Proc.NatL Acad. Sci. USA
`Vol. 79, pp. 4020~4024, July 1982
`Biochemistry
`
`Genes encoding Escherichia" coli aspartate transcarbamoylase:
`The pyrB-pyrl operon
`(transcriptional regulation/cloning pyrB and pyrl genes/map position of gene encoding regulatory chains/intercistronic region)
`C. DAVID PAUZA*, MICHAEL J. KARELS, MARC NAVRE, AND H. K. SCHACHMANt
`Department of Molecular Biology and Virus Laboratory, Wendell M. Stanley Hall, University of California, Berkeley, California 94720
`
`Contributed by Howard K. Schachman, Apri/9, 1982
`
`In both Escherichia coli and Salmonella typhi(cid:173)
`ABSTRACT
`murium there is approximately balanced synthesis of the six cat(cid:173)
`alytic and six regulatory polypeptide chains of the regulatory en"
`zyme aspartate transcarbamoylase (carbamoylphosphate:L-as(cid:173)
`partate carbamoyltransferase, EC 2.1.3.2). This control is achieved
`by the contiguous pyrB and·pyrl genes, which encode the catalytic
`and regulatory chains, respectively. Evidence for this single tran(cid:173)
`scriptional unit was obtained by a study of various deletion mu(cid:173)
`tations and from the DNA sequence of the intercistronic region
`between pyrBand pyrl. One pyrB deletion, pyrB748, produced
`a normal level of regulatory chains even though.it removed a sub(cid:173)
`stantial portion of the pyrB gene. Another deletion, pyrB740,
`shares a similar terminus at one end within pyrB, but the promoter
`region is removed.· ln·this deletion mutation, there is no produc(cid:173)
`tion of the regulatory polypeptide, indicating that a single region
`adjacent to pyrB·controls transcription of pyrl as well. Molecular
`cloning and subsequent DNA analysis demonstrated that the pyrB
`and pyrl genes are contiguous with pyrl as the distal gene in the
`operon. The cistrons are separated by a IS-nucleotide untrans(cid:173)
`lated region containing a sequence capable of interacting with the
`16S ribosomal RNA and allowing translation of the pyrl cistron.
`
`It has been known for many years that the allosteric enzyme
`aspartate transcarbamoylase (ATCase; aspartate carbamoyl(cid:173)
`transferase; carbamoylphosphate:L-aspartate carbamoyltrans(cid:173)
`ferase; EC 2.1.3.2) from Escherichia coli is an oligomer com•
`posed of six catalytic (c) and six regulatory (r) polypeptide chains
`(1-6). Moreover, there is approximately balanced biosynthesis
`of the chains in both E . coli (7) and Salmonella typhimurium
`(8). These observations led to the suggestion that the structural
`genes for the c and r chains (pyrB and pyrl, respectively) are
`organized as an operon (8) and that biosynthesis invol~es a
`polycistronic messenger RNA (7). However, precise knowledge
`of the location of the pyrl gene and definitive evidence that both
`genes are subject to the same transcriptional control have not
`been available.
`ATCase of both E. coli and S. typhimurium is composed of
`two trimeric catalytic (C) subunits and three dimeric regulatory
`(R)subunits (1~6, 9, 10). Isolated C trimers of molecular weight
`100,000 are catalytically active but insensitive to· the feedback
`inhibitor, CTP, and the activator, ATP (1), whereas the freeR
`dimers of molecular weight 34,000 are devoid of catalytic ac(cid:173)
`tivity but still bind both CTP and ATP with high affinity (1).
`When C and R subunits are mixed, reconstituted ATCase of
`molecular weight 300,000 is formed in high yield, and the com(cid:173)
`plex exhibits the allosteric properties of native ATCase (1).
`Several lines of evidence indicate that the pyrl gene is in
`close proximity to the pyrB gene. Single deletions in S. typhi(cid:173)
`murium eliminate production of both c and r polypeptide chains
`
`The publication costs of this article were defrayed in part by page charge
`payment. This article must therefore be hereby marked "advertise·
`ment" in accordance with 18 U. S. C. § 1734 solely to indicate this fact.
