Mol Gen Genet (1982) 187:391-400 NIG'G © Springer-Verlag 1982 The Organization and Regulation of the pyrBI Operon in E. coli Includes a Rho-Independent Attenuator Sequence William D. Roof 1, Karen F. Foltermann 1, and James R. Wild L 2 1 Department of Biochemistry and Biophysics, and 2 Faculty of Genetics, Texas A&M University, College Station, Texas 77843 USA Summary. 1. The two polypeptide chains that comprise as- partate carbamoyltransferase in Escherichia coli are encoded by adjacent cistrons expressed in the order, pro- moter-leader-catalytic cistron-regulatory cistron (p-leader- pyrB1). These two cistrons and their single control region have been cloned as a 2,800 base pair (bp) fragment (The minimal coding requirement for the catalytic and regulatory polypeptides is about 1,350 bp plus control regions). The genes contained by this fragment are subject to normal re- pression controls and thus possess the intact control regions. 2. By deleting an internal fragment with specific restric- tion endonucleases, it was possible to construct shortened fragments which no longer produced the regulatory poly- peptide. In these cases the expression of the catalytic cistron was normal and subject to repression upon growth in the presence of uracil. Since the pyrB cistron retained transcrip- tional control, the regulatory polypeptide was not required for expression or control of the catalytic cistron. As ex- pected, the catalytic trimer (Mr=100,000 daltons) from these deletion mutants had no effector response nor did it exhibit homotropic kinetics for aspartate. The enzyme was identical to the % trimer purified from the native ho- loenzyme by neohydrin dissociation. 3. Insertion of Mu d~(lac Ap r) into the structural region of pyrB had a negative effect on the expression of pyrI. This supports the idea that the catalytic and regulatory polypeptide chains of aspartate carbamoyl-transferase are encoded by a single bicistronic operon. Detailed restriction analysis of the cloned pyrBI region has produced a genetic map of restriction sites which is colinear with the published amino acid sequences of the two polypeptides. These maps indicate that the 3'-terminus of the catalytic cistron is adja- cent to the Y-terminus of the regulatory cistron and sepa- rated by 10-20 bp. 4. DNA sequence analysis of the Y-proximal regions of pyrBI revealed that an extensive leader sequence sepa- rated the promoter and first structural gene pyrB. This lead- er of approximately 150 bp contains an attenuator sequence and the translational signals required for the production of a leader polypeptide of 43 amino acids. In this paper we describe the structural organization of pyrBI, and provide a detailed analysis of its regulatory region including its DNA sequence. Offprint requests and all correspondence to: J.R. Wild Introduction De novo pyrimidine (UMP) biosynthesis (see Fig. 1) in both Escherichia coli and Salmonella typhimurium is regulated allosterically at aspartate carbamoyltransferase (ATCase; Gerhart and Pardee 1962) and carbamoylphosphate synthe- tase (CPSase; Pierard et al. 1965; Anderson and Meister 1965) or transcriptionally throughout (carAB to pyrF) in response to fluctuations in endogenous nucleotide pools (Williams and O'Donovan 1973; Kelln etal. 1975; Schwartz and Neuhard 1975). While the evidence for the involvment of various nucleotide pools in the repression/ derepression of the pyr genes is clear, extensive searches for a suitable aporepressor have not been successful (O'Donovan and Neuhard 1970; Kelln and O'Donovan 1976). O'Donovan and Gerhart (1972) reported a putative pyrR which resulted in derepression of the pyr genes, but upon subsequent analysis it was found to be a mutation in UMP kinase (pyrH) (Justesen and Neuhard 1975). This leaky mutant produced greatly reduced levels of UDP and UTP, thereby causing derepression of the de novo pathway. Kelln has isolated cis-acting