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Mechanism of UTP-Modulated Attenuation at the Pyre Gene Of Tlhe EMBO Journal vol.3 no.12 pp.2857-2861, 1984 Mechanism of UTP-modulated attenuation at the pyrE gene of Escherichia coli: an example of operon polarity control through the coupling of translation to transcription 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 terminator 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.
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