`
`(8). In addition, specialized A phages capable of transducing the
`pyrB gene also encode the r chain (11), but the distance between
`pyrB and pyrl is uncertain. Although the genes for the biosyn(cid:173)
`thetic pathway (pyrB, pyrC, pyrD, pyrE, and pyr:F) respond
`in a coordinate manner to uracil starvation (12), they map at
`different positions (13). The increase in ATCase activity, re(cid:173)
`flecting the elevated and balanced expression of pyrB and pyrl,
`is at least 10-fold greater than that of the other enzymes (12, 14).
`Support for the suggestion that pyrB and pyr.l are part of the
`same transcription unit stems from two independent sets of
`experiments. Perbal and Herve (7) determined the kinetics of
`incorporation of radioactively labeled amino acids into the c and
`r chains during and immediately after uracil starvation, and on
`the basis of the results they proposed that synthesis of both
`types of polypeptide chains was directed by a polycistronic
`messenger RNA in E. coli. Further evidence for an operon
`structure was provided by Syvanen and Roth (8) in their dem(cid:173)
`onstration of the polarity of pyrB chain-terminating mutations
`on the synthesis of r chains in S . typhimurium.
`In order to eliminate uncertainty regarding the organization
`of the pyrB and pyrl genes and the control of their expression,
`we have analyzed the effect of pyrB deletion mutations on the
`synthesis of the r polypeptide chains. These studies have shown
`that pyrl is subject to the same transcriptional regulation as
`pyrB even in the absence of the pyrB product. In addition, we
`have determined the position of the pyrl gene by DNA se(cid:173)
`quence analysis of the intercistronic region between pyrB and
`pyrt These results establish that the contiguous pyrB and pyrl
`genes constitute a single transcriptional unit encoding the c and
`r chains of E. coli ATCase.
`
`MATERIALS AND METHODS
`Bacterial Strains and Their Construction. All S . t!Jphimu(cid:173)
`rium strains listed in Table 1 are derivatives of LT2 except
`TR3200, which was derived from LTI. The pyrH700 mutation
`results in a partially defective UMP kinase; as a consequence
`the levels of UDP and UTP are reduced, resulting in greatly
`increased production ofATCase (9, 15-17). Mutation pyrB655
`is a deletion that removes all of pyrB (8). The F393 episome is
`derived from F128 (F' pro lac) of E. coli K-12 and contains the
`E. coli pyrB (17). Media, transductional methods, and conju(cid:173)
`gational transfers were described earlier (17). High-titer phage
`P22lysates for mapping experiments were concentrated 10-fold
`to yield titers of about IOn plaque-forming units/ml; they were
`stored in T-2 buffer (18).
`
`Abbreviations: ATCase, aspartate transcarbamoylase; c, catalytic poly(cid:173)
`peptide chain; r, regulatory polypeptide chain; C, catalytic subunit; R,
`regulatory subunit; Pyr+,Jyrimidine-independent; kb, kilobase.
`*Present address: Medic Research Council Laboratory of Molecular
`Biology, Cambridge, England:
`t To whom .reprint requests should be addressed.