mutations ("operator-like") which are derepressed for pyrB (R.A. Kelln, personal com- munication) and recently, Jensen et al. (1982) reported the derepression ofpyrB in a strain of S. typhimurium possess- ing an altered RNA polymerase (mapping in the rpoBC gene cluster). While this observation suggests a regulatory role for RNA polymerase in the expression of pyrB, the regulatory control ofpyrBI remains undefined. The present study details the structural organization of the pyrBI region of the E. eoli chromosome and presents evidence consistent with sequential expression of the catalytic and regulatory polypeptides from a bicistronic operon (po pyrB, pyrI). Sim- ilar results describing a bicistronic operon encoding ATCase have been obtained for S. typhimurium (G. Mi- chaels and R.A. Kelln submitted to Mol. Gen. Genet). The aspartate carbamoyltransferase (EC 2.1.3.2) of E. coli is a multimeric enzyme possessing allosteric control sites on regulatory polypeptides which are distinct from the cata- lytic subunits (Gerhart and Schachman, 1965). The ATCase of E. coli and other enteric bacteria (Wild et al. 1980) and yeast phosphofructokinase (EC 2.2.1.11; Laurent et al. 1978) are the only reported multimeric enzymes comprised of catalytic subunits and separable regulatory subunits. The native holoenzyme of E. coli is a dodecamer composed of six identical catalytic polypeptides (functional as a trimer, %) and six identical regulatory polypeptides (functional as 0026-8925/82/0187/0391/$02.00
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`392 pyrG CTP ~ UTP glu gin UDP carAB gin glu cp 2ATP D argA argB-E GLU ~-~NAG-~ ORN Pi II AcCoA CoA pyrBI pyrC-F pyrH ASP Pi PRPP PPi CAN 4 ~ UMP CIT argG-H asp fum Fig. 1. De novo biosynthetic pathways for pyrimidine and arginine with genetic loci of E. coli indicated. GLU, glutamate; NAG, N- acetyl-glutamate; ORN, ornithine; CIT, citrulline; asp, asparate; fum, fumarate; ARG, arginine; HCO3-, bicarbonate; gln, gluta- mine; glu, glutamate; ATP, adenosine-5'-triphosphate; ADP, adenosine-5'diphosphate; CP, carbamoylphosphate; ASP, aspar- tare; CAA, carbamoylaspartate; UMP, uridine-5'monophosphate; UDP, uridine-5'diphosphate; UTP, uridine-5'-triphosphate; CTP, cytidine-5'-triphosphate; PRPP, 5'-phosphoribosyl-3'-pyrophos- phate; Pi, inorganic phosphate; PPi, inorganic pyrophosphate. The genetic loci encode the respective biosynthetic enzymes : argA, ami- no acid acetyltransferase (EC 2.3.1.1.); argB acetylglutamate ki- nase (EC 2.7.2.8); argC, N-acetyl-7-glutamyl-phosphate reductase (EC 1.2.1.38) ; argD, acetylornithine aminotransferase (EC 2.6.1.11); argE, acetylornithine deacetylase (EC 3.5.1.16); argL argF, duplicate genes for ornithine carbamoyltransferase (EC 2.1.3.3); argG, argininosuccinate synthetase (EC 6.3.4.5); argH, argininosuccinate lyase (EC4.3.2.1); earAB, glutamine (light) and ammonia (heavy) subunits of carbamoylphosphate syn- thesase (EC 6.3.5.5 or EC 6.3.4.16);pyrBI, catalytic and regulatory subunits of aspartate carbamoyltransferase (EC 2.1.3.2);pyrC di- hydroorotase (EC 3.5.2.3); pyrD, dihydroorotate oxidase (EC 1.3.3.1); pyrE, orotate phosphoribosyltransferase (EC 2.4.2.10) ; pyrF, orotidine-5'phosphate decarboxylase (EC 4.1.1.23); pyrG, CTP synthetase, (EC 6.3.4.2); pyrH, UMP kinase (EC 2.7.4.4). The genetic nomenclature is that of E. coli (Bachmann and Low 1980) and the enzyme nomenclature is from Dixon and Webb (1979). Multiple biosynthetic steps are indicated as CAA to UMP in 4 steps catalyzed by enzymes encoded by genes pyrC to pyrF a dimer, r2) (Weber 1968). Only the catalytic trimer is re- quired for the transcarbamoylation reaction: carbamoyl- phosphate + aspartate ~ carbamoylaspartate + Pi. Such ca- talysis is not subject to allosteric modification. The structur- al architecture of the holoenzyme is described as 2(c3): 3(r2) in which each catalytic site in one trimer is cooperatively associated with a