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, April 9, 1982 ABSTRACT In both Escherichia coli and Salmonella typhi- (8). In addition, specialized A phages capable oftransducing the murium there is approximately balanced synthesis of the six cat- pyrB gene alsoencode the r chain (11), but the distance between alytic and six regulatory polypeptide chains of the regulatory en- pyrB and pyrI is uncertain. Although the genes for the biosyn- zyme aspartate transcarbamoylase (carbamoylphosphate:L-as- thetic pathway (pyrB, pyrC, pyrD, pyrE, and pyrF) respond partate carbamoyltransferase, EC 2.1.3.2). This control is achieved. in a coordinate manner to uracil starvation (12), they map at by the contiguous pyrB and pyrl genes, which encode the catalytic different positions (13). The increase in ATCase activity, re- and regulatory chains, respectively. Evidence for this single tran- flecting the elevated and balanced expression ofpyrB and-pyrI, scriptional unit was obtained by a study of various deletion mu- is at least 10-fold greater than thatofthe other enzymes (12, 14). tations and from the DNA sequence of the intercistronic region Support for the suggestion that pyrB and pyrI are part ofthe between pyrB and pyrI. One pyrB deletion, pyrB748, produced two independent sets of a normal level ofregulatory chains even though it removed a sub- same transcription unit stems from stantial portion of the pyrB gene. Another deletion, pyrB740, experiments. Perbal and Herve (7) determined the kinetics of shares a similar terminus atone endwithin pyrB, butthe promoter incorporation ofradioactively labeled amino acids into the c and region is removed. In this deletion mutation, there is no produc- r chains during and immediately after uracil starvation, and on tion of the regulatory polypeptide, indicating that a single region the basis of the results they proposed that synthesis of both adjacent to pyrB controls transcription ofpyrI as well. Molecular types of polypeptide chains was directed by a polycistronic cloning and subsequent DNA analysis demonstrated that the pyrB messenger RNA in E. coli. Further evidence for an operon and pyrI genes are contiguous with pyrI as the distal gene in the structure was provided by Syvanen and Roth (8) in their dem- operon. The cistrons are separated by a 15-nucleotide untrans- onstration of the polarity of pyrB chain-terminating mutations lated region containing a sequence capable ofinteracting with the on the synthesis of r chains in S. typhimurium. 16S ribosomal RNA and allowing translation of the pyrI cistron. In order to eliminate uncertainty regarding the organization ofthe pyrB and pyrI genes and the control oftheir expression, It has been known for many years that the allosteric enzyme we have analyzed the effect ofpyrB deletion mutations on the aspartate transcarbamoylase (ATCase; aspartate carbamoyl- synthesis ofthe r polypeptide chains. These studies have shown transferase; carbamoylphosphate.L-aspartate carbamoyltrans- that pyrI is subject to the same transcriptional regulation as ferase; EC 2.1.3.2) from Escherichia coli is an oligomer com- pyrB even in the absence ofthe pyrB product. In addition, we posed ofsix catalytic (c) and six regulatory (r) polypeptide chains have determined the position of the pyrI gene by DNA se- (1-6). Moreover, there is approximately balanced biosynthesis quence analysis of the intercistronic region between pyrB and of the chains in both E. coli (7) and Salmonella typhimurium. pyrL These results establish that the contiguous pyrB and pyrI (8). These observations led to the suggestion that the structural genes constitute a single transcriptional unit encoding the c and genes for the c and r chains (pyrB and pyrI, respectively) are r chains of E. coli ATCase. organized as an operon (8) and that biosynthesis involves a polycistronic messenger RNA (7). However, precise knowledge MATERIALS AND METHODS ofthe location ofthe pyrI gene and definitive evidence thatboth Bacterial Strains and Their Construction. All S. typhimu- genes are subject to the same transcriptional control have not rium strains listed in Table 1 are derivatives of LT2 except been available. TR3200, which was derived from LT7. The pyrH700 mutation ATCase of both E. coli and S. typhimurium is composed of results in a partially defective UMP kinase; as a consequence two trimeric catalytic (C) subunits and three dimeric regulatory the levels of UDP and UTP are reduced, resulting in greatly (R).subunits (1-6, 9, 10). Isolated C trimers ofmolecular