`
`4020
`
`Merck Ex. 1050, pg 1314
`
`

`
`Biochemistry: Pauza et al
`
`Proc. Natl Acad. Sci. USA 79 (1982)
`
`4021
`
`Genotype
`
`Table 1. Bacterial strains
`Strain
`.8. typhimurium
`HS2230 argl2002 fol-101 leuD798 proAB47 pyrB655 pyrH700
`HS2231 arg/2002 fol-101 leuD798 proAB47 pyrB655 pyrH700/
`F393tac• pro• argF280 (P22 pyrB•)
`HS2255 argl2002 fol,101 leuD798 proAB47 pyrB655 pyrH700/
`F393tac• pro• argF282::Tn10 (P22 pyrB740)
`HS2256 argl2002 fol-101 leuD798 proAB47 pyrB655 pyrH700/
`F393lac+ pro• argF282::Tn10 (P22pyrB•)
`HS2273 argl2002 fol-101leuD798 proAB47 pyrB655 pyrH700/
`F393tac• pro• argF282::Tn10 (P22 pyrB748)
`HS2278 argl2002 fol-101 leuD798 proAB47 pyrB655 pyrH700/
`F393lac+ pro• (1>22 pyrB754)
`fol-101 leuD798 proAB47 pyrB655 pyrH700/
`F393tac• pro• argF282::Tn10 (P22 pyrB748)
`fol-101 leuD798 proAB47 pyrB655/
`F393 lac• pro• argF282::TnlO (P22 pyrB748)
`TR3200* amtA1 proAB47 pyrB655 trp-130
`
`HS2306
`
`HS2307
`
`E. coli
`AT2535t pyrB59, argH2, thi-1, his-1,purF1, mtl-2, xyl-7, malA1,
`ara-13, lacY1 or lacZ9, rps-18, 9 or 14, ton.A2 or ton-
`14, tsx-23 or tsx-25, Ar, A-, supE44
`* Kindly provided by J. R. Roth.
`t Obtained from the E. coli·Genetic Stock Center (New Haven, CT).
`
`Isolation and Mapping of Deletion Mutants. Deletion mu(cid:173)
`tants HS2255 (pyrB740), HS2273 (pyrB748), and HS2278
`(pyrB754) are spontaneous derivatives of HS2256 obtained by
`selecting for pyrB mutants as suppressors of arginine auxotro(cid:173)
`phy (17). These deletion mutants are completely auxotropic for
`pyrimidines. Mapping was performed by transductional crosses
`using high-titer P22 stocks grown on point mutants and select(cid:173)
`ing for the ability of the deletion mutants to form pyrimidine(cid:173)
`independent (Pyr+) transductants in spot tests on selective me(cid:173)
`dia. Endpoints were confirmed by crosses using 100-200 11-l of
`high-titer P22 stocks and 100 11-l of recipient cells that had been
`concentrated 10-fold after overnight growth in nutrient broth
`supplemented with a 2% vol of E medium (at 50 times the nor(cid:173)
`mal concentration). The crosses were scored after 48 hr at 37"C.
`In crosses in which recombination was possible at least 100 col(cid:173)
`onies resulted, whereas negative results were indicated by a
`complete absence of Pyr+ transductants.
`Preparation of Cell Extracts. Bacteria for assays of R subunit
`were grown to late logarithmic phase in 25-ml cultures of E
`medium supplemented with 0.25% glucose, uracil at 20 WU
`ml, and required amino acids at 100 1£g/ml. Extracts were pre(cid:173)
`pared as described by Syvanen and Roth (8). Protein concen(cid:173)
`trations in the extracts were 0.8-1.5 mg/ml.
`Preparation of Radioactively Labeled C Subunit. 1251-La(cid:173)
`beled C subunit was prepared by the method of Syvanen et aL
`(19}, using 0.5 mCi (1 Ci = 3. 7 X 1010 becquerels) of Na125I
`(Amersham) and 40 11-g of purified C subunit from E. coli ATC(cid:173)
`ase. The specific activity of the labeled protein was 4.2 x 106
`cpm/1£g.
`Assay for R Subunit in Cell Extracts. The amount of R sub(cid:173)
`unit in the extracts was measured by the procedure (assay II)
`of Syvanen and Roth (8). Cell extracts (50 11-l) were treated with
`0.2&..~tmol of neohydrin (K & K) to dissociate any ATCase into
`free R and C subunits (1). After 2 min the bisubstrate analog
`N-(phosphonacetyl)-L-aspartate was added to yield a concen(cid:173)
`tration of 20 1£M, followed by the addition of 1.5 ~£mol of 2-
`mercaptoethanol and 125I-labeled C subunit. The amount of
`125I-labeled C subunit was varied between 2 and 8 11-g (1.5 X
`lOS cpm per tube), depending upon the amount of R subunit
`
`found to be present in the extracts in preliminary experiments.