catalytic site in the other trimer through a regulatory dimer (Cohlberg et al. 1972). The interaction of these two types of polypeptides (defined as the r:c domain of bonding) provides the communication essential for the allosteric regulation of the enzyme and its homo- tropic ligand interactions. Structural analyses of the puri- fied enzyme have established that the regulatory polypep- tide is composed of 152 amino acids (Weber 1968) and the catalytic polypeptide is composed of 306 amino acids + 1% (Monaco et al. 1978). (An unpublished amino acid se- quence from W. Konigsberg identifies approximately 300 residues). High resolution X-ray diffraction studies have produced an elaborate model for this allosterically regu- lated enzyme, and biochemical studies have detailed the nature of the multiple active sites and the R~T transitions of the enzyme (Kantrowitz et al. 1981 ; Monaco et al. 1978). Despite the extensive studies with ATCase, virtually nothing is known about the structural organization and -- regulation (i.e. the genetic control) of the cistrons encoding the two polypeptides of the enzyme. It has been shown (Wild et al. 1981) that the regulatory cistron is closely linked to the catalytic cistron (designated pyrB, map position 96 minutes, aspartate carbamoyltransferase, EC 2.1.3.2, cata- lytic subunit) (Bachmann and Low 1980). The regulatory cistron (designated pyrl) encodes the regulatory polypeptide and was first identified by Feller et al. (1981) when they described an altered ATCase defective in its regulatory po- lypeptide. In this paper we provide a definition of the regu- latory and catalytic cistrons of ATCase and initiate an anal- ysis of the control of their genetic expression. Materials and Methods Preparation of Bacterial Strains and Plasmids F'-plasmid DNA was purified from E. coli K-12 (KLF17/ KLI32) carrying F'117 (F'pyrB +, argI + ; ECGSC # 4255) according to the procedures of Deonier and Mirels (1977). Recombinant DNA plasmids of various sizes were pro- duced by ligation of restriction endonuclease-produced fragments of F'117 into pBR322 (Bolivar et al. 1977). The chimeric plasmids were transformed into appropriate recipi- ent strains as described previously (Dagert and Ehrlich 1979). Transformed strains were identified by a rapid screening procedure (Davis et al. 1980) and plasmid DNA was prepared according to the cleared lysate method (Katz et al. 1973). E. coli TB2 was derived from insertion and subsequent excision of Mu dl(lac Ap') (Casadaban and Cohen 1979) in wild-type K-12. The resultant strain was lacking OTCase and ATCase. Thus the parental strain is presumed to have been initially argF-, argI +. Some E. coli K-12 strains possess two structural genes for OTCase (Glansdorff et al. 1967). Upon excision of Mu dl(lac Ap r) (Bukhari 1976) the strain became Ap S and non-revertible for argI, pyrB and pyrI (the simplest explanation is that imprecise excision removed part of the pyrB/argI region). Restriction endonucleases were obtained from Bethesda Research Labs (Rockville, Maryland), New England Biolabs (Beverly, Massachusetts) and Boehringer-Mann- heim (Indianapolis, Indiana) and they were used according to the suppliers' recommendations. Fragment digests were subjected to electrophoresis on vertical acrylamide gels (6-10%) or horizontal submersible agarose gels (0.7%) pre- pared in Tris-borate buffer, pH 8.3 (0.089 M Tris base, 0.089 M boric acid, and 2.5 mM Na 2 EDTA). After electro- phoresis, the gels were stained with ethidium bromide (0.5 gg/ml) and the DNA fragments visualized in UV-light (302 rim). Molecular sizes of recombinant fragments were estimated relative to known digests. The following plasmid nomenclature describes the nature of the resident DNA fragment (see Table 1): pPB-hl04 plasmid pyrB holoenzyme-isolate 104, pPB-c201 plasmid pyrB catalytic cistron-isolate 201, pAI-cl01 plasmid argI catalytic cistron-isolate 101.
