979) J. Bacteriol., 138, 731-738. translation and transcription. Jensen,K.F., Neuhard,J. and Schack,L. (1982) EMBO J., 1, 69-74. Jensen,K.F., Larsen,J.N., Schack,L. and Siversten,A. (1984) Eur. J. Bio- chem., 140, 343-352. Materials and methods Kelln,R.A., Kinahan,J.J., Foltermann,K.F. and O'Donovan,G.A. (1975) J. Materials Bacteriol., 124, 764-774. Maniatis,T., Fritsch,E.F. and Sambrook,J. (1982) Molecular Cloning. A IPTG, o-nitrophenyl-j3-D-galactoside (ONPG), 5-bromo-4-chloro-3-indolyl- Laboratory Manual, published by Cold Spring Harbor Laboratory Press, 3-D-galactoside (XG), 5' UMP and antibiotics were obtained from Sigma. NY. Restriction enzymes, DNA polymerase I (large fragment) and T4 DNA ligase Miller,J.H. (1972) Experiments in Molecular , published by Cold were purchased from New England Biolabs, and exonuclease SI was purchas- Spring Harbor Laboratory Press, NY. ed from Boehringer. Nowlan,S.F. and Kantrowitz,E.R. (1983) Mol. Gen. Genet., 192, 264-271. Strains and plasmids Pierard,A.N., Glansdorff,N., Gigot,D., Crabeel,M., Halleux,P. and Thiry, The construction of strain S(1256 (araDl39,A(lac)169,thi,rpsL,pyrB) has L. (1976) J. Bacteriol., 127, 291-301. previously been described by Jensen et al. (1984). Strain NF1815 is a recA Poulsen,P., Jensen,K.F., Valentin-Hansen,P., Carlsson,P. and Lundberg, derivative of MCI000, while strain NF1830 is NF1815 containing the episome L.G. (1983) Eur. J. Biochem., 135, 223-229. F'laclqfZ::Tn5. Plasmids pPP2 and pPPl both contain the pyrE operon Poulsen,P., Bonekamp,F. and Jensen,K.F. (1984) EMBO J., 3, 1783-1790. cloned as a 2.0-kb Pvull-PvuII fragment into pBR322, but in different orien- Roof,W.D., Foltermann,K.F. and Wild,J. (1982) Mol. Gen. Genet., 187, tations (Poulsen et al., 1983, 1984). Plasmid pMLB1034 was previously 391400. described by Weinstock et al. (1983); plasmid pUC9 by Vieira and Messing Schwartz,M. and Neuhard,J. (1975) J. Bacteriol., 121, 814-822. (1982), and pBR327 by Soberon et al. (1980). Soberon,X., Covarrubias,L. and Bolivar,F. (1980) Gene, 9, 287-305. Tumbough,C.L., Jr. (1983) J. Bacteriol., 153, 998-1007. Plasmid constructions Turnbough,C.L., Jr., Hicks,H.L. and Donahue,J.P. (1983) Proc. NatI. Plasmids were constructed as described in the legends to Figures 3 and 4 using Acad. Sci. USA, 80, 368-372. strain NF1815 as the host strain. Plasmid DNA was prepared as described by Vieira,J. and Messing,J. (1982) Gene, 19, 259-268. Maniatis et al. (1982). Restriction enzyme digestions were carried out as Weinstock,G., ap Rhys,C., Berman,M., Hampar,B., Jackson,D., Silhavy, recommended by the manufacturer. All DNA fragments used for cloning T., Weisemann,J. and Zweig,M. (1983) Proc. NatI. Acad. Sci. USA, 80, were purified by agarose gel electrophoresis, cut out from the gel, and electro- 44324436. eluted into dialysis bags as described by Maniatis et al. (1982). Nuclease S5 was used as recommended by the manufacturer. Transformation and conju- Received on 6 August 1984; revised on 10 September 1984 gational procedures were essentially as described by Miller (1972). The presence of a functional lacZ gene on the plasmids was scored on agar plates containing XG (0.005%). Media and conditionsfor growth Isolation of strains was performed on NY medium, which is the same as L-broth (Miller, 1972) except that NZ amine is used instead of Bacto Tryp- tone. Ampicillin was added to a final concentration of 200 jig/ml and XG to 0.005%. For liquid cultures the (A + B) medium of Clark and Maal1e (1967) was used. Glucose and casamino acids were added to a final concentration of 0.2%. Other nutritional supplements were added at the following concen- trations (ug/ml): uracil, 25, 5' UMP, 100, and thiamine, 1. Antibiotics were added at 25 ug/ml of ampicillin and 30 ,g/ml of kanamycin. Depletion of uracil was obtained by growing the cultures with a limited amount (1.3 - 1.4 pg/ml) of uracil together with excess UMP. Shift from UMP to uracil as the pyrimidine source was obtained by adding uracil to a UMP grown culture at a final concentration of 25 pg/ml. Cultures were grown with shaking at 37°C and growth was monitored by measuring absorbance at 436 nm in an Eppendorf photometer. Induction of (3-galactosidase synthesis 60 ml cultures were inoculated from exponentially growing overnight cultures at an OD4N = 0.05. At OD436 = 0.15, (3-galactosidase synthesis was induced by addition of IPTG to a final concentration of 100 pg/ml. During 120- 150 min after induction, samples (0.5 ml) were withdrawn from the cultures to 50 pl chloramphenicol (2 mg/ml) and left on ice for subsequent (3-galacto- sidase assay. The cells were disrupted by ultrasonic treatment for I min, and after centrifugation the supernatant was used directly in the ,B-galactosidase assay which was performed essentially as described by Miller (1972). One unit is defined as the amount of enzyme that converts 1 nmol substrate into product per min. The stability of the hybrid proteins was proved by monitor- ing ,B-galactosidase activity at different times. No decrease in enzyme activity was found during at least 2 days after sonication (data not shown). 2861