`This procedure was followed in order to obtain the maximal
`sensitivity consistent with a broad range of detection. The re(cid:173)
`constitution of ATCase was achieved by incubating the mixtures
`of subunits for 30 min at 30°C. In all experiments the labeled
`C subunit was in excess of any unlabeled C subunit in the ex(cid:173)
`tracts, and not more than 50% of the 125I-labeled subunit was
`converted to radioactively labeled ATCase. Unlabeled ATCase
`and C subunit were added as markers and the samples were
`subjected to electrophoresis in 5-cm polyacrylamide gels (5%).
`After electrophoresis the gels were stained and sliced, and the
`distribution of radioactivity in ATCase and C subunit was
`determined.
`Plasmid Construction. Restriction endonucleases were ob(cid:173)
`tained from New England BioLabs and used according to the
`supplier's specifications. The bacterial strain AD llm5, carrying
`the ,\specialized transducing phage ykl4m5 (.\d pyrB argl valS)
`as a prophage (20) was kindly provided by Aki Kikuchi. Bac(cid:173)
`teriophage DNA was isolated by the procedure ofWu et al (21).
`Plasmid DNA was isolated by the alkaline-NaDodS04 extrac(cid:173)
`tion method ofBirnboim and Doly (22), and the DNA was pu(cid:173)
`rified further by centrifugation in a CsCl/ ethidium bromide
`gradient (23).
`A molecular recombinant plasmid containing the pyrB gene
`was constructed by digestion of ykl4m5 and pBR322 (24) DNA
`with the restriction endonucleases Pst I and EcoRI and sub(cid:173)
`sequent ligation of the fragments with phage T4 DNA ligase.
`Plasmids were used to transform strain AT2535 (carrying the
`pyrB59 allele) made competent by treatment with CaC12 (25).
`The resulting transformants were selected as Pyr + tetracycline(cid:173)
`resistant colonies. One isolate was selected and the 7. 4-kilobase
`(kb) plasmid, pDP7, was purified and analyzed by restriction
`endonuclease digestion and electrophoresis in 0. 7% agarose
`gels.
`The size of the DNA fragment containing the pyrB gene was
`reduced by digesting plasmid pDP7 with EcoRI and treating
`the linear DNA with Bal31 double-stranded exonuclease (26)
`for 30 min at 30°C. Incubation of the resulting DNA overnight
`with T4 DNA ligase yielded plasmid pDP8, which was used to
`transform AT2535 cells, selecting for pyrimidine prototrophy.
`Isolates were screened to ensure that they were tetracycline
`sensitive because exonucleolytic digestion of pDP7 from the
`EcoRI site should result in the loss of tetracycline resistance
`(27). The ability of plasmid pDP8 to encode both c and r poly(cid:173)
`peptides was tested as follows: A derivative ofTR3200 (carrying
`the pyrB655 deletion) containing pDP8 was constructed. Cel(cid:173)
`lular extracts were prepared and subjected to electrophoresis
`in 5% polyacrylamide gels. The mobilities of the enzymically
`active proteins were determined by using the activity stain de(cid:173)
`scribed by Bothwell (28). As little. as 1 ng of active protein (ATC(cid:173)
`ase or G subunit) can be detected readily by this procedure.