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`393 TabLe 1. Genetic characterization of overlapping recombinant DNA fragments detailing the pyrBI-argI region of the E. coli K-12 genome. Plasmid DNA was purified from E. coli K-12 (KLF17/KL132) carrying F'pyrBI-argI (ECGSC # 4255) and various restriction endonucle- ases were utilized to produce overlapping restriction fragments which were cloned into pBR322. The plasmid pPB-hl04 was modified by the removal of additional fragments as represented in Figure 1. The plasmids pPB-e201 and pPB-e202 are internal deletions of pPB-hl04 in which Bgl II and Hpa I have been utilized to form the deletions. The argI + plasmids were created from pPB-hl02. Plasmid pAI-101 was created following partial restriction with Pst I and pAI-102 was recovered from a complete Hpa I digestion. The overlapping fragments are represented in Fig. 3. The molecular size of the cloned fragment was determined by agarose or acrylamide gel electrophoresis as described in the text. The genotypes were determined by the transformation of appropriate deletion mutants to prototrophy in the case of argI and pyrB. The presence of pyrI was determined biochemically or irnmunologically as described in the text. The enzyme structure was deduced from molecular weight determinations on Sephadex G-200 (Wild et al. 1980) Plasmid * Restriction site + Fragment Genotype Enzyme Structure Molecular size ATCase OTCase (kb) pPB-hl01 HindIII-HindlII 14 pyrB + , pyrI + , argI + 2(%): 3(r2) c 3 pPB-hl02 EcoRI-EcoRI 12 pyrB + , pyrI + , argI + 2(%) : 3(r2) c 3 pPB-hl03 SaII-SalI 10 pyr B + , pyr I + , argI + 2(%): 3(rz) c3 pPB-hl04 PstI-PstI 6.0 pyrB + , pyrI +, 2(c3) : 3(r2) pPB-ht05 SalI-PstI 2.8 pyrB + , pyrI +, 2(c3) : 3(r2) pPB-c201 PstI-PstI A( Bg l II-Bg l II) 5.3 p yr B + , c3 - pPB-c202 PstI-PstIA(HpaI-HpaI) 3.5 pyrB +, c3 pAI-10/ PstI-PstI 5.0 argI +, - c3 pAI- ~ 02 HpaI-HpaI 3.0 argI + , - c 3 pPB-c201 was produced by the deletion of a 650 bp frag- ment from pPB-M04 with BglII. Similarly, pPB-c202 re- sulted from the deletion of a 2.5 kb fragment with HpaI (see Fig. 2). Enzymatic assay, purification, and molecular weight determination were performed to verify the nature of various cistronic products. Enzyme assays for ATCase in cell-free extracts of E. coli cells have been described earlier (Wild et al. 1980). Molecular weights of ATCase were estimated by ascending G-200 chromatography. The enzyme was purified according to the procedures of Gerhart and Holoubek (1967) and was separated into catalytic and regulatory subunits by exposure to the mercurial, neohydrin (Yang et al. 1978). The phenotypes resulting from the various cloned frag- ments can best be defined by the resulting enzymatic struc- tures. Thus plasmid pPB-hl04, containing pyrB, pyrI, and argI, produces the intact holoenzyme for ATCase [Mr= 300,000, 2(c3):3(r2) ] and the typical OTCase trimer [M r = 100,000; %]. Similarly, plasmids such as pPB-c201 (pyrB +) produced only the catalytic trimer (%) of ATCase and lacked the regulatory dimer (r2) and OTCase. The presence of the regulatory subunit in cell-free extracts was verified by specific antibody precipitation or titration against func- tional catalytic subunit (c3) according to the technique of Perbal and Herv6 (1972). DNA Sequence Determination DNA fragments used for sequence determination were iso- lated from preparative polyacrylamide gels by the methods of Maxam and Gilbert (1980). Fragments were dephosphor- ylated and then labeled at their 5' ends by using bacterial alkaline phosphatase and polynucleotide kinase (Bethesda Research Laboratories, Rockville, Maryland) and [y_32p] ATP (Schwartz-Mann). The labeling reaction was followed by digestion with an appropriate restriction enzyme, and the desired fragments were isolated by preparative poly- acrylamide gel electrophoresis. Sequence determination of the isolated fragments was accomplished by using the C, C + T, A > C, A + G, and G specific-cleavages described by Maxam and Gilbert (1980). pyrB pyrl argl valS co rB~.J-3~ (96.5) (96) ~,,,~ /carAB(1) (70) .argR "~ /t'- (68) argG ~ / (6~54rp~r;~ 5P J~ p.yrD (21) pyrC (23) pyrF (25) Fig. 2. The genetic map of E. coli K-12, highlighting the gene