weight increased production ofATCase (9, 15-17). Mutation pyrB655 100,000 are catalytically active but insensitive to the feedback is a deletion that removes all ofpyrB. (8). The F393 episome is inhibitor, CTP, and the activator, ATP (1), whereas the free R derived from F128 (F' pro lac) ofE. coli K-12 and contains the dimers of molecular weight 34,000 are devoid of catalytic ac- E. coli pyrB (17). Media, transductional methods, and conju- tivity but still bind both CTP and ATP with high affinity (1). gational transfers were described earlier (17). High-titer phage When C and R subunits are mixed, reconstituted ATCase of P22 lysates for mapping experiments were concentrated 10-fold molecular. weight 300,000 is formed in high yield, and the com- to yield titers ofabout 1011 plaque-forming units/ml; they were plex exhibits the allosteric properties of native ATCase (1). stored in T-2 buffer (18). Several lines of evidence. indicate that the pyrI gene is in deletions in S. close proximity to the pyrB gene. Single typhi- Abbreviations: ATCase, aspartate transcarbamoylase; c, catalytic poly- murium eliminate production ofboth c and r polypeptide chains peptide chain; r, regulatory polypeptide chain; C, catalytic subunit; R, regulatory subunit; Pyr , pyrimidine-independent; kb, kilobase. The publication costs ofthis article were defrayed in part by page charge * Present address: Medical Research Council Laboratory of Molecular payment. This article must therefore be hereby marked "advertise- Biology, Cambridge, England. ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. t To whom reprint requests should be addressed. 4020 Downloaded by guest on September 27, 2021 Biochemistry: Pauza et al. Proc. Natl. Acad. Sci. USA 79 (1982) 4021 Table 1. Bacterial strains found to be present in the extracts in preliminary experiments. Strain Genotype This procedure was followed in order to obtain the maximal sensitivity consistent with a broad range of detection. The re- S. typhimurium constitution ofATCase was achieved by incubating the mixtures HS2230 arg12002 fol-101 leuD798 proAB47pyrB655 pyrH700 of subunits for 30 min at 30TC. In all experiments the labeled HS2231 arg12002 fol-101 leuD798 proAB47pyrB655 pyrH700/ C subunit was in excess of any unlabeled C subunit in the ex- F393 lac+ pro+ argF280 (P22 pyrB+) HS2255 argI2002 fol-101 leuD798 proAB47pyrB655 pyrH700/ tracts, and not more than 50% of the "2I-labeled subunit was F393 lac+ pro' argF282::Tnl0 (P22 pyrB740) converted to radioactively labeled ATCase. Unlabeled ATCase HS2256 argl2002 fol-101 leuD798 proAB47pyrB655 pyrH700/ and C subunit were added as markers and the samples were F393 lac+ pro' argF282::TnlO (P22 pyrB+) subjected to electrophoresis in 5-cm polyacrylamide gels (5%). HS2273 argI2002 fol-101 leuD798 proAB47pyrB655 pyrH700/ After electrophoresis the gels were stained and sliced, and the F393 lac+ pro' argF282::Tnl0 (P22 pyrB748) distribution of radioactivity in ATCase and C subunit was HS2278 argI2002 fol-101 leuD798 proAB47 pyrB655 pyrH700/ determined. F393 lac+ pro+ (P22 pyrB754) Plasmid Construction. Restriction endonucleases were ob- HS2306 fol-101 leuD798 proAB47pyrB655 pyrH700/ tained from New England'Biobabs and used according to the F393 lac+ pro' argF282::TnlO (P22 pyrB748) supplier's specifications. The bacterial strain AD11m5, carrying HS2307 fol-101 leiID798 proAB47pyrB655/ the A specialized transducing phage ykl4m5 (Ad pyrB argl valS) F393 lac+ pro' argF282::TnlO (P22 pyrB748) as a prophage (20) was kindly provided by Aki Kikuchi. Bac- TR3200* amtAl proAB47pyrB655 trp-130 teriophage DNA was isolated by the procedure ofWu et al. (21). Plasmid' DNA was isolated by the alkaline-NaDodSO4 extrac- E. coli tion method of Birnboim and Doly (22), and the DNA was pu- AT2535t pyrB59, argH2, thi-1, his-i, purFl, mtl-2, xyl-7, malAl, rified further by centrifugation in a CsCI/ethidium bromide ara-13, lacYl or lacZ9, rps-18, 9 or 14, tonA2 or ton- gradient (23). 14, tsx-23 or tsx-25, Ar, A-, supE44 A molecular recombinant plasmid containing the pyrB gene * Kindly provided by J. R. Roth. was constructed by digestion ofykl4m5 and pBR322 (24) DNA t Obtained from the E. coli'Genetic Stock Center (New Haven, CT). with the restriction endonucleases Pst I and EcoRI and sub- sequent ligation of the fragments with phage T4 DNA ligase. Isolation and Mapping of Deletion Mutants. Deletion mu- Plasmids were used to transform strain AT2535 (carrying the tants HS2255 (pyrB740), HS2273 (pyrB748), and HS2278 pyrB59 allele) made competent by