`Sequence Determination. A DNA fragment approximately
`650 base pairs in length was derived by digestion of pDP8 with
`the restriction endonuclease Msp I (Fig. 1) and was labeled at
`the 5' termini with [ -y-32P]ATP (Amersham, >5,000 Ci/mmol)
`and polynucleotide kinase (P-L Biochemicals). The DNA
`strands were separated according to the procedure of Maxam
`and Gilbert (29). In other experiments the plasmid was digested
`with Bgl II, and the 3' ends were labeled with the large frag(cid:173)
`ment of DNA polymerase (Boehringer Mannheim) and a-
`32P-labeled deoxyribonucleoside triphosphates (Amersham,
`>3,000 Ci{mmol) followed by digestion with Pvu II. There(cid:173)
`sulting 950-base-pair fragment was isolated by polyacrylamide
`gel electrophoresis (29). Nucleotide sequences of the Bgl II/
`Pvu II fragment and both strands of the Msp I ·fragment were
`
`Merck Ex. 1050, pg 1315
`
`

`
`4022
`
`Biochemistry: Pauza et al
`
`Proc. Natl Acad. Sci. USA 79 (1982)
`
`Pst I
`
`pyrB
`
`P~ull
`
`0.5 kb
`
`t
`
`pyr/
`
`II
`
`Jnct
`
`FIG. 1. Structure of region of plasmid pDP8 containing pyrB and
`pyrl. The plasmid was derived from pDP7 (containing pyrB and pyrl
`on an EcoRI/Pst I fragment from the .\ specialized transducing phage
`yk14m5) by linearization of the molecule by EcoRI digestion, treat(cid:173)
`ment with Bal 31 double-stranded uonuclease, and ligation with T4
`DNA ligase. Only the segment containingpyrB and pyrl is shown. The
`junction with pBR322 after Bal31 digestion is indicated by Jnct, and
`the restriction sites utilized to produce fragments for nucleotide se(cid:173)
`quence determinations are shown. Arrows represent the sites cleaved
`by Msp I to yield the fragment containing 650 base pain. The fragment
`produced by Bgl IT/Pvu IT contained 950 base pairs.
`
`determined by the method of Maxam ·and Gilbert (29) with the
`modifications described by Smith and Calvo (30).
`
`RESULTS
`Mapping of Deletion Mutations. Twenty-four pyrB deletion
`mutants have been isolated as part of the construction of a de- .
`tailed recombination map of the pyrB locus; only three are de(cid:173)
`scribed here. These deletions, pyrB740, pyrB748, and.pyrB754,
`were mapped by transductional crosses with point mutations
`whose map positions had been determined independently (31)
`and are shown in Fig. 2; In this map, pyrB554 is the most pro.
`mater-proximal point mutation that has been mapped (31).
`Deletions pyrB740 and pyrB748 fail to recombine with point.
`mutations from pyrB554 through pyrB717. However these two
`deletions recombine with all point mutations from pyrB727
`through the end of pyrB. A pyrB754 strain is able to form Pyr+
`transductants when donors carry point mutations from pyrB554
`through pyrB731, but pyrB754 ·does not recombine with
`pyrB713 or pyrB730. These re!mlts are summarized by the map·
`shown in Fig. 2.
`The deletion pyrB754 extends at least to argF on the F393
`episome because it has simultaneously become Pyr- and tet(cid:173)
`racycline sensitive. (the transposon TnlO in argF of the parent
`strain confers tetracycline resistance). Because of the deletion,
`argF has become 100% linked with pyrB by P22-mediated
`transduction (342 of 342 Pyr+ transductants had become argi(cid:173)
`nine independent), as contrasted with the original cotransduc(cid:173)
`tion frequency of 7% (for pyrB554, only 24 of 342 Pyr+ trans-
`
`<t on on
`
`pyrB
`
`740
`748
`
`pyri
`
`754
`
`FIG. 2. Map of pyrB.deletions. Relevant point mutations from the
`fine structure recombination map of the pyrB gene obtained by three(cid:173)
`factor crosses (31) are presented at the top. Shown below are the three
`partial deletions of pyrB utilized ·in this. work; endpoints are def"med
`by the nearest point mutations. Both pyrB740 and pyrB748 have their
`right endpoints between pyrB717 and pyrB727~ Because pyrB727 re(cid:173)
`combines with pyrB748 at. a lower frequency than with pyrB740, the
`end of deletion pyrB748 is probably closer to pyrB727 than is the end(cid:173)
`point of pyrB740. Deletions pyrB740 and pyrB748 fail to recombine
`with pyrB554, the most promoter-proximal point mutation that has
`been mapped. Deletion pyrB740 continues leftward through the pro(cid:173)
`moter for pyrB, whereas deletion pyrB748 does not (see text). Point
`mutation pyrB231 has been shown to correspond to amino acid residue
`125 in the c polypeptide chain (32). Mutation pyrB754 deletes the car(cid:173)
`boxyl-terminal coding region of pyrB, all of pyrl, and the flanking re(cid:173)
`gion at least to argF. Transcription is from left to right.