loci involved in the de novo biosynthesis of arginine and pyrimidines. A description of the genetic nomenclature is presented in the legend to Fig. 1. Additional gene loci include corB, argR, and valS which encode respectively cobalt resistance (magnesium transport), the aporepressor for arginine, and the tRNA synthetase for valine (Bachmann and Low 1980) Results Cloning and Sublconing of pyrB, pyrL and argI The pyrB[/arg[ region of the E. coli K-12 chromosome has been subcloned into pBR322 from F'117 (F'pyrB+,pyr - [ +, argI +) using several restriction endonucleases (see Ta- ble 1). The genetic map orE. coli K-12 is presented in Fig. 2 and the genetic loci involved in arginine and pyrimidine biosynthesis are highlighted. The biosynthetic enzymes
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`394 18,000 ] 16,000 14,000 12,000 10,000 8,000 r ] r I I I I I I 6,000 4,000 2,000 0 ] I I I I r I I [-- i101 -- I~ i102 h103 h102 I I h104 -- h105 --1 Sail BstEII BstEII Hpal Pstl Hpal -t--1-- v / arg I Bst Eli Bst Eli H~I Sa__~l I EcoRI Pst I rm~ pyr BI Fig. 3. Analysis of the pyrBI-argI region of the E. coli by restriction endonuclease fragment mapping. Overlapping restriction endonuclease fragments were used to define the location of pyrB, pyrI, and argI loci relative to one another. The various DNA fragments, cloned into pBR322 and described in Table 1, are represented in numbers of base-pairs ((~18,000) (It should be noted that pyrB and argI encode respective catalytic polypeptides of ATCase and OTCase while pyrI codes for the regulatory polypeptide of ATCase. There is no arginine locus analogous to pyrl). The gene loci are oriented on the E. coli map in such a manner that argI is close to 0 minutes and that expression is counterclockwise for both, presumably, therefore from the same strand as suggested by Syvanen & Roth (1973) for S. typhimurium unique to each pathway (Fig. 1) are dispersed throughout the chromosome, yet the appropriate gene loci are subject to cooperative regulatory controls upon repression/dere- pression (Beckwith et al. 1962; Williams and O'Donovan 1973; Kelln et al. 1975; and Schwartz and Neuhard 1975 for pyrimidines/and Vogel 1961 ; Elseviers et al. 1972; Vogel et al. 1971; Leisinger and Haas 1975 for arginine). The pyrBI and argI gene loci are characterized by the dramatic levels of derepression which can be obtained (the derepres- sion/repression ratio may approach 500 for these three loci) while the other enzymes of the pathways vary only 2-4 fold (Vogel et al. 1971; Williams and O'Donovan 1973; Kelln et al. 1975; Schwartz and Neuhard 1975). By using overlapping restriction mapping and secondary digestion of cloned fragments, it has been possible to characterize the genetic organization of the cistrons encoding ATCase (pyrB1) and OTCase (argl). As summarized in Fig. 3, the following observations can be made: (i) The pyrBI gene region is located approximately 3,000 bp from argI gene; (ii) The pyrB and pyrI cistrons are immediately adjacent, with pyrI distal from argI; (iii) There is a dramatic similarity in the restriction pat- terns ofpyrBI and the argI gene region. (The PstI - BstEII - HpaI recognition sites are virtually identical in location for both regions). It has been shown already that the argI and pyrB polypeptides possess some N-terminal sequence homologies indicative of gene duplication (Gigot et al. 1977). Thus, the organization of the pyrBI/argI region may be interpreted to support the suggestion that this region of the E. coli represents an evolutionary duplication or transposition (Legrain et al. 1976). Colinear Comparison of the Restriction Map of pyrB and pyrI with Reported Amino Acid Sequences It has been possible to construct comparative maps of the pyrBI region using the restriction endonuclease sites and the reported amino acids sequences of the ATCase cistrons. The precise limits of the genes can be determined from the sizes of their polypeptide products: 300 amino acids for the pyrB product, 152 amino acids for the pyrI product and approximately 300 amino acids for the argI product. The colinear maps of the restriction sites ofpyrBI and corre- sponding amino acid sequences are presented in Fig. 4. Since all