`
`Table 2. Production of R subunit in various strains
`
`R
`subunit,•
`Relevant genotype
`Strain
`p.g/mg
`HS2230
`<0.2
`argl2002 pyrB655 pyrH700
`argl2002 pyrB655 pyrH700 /F' pyrB+
`HS2231
`12.9
`HS2255
`<0.2
`argl2002 pyrB655 pyrH700/F' pyrB740
`HS2273
`argl2002 pyrB655 pyrH700/F' pyrB748
`11.7
`HS2278
`<0.2
`argl2002 pyrB655 pyrH700/F' pyrB754
`7.5
`HS2306
`pyrB655 pyrH700/F' pyrB748 ·
`<0.2
`HS2307
`pyrB655/F' pyrB748
`• Amounts Qf R subunit per mg of cell protein in the cell extracts were
`determined by the procedure of Syvanen and Roth (8). Amounts less
`than 0.2 p.g/mg were below the level of detection.
`
`ductants were arginine independent). These results confirm the
`extent of the deletion pyrB754.
`Production of R Subunit in pyrB Deletion Strains. The dif(cid:173)
`ferent strains containing deletions in pyrB were assayed for the
`productionofRsubunit(asdescribedinMaterialsandMethods)
`in order to determine which regions were necessary for pyrl
`expression. Table 2 summarizes the results of these assays in
`terms of the amount of R subunit- in crude .extracts.
`HS2230, carrying only the deletion pyrB655, produced no
`detectable R subunit, as expected from the results of Syvanen
`and Roth (8). In contrast, HS2231, with a normal pyrB gene on
`the episome, produced a large amount of R subunit-because of
`the effect of the pyrH700 mutation (17). Deletions pyrB7 40 and
`pyrB754 eliminated the production of R subunit.* However,
`a large quantity of R subunit was produced in HS2273, which
`carries the deletion pyrB748. This mutation does not interfere
`with the normal control ofR subunit synthesis, as shown by the
`results with HS2307. In this pyrH+ derivative virtually no R
`subunit is detectable. The effect of pyrH700 on the production
`of R subunit in the absence of the pyrB product is consistent
`with the increase observed in ATCase production in pyrB+
`strains caused by pyrH700 (17). The overproduction of R sub(cid:173)
`unit in HS2273 ·is due primarily to the pyrH700 allele rather
`than argl2002, as shown by the comparison of the amounts of
`R subunit in the extracts of HS2306 and HS2307:
`Structure of pDP8. The plasmid pDP8 was mapped by diges(cid:173)
`tion with the Pst I, Bgl II, and Pvu II restriction endonucleases.
`The extent of the Bal 31-induced deletion was estimated by
`digestion with Hin£1 and Taq I, placing the endpoint between
`nucleotides 851 and 1,005 of pBR322 (33). Nucleotide sequence
`determinations (34) show the junction to be at nucleotide 874
`of the pBR322 sequence .(27). The total size· of the deletion. is
`2 kb, indicating that pDP8 is 5.4 kb. Polyacrylamide gel elec(cid:173)
`trophoresis of cellular extracts from a TR3200 derivative con(cid:173)
`taining pDP8 revealed a single band when the gels were stained
`for enzyme activity (ref. 28; see Materials and Methods). The
`protein responsible for the activity had the electrophoretic mo.
`bility of native ATCase rather than free C subunit. Thus the
`ability of pDP8 to direct the synthesis of intact ATCase in the
`TR3200 background was the result of the presence of both pyrB
`and pyrl genes in the plasmid.