of these restriction enzymes are class II endonucle- ases which recognize specific base sequences, it is possible to determine the corresponding cognate amino acids (see Legend for Fig. 4). These colinear maps reveal several addi- tional structural details of the pyrBI gene region: (i) The reported amino acid sequences approximate the restriction site map; (ii) The N-terminus of the regulatory polypeptide encoded by pyrI lies alongside the C-terminus of the cata- lytic polypeptide, encoded by pyrB; approximately 10-20 bp separate the two coding regions. Organization of pyrB and pyrI into an Operon The structural arrangement of pyrBI region suggests that the two cistrons are organized as a bicistronic operon. Fur- ther evidence to support this contention was obtained by the concommitant loss of both the catalytic and regulatory polypeptides upon insertion of Mu dl(lac Ap r) into pyrB of the chromosome. This Mu lysogen was Ap% pyrB-, and pyrI-, yet argI +. Following incubation at 42 ° C, single colony isolates were screened for pyrBI- and Apt It was possible to recover argI- strains, such as TB2, amongst these isolates. While it appears that a Mu insertion into pyrB does not affect the expression of argI, imprecise ex- cision of Mu from pyrB can result in the elimination of argI and pyrB. Perbal et al. (1977) have shown that the production of the regulatory polypeptide (r) is stoichiomet- ric for the catalytic polypeptide (c) and the production of r seems to be dependent upon the biosynthesis of c. They have proposed that a zinc-dependent association of cata-
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`HinF I HinFI HinFI 15 120 300 3011 115 /3 255 Bgl II Taql HinF, 1 H~, I; / Alu, 1 90 18° [8° 1601 75 I 7s I pvrB CISTRON ~ CISTRON 395 Leu-Ala Gly-Asp-Leu Arg-Va!-GIn Met Asp-Leu ]lie-Asp Asn Met-Ala; ;Arg-Thr 1 SeiLeu Ser;Leu 1 ke~/I Ser-L_eu; i [ ; 1 VaiAsn ~ Arg-Arg 15 54 159 169 208 219 300 0 30 57 83 104 128 153 16 55 16-7- 17~ 209 221 31 58 84 105 129 CATALYTIC POLYPEPTIDE REGULATORY POLYPEPTIDE Fig. 4. The colinear map of the pyrB-pyrI region and its polypeptides. Since class II restriction endonucleases recognize specific inverted palindromic sequences, it is possible to determine the corresponding di- and tri-peptides encoded by such DNA segments. For example: The Hpa I restriction site, 5'-GTTAAC-3', has the potential to encode N-Val-Asn when transcribed and translated in the first position reading frame; N-Leu-Thr in the second reading frame or a chain-terminating UAA in the third. Only one Val-Asn sequence is observed for the amino acid sequence of the regulatory polypeptide at residues 83/84 and the Leu-Thr combination is not observed. Similar logic applies for the Bgl I! restriction sites, 5'-AGATCT 3', which unequivocally designates Asp-Leu at amino acid residues 57/58. Theoretically, the restriction of the DNA with Bgl II and Hpa I should produce a fragmefit of 80 bp in length. This is presented in the top schematic figure. Thus the locations of the regulatory and catalytic cistrons are established accurately. It was possible to locate the unique position for all restriction endonucleases recognizing 6 or more base-pairs. Using endonucleases that recognize 5 bp sites it was possible to estimate the location relative to DNA fragment sizes. By comparing the known polypeptide map to the restriction endonuclease map it was possible to determine that between 10-20 base pairs separate the catalytic and regulatory cistrons lytic trimers and regulatory dimers occurs only in the cyto- plasm and not on transcriptionally active polysomes. Fur- thermore, the same study showed that there is less than five percent production of r in the absence of c. The results of our study support the earlier suggestion by Perval and Herve (1972) that the pyrB and pyrI cistrons comprise a bicistronic operon in which the catalytic cistron is promot- er-proximal. The Regulatory Polypeptide has no Role in the Expression of the Catalytic Cistron The plasmid pPB-hl04 was subjected to internal deletion by Hpa[ which removed approximately 2,500 bp including the carboxy-terminal region ofpyrL In a separate deletion, 650 bp including the last two-thirds (95 amino acid