`Sequence of the Intercistronic Region. The nucleotide se(cid:173)
`quence of parts of the pyrB and pyrl structural genes and the
`intercistronic region between them is shown in Fig. 3. Begin(cid:173)
`ning with the initiation trinucleotide; ATG, the predicted se-
`* Similar results were obtained for nine additioaal deletions. Five dele(cid:173)
`tions extend in the same direction as pyrB740 with right endpoints
`at five widely separated locations in pyrB. The other four delete dif(cid:173)
`ferent amounts of the promoter-distal end of pyrB.
`
`Merck Ex. 1050, pg 1316
`
`

`
`Biochemistry: Pauza et al
`
`Proc. NatL Acad. Sci. USA 79 (1982)
`
`4023
`
`AI a-Leu-Val- Leu-Asn-Arg-Asp-Leu-Vai-Leu-Stop
`
`Met-Thr-His- Asp-An-Lys-Leu-GI n-Val
`
`GCA-tTG-GTT-CTG-AAT-CGC-GAT-CTG-GTA-CTG-TAAGGGGAAATAGAGATG-ACA-CAC-GAT-AAT-AAA-TTG-CAG-GTT
`
`FIG. 3. Partial nucleotide sequence of the 650-base DNA fragment containing parts of pyrB and pyrl and the intercistronic region. The sequence
`reads from the 5' direction on the left to the 3' direction on the right. Amino acids shown above the DNA sequence represent the carboxyl-terminal
`region of the c chain and the amino-terminal region of the r chain.
`
`quence of amino acids (shown above the DNA sequence in the
`5' -to-3' direction) is identical to the amino terminus of the r
`polypeptide chain determined by Weber (2). The nucleotide
`sequence preceding the termination codon, TAA, is thought to
`constitute the coding region for the carboxyl terminus of the c
`polypeptide chains for the following reasons: First, the terminal
`amino acid corresponding to the trinucleotide CTG is leucine,
`which has been identified as the carboxyl-terminal amino acid
`in the c chains {2). Second, the 650-base-pair Msp I fragment
`contains a nucleotide region encoding the peptide Arg-Leu(cid:173)
`Asp-Pro-Ser-Glu-Tyr-Ala-Asn-Val-Lys, described by Landfear
`et aL {35) as the amino acid residues from positions 207 to 217
`in the c chains. This nucleotide sequence is in the same reading
`frame 192 base pairs from the carboxyl terminus in Fig. 3. Third,
`a DNA fragment adjacent to the Msp I fragment described here
`has a nucleotide sequence {34) that would encode a peptide
`identical to the 38 amino acid polypeptide whose sequence was
`determined by Wall and Schachman {32).
`Although the complete nucleotide sequence of the pyrB gene
`and the amino acid sequence of the c chains are not as yet avail(cid:173)
`able, the evidence cited here is sufficient to warrant the con(cid:173)
`clusion that the nucleotide sequence in Fig. 3 includes regions
`of the pyrB and pyrl structural genes as well as the intercistronic
`region.
`
`DISCUSSION
`Studies aimed at demonstrating an operon encoding the c and
`r chains of ATCase (7, 8) have been hindered by the lack of direct
`information concerning the linkage of the pyrB and pyrl genes
`and the inability to isolate regulatory mutants of the cis-domi(cid:173)
`nant type. From a study of various deletion mutations we have
`established that a region genetically linked to pyrB regulates
`the production of both c and r polypeptide chains in a single
`transcriptional unit. Moreover, the structural organization of
`the pyrB and pyrl genes was determined by analysis of the DNA
`sequence of the intercistronic region.