resi- dues) ofpyrI were removed. In each case, pyrB was respon- sive to typical repression conditions in the presence/absence of exogenous uracil (50 I~g/ml) for both plasmids (Table 2). Relevant plasmids (Table 1) were characterized by acrylam- ide gel electrophoresis and by auxotrophic characteristics. After the internal fragments were removed, the resulting plasmids were transformed into competent TB2 cells. The molecular weights of ATCase from the various plasmids were determined by chromatography on Sephadex G-200 as described in earlier reports (Wild et al. 1980). The strains containing the catalytic pyrB and partial deletion of the pyrI cistron produced only catalytic polypeptides and all enzymatic activity was recovered as catalytic trimers (Mr = 100,000 daltons. Thus, the presence of a functional pyrI gene product is not required for normal pyrB expression and regulation. Analysis of the Regulatory Region of pyrBI by DNA Sequence Determination The DNA sequence of the promoter region of pyrBI is presented in Fig. 5. The nucleotide sequence contains three Table 2. Repression of ATCase formation in various plasmid con- structs of E. coli. The specific activities are expressed as micromoles of carbamoylaspartate produced per minute reaction time per milli- gram of protein from a cell-free extract. The strains and plasmids are described in the text and Table 1. Repression index is calculated as the ratio of specific activity without uracil/specific activity with uracil for each strain Strain Genotype Specific Repression activity index K12 (min) pyrB +, pyrI + 12 0 K12 (+U) pyrB ÷, pyrI + 4.0 3.0 TB2 (min) pyrB-, pyrI- <0.1 TB2:pPBhl04 (min) pyrB +, pyrI + 188 0 TB2:pPBhI04 (+U) pyrB +, pyrI + 70 2.7 TB2:pPBc201 (rain) pyrB +, pyrI- 225 0 TB2:pPBc201 (+U) pyrB+,pyrI - 134 1.7 TB2: pPBc202 (min) pyrB +, pyrI- 151 0 TB2:pPBc202 ( + U) pyrB +, pyrI- 39 3.8 sites that are organized spatially along the DNA in agree- ment with the consensus "idealized" promoter sequence (Pribnow 1975; Gilbert 1976; Rosenberg and Court 1979). A classic RNA polymerase recognition site (Re) is centered ten base pairs (-10) from the presumptive transcriptional initiation site designated I (bp = + 1). A sigma "recognition site" (Ro) is located approximately thirty-five base pairs (-35) preceding the transcriptional initiation site. The Ro sequence, TATAATG, represents the "idealized" Pribnow box which serves as the base specific contact sequence for the RNA polymerase core (Pribnow 1979). Twelve base pairs separate the Rc sequence from R, thus defining an ideal promoter sequence covering approximately 40 bp. This precedes the translation initiation of the catalytic poly- peptide of ATCase (+ 153) which can be identified by fitting
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`396 XXX TCA AAT AAA AAT GCA AAT ACC TTG ACT TTT - 110 - 100 -90 --80 AAT TCA AAT AAA CCG TTT GCG CTG ACA -70 -60 S~maRecognition AAT ATT GCA TCA AAT CTT GCG CCG CTT' --50 --40 -30 Pribnow Box I CTG ACG ATG A T ATA AT CCG GAC AAT TTG CCG -20 -10 +1 SD-1 Leader Polypeptide GGA GGA TGT ATG GTT CAG TGT GTT CGA TAT TTT met. val. gln. cys. val. arg. tyr. phe. GTC TTA CCG CGT CTG AAA AAA GAC GCT vaL leu. pro. arg. leu. lys. lys. asp. ala +10 +20 +30 +40 +50 +60 Attenuator i GGC CTG CCG TTT TTC TTC CCG TTG ATC ACC CAT TCC CAG CCC CTC AAT CGA GGG GCT TTT gly. leu. pro. phe. phe. phe. pro. leu. ile. thr. his. ser. gln. pro. leu. ash. arg. gly. ala. phe + 70 +80 +90 + 100 + 110 + 120 SD-2 Catalytic Polypeptide TTT T)tC CTA GG'C AGG AGP~ TAA AAG ATG GCT AAT CCG CTA TAT CAG AAA CAT ATC ATT TCC phe. tyr. leu. gly. arg. arg (met) aia. asn. thr. leu. tyr. gln. lys. his. ile. ile. set + 130 + 140 + 150 + 160 + 170 + 180 Fig. 5. The nucleotide sequence of the pyrBI initiation region. The presumptive location of the transcriptional initiation is indicated by '°I" and orients the sequence from +1. The Pribnow Box, Ro, is shown enclosed in a box at -10; R is bracketed at -35. G Two possible Shine-Dalgarno sequences for ribosomal association are indicated as SD 1 at + 10 and SD 2 at + 145. A possible leader polypeptide initiation site (ATG) is indicated at +20 and the translational initiation site for the catalytic polypeptide of