`The pyrB748 mutation, as seen in Fig. 2, removes a large
`portion of the pyrB gene. Nonetheless there is normal synthesis
`of the r polypeptide. In a pyrB748 strain carrying the unlinked
`pyrH700 mutation there is a large production ofR subunit (Ta(cid:173)
`ble 2) despite the lack of synthesis of C subunit. In contrast, the
`synthesis of R subunit is very low in a pyrB748 strain lacking
`the pyrH700 mutation (strain HS2307). These observations on
`the production of R subunit are very similar to those for R pro(cid:173)
`duction in comparable backgrounds of pyrB+ strains, in which
`the R subunit exists as ATCase {17). We conclude from these
`results that the normal function of the regulatory region for pyrl
`is unaffected by pyrB748 and that possible trans effects of the
`c polypeptide on pyrl expression are highly unlikely.
`As shown in Table 2, both pyrB754 and pyrB740 mutations
`eliminate production of the R subunit. In the case of pyrB754
`the absence of the r polypeptide is attributed to the deletion
`of the pyrl structural gene {Fig. 2). In pyrB740, however, the
`pyrl structural gene is intact. Thus the elimination ofR subunit
`production must be caused by the removal of the promoter of
`the pyrB-pyrl operon in the pyrB740 deletion with the con(cid:173)
`sequent lack of pyrl expression.
`The DNA sequence determination {Fig. 3) reveals that the
`pyrB gene is promoter-proximal to pyrl in E. coli and that pyrB
`
`and pyrl are contiguous genes. These results account for the
`polarity data of Syvanen and Roth {8) for S. typhimurium and
`the observations with the various deletions discussed above.
`Additional sequence information {34) confirms the gene order
`shown in Fig. 1 with the pyrB cistron closer to the Pst I en(cid:173)
`donuclease recognition site and transcription away from that
`site. Because pyrB was isolated on a Pst I/EcoRI fragment from
`the transducing phage ykl4m5 (see Materials and Methods) and
`a 16.4-kb EcoRI fragment of this phage contains both the pyrB
`and argl genes {34), the Pst I end of the cloned fragment must
`be closer to argl. Therefore, as reported for S. typhimurium
`{8), transcription proceeds away from argl, in the so-called
`counterclockwise direction on the E. coli chromosome {36).
`The IS-nucleotide untranslated region separating pyrB and
`pyrl differs from those for trpE-trpD {37) and trpB-trpA {38),
`whose products are needed in stoichiometric amounts to form
`anthranilate synthase and tryptophan synthase, respectively.
`For both of these pairs of genes the intercistronic region con(cid:173)
`tains only two untranslated nucleotides. Oppenheim and Yan(cid:173)
`ofsky {39) suggested that the small intercistronic regions would
`allow "translational coupling" of the genes by a mechanism in
`which one of the ribosomal subunits remains associated with
`the mRNA throughout translation of both structural genes,
`thereby leading to stoichiometric production of the polypep(cid:173)
`tides. In the case of the pyrB-pyrl operon the intercistronic
`region
`
`***** *
`T-A-A-G-G-G-G-A-A-A-T-A-G-A-G
`
`contains six nucleotides {indicated by asterisks) that have a se(cid:173)
`quence complementary to that of the 16S ribosomal RNA con(cid:173)
`sidered to be important in the initiation of translation. This re(cid:173)
`sult suggests that translational coupling probably does not occur
`in the pyrB-pyrl operon by the same mechanism as in the trp
`operon, even though the products of these genes are all needed
`in stoichiometric amounts to form the oligomeric complexes.
`Our findings demonstrate that the pyrB and p.yrl genes are
`contiguous and are transcribed under the control of the same
`promoter. They are separated by a short intercistronic region
`containing the translational terminator for pyrB and a potential
`ribosome binding site for pyrl. These results unambiguously
`establish that pyrB and pyrl make up an operon encoding the
`catalytic and regulatory chains of ATCase. Completion of the
`nucleotide sequence for this operon should yield additional in(cid:173)
`formation on the structure of ATCase and the regulation of its
`synthesis.
`
`We thank Peter Bullock for valuable advice and Lily Wu for excellent
`technical assistance. This work was supported by National Institute of
`General Medical Sciences Research Grant GM 12159 and by National
`Science Foundation Research Grant PCM76-23308.
`
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