ATCase is shown at +155. The possible leader polypeptide is indicated with italicized amino acid sequence deduced from the DNA sequence beginning with ATG encoding met at +20 and continuing until the chain terminating codon at +147. The amino-terminus of the catalytic polypeptide begins in the same reading frame with ala.asn.thr at 157. A strong, Rho-independent altenuator is indicated from -t-105 to +135. An additional region of inverted symmetry is indicated from +58 to +86. An inverted palindromic repeat covering 16 base pairs overlaps the site of transcriptional initiation (+ 1) and is centered around + 2, + 3 the DNA sequence to the known N-terminal amino acid sequence of the pyrB product as determined by Gigot et al. (1977). An unexpected feature of this promoter is its spatial separation from the translational initiation site of the cata- lytic polypeptide. This promoter region is characterized by an extensive leader which contains: 1) a strong, Rho-independent attenuator sequence cen- tered about + 115, 2) a weaker palindromic sequence centered about + 70, 3) two potential ribosomal binding sites with sequences complementary to 3' sequence of the 16S ribosomal RNA (SD1 and SDz in Fig. 5), (Shine and Dalgarno 1974), 4) a potential leader polypeptide of 43 amino acids initi- ating translation at + 18, and 5) a 16 bp region of dyad symmetry centered at + 2, + 3 which resembles a repressor binding site. Effect of Amino Acid Supplement on the Expression of pyrBI in vivo The specific activity of ATCase increases three-fold when E. eoli K-12 is grown in M-56 minimal medium supple- Table 3. Effect of amino acid supplement on the expression of ATCase in E. eoli K-12. Bacteria were grown in minimal medium M-56 supplemented with 0.2% glucose, 50 gg uracil (U) per ml, 0.1% acid hydrolyzed casein (CAS, casamino acids), and/or 100 gg L-arginine (ARG) per ml. Specific activity is presented as described in the text as micromoles carbamoylaspartate produced per minute reaction time per milligram protein from a cell-free extract. Repres- sion index is presented as specific activity in absence of uracil divided by the specific activity in the presence of uracil. In order to evaluate the overall effect of supplements, the percentage of specific activity relative to minimal +U (repressed conditions) is calculated in column four Growth conditions Specific Repres- Percentage activity sion minimal + U index Minimal (M56) 16.7 420 Minimal + U 4.0 4.2 J 00 Minimal + CAS 46.9 1,170 Minimal + CAS + U 7.9 5.9 198 Minimal + CAS + ARG 49.2 1,230 Minimal + CAS + ARG + U 11.5 4.3 287
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`Merck Ex. 1052, pg 1327
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`397 mented with 0.1% casamino acids and 100 gg L-arginine per ml as compared to growth in the unsupplemented minimal medium (Table 3). In fact, wild-type strains of E. coli contain about the same specific activities for ATCase in cultures grown in the presence of uracil supplemented with casamino acids and arginine compared to unrepressed conditions (minimal medium without uracil; 11.5 vs 16.7 ~tmoles carbamoylaspartate/min/mg protein). This supplement has little effect on growth rate of prototrophic strains (less than 20%, unpublished observation) but does have a significant effect on the specific activity of ATCase. Repression by growth in uracil is not overcome by amino acid supplement, but the baseline level of ATCase produc- tion is increased. Discussion The control regions of several amino acid biosynthetic operons are characterized by leader regions similar to that reported for pyrBI in this paper. The DNA sequences of these leader regions usually contain two or more sequences of dyad symmetry which can form mutually exclusive sec- ondary structures in the RNA transcript (Yanofsky 1981; Blasi and Bruni 1981; Keller and Calvo 1979). One of the sequences is homologous with Rho-independent site for transcriptional termination, called the termination structure (Yanofsky 1981; Rosenberg and Court 1979; Adhya and Gottesmann 1978). Various other secondary symmetries provide transcriptional or translational pause sites allowing alternate hairpin interactions with the termina