Synthesis of aspartate transcarbam Transcriptional regulation of the pnh

The first committed reaction in pyrimidine biosynthesis in Escherichia coli and Salmonella typhimurium is catalyzed by the allosteric enzyme aspartate transcarbamoylase (aspartate carbamoyltransferase; carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2), the product of the pyrB-pyrl operon. Regulation of the pyrimidine pathway is achieved in part by changes in the enzyme's catalytic activity as a function of the concentration of substrates and other metabolites as well as by variations in enzyme synthesis in response to changes in cellular levels of pyrimidine nucleotides. Although there is substantial evidence that UTP concentration has a marked influence on expression of the pyrB-pyrl operon, the mechanism of this control is not known. We have cloned the operon and determined the nucleotide sequence of the region preceding the first structural gene (pyrB). These studies show two regions sharing considerable homology with the consensus sequence of E. coli promoters, a segment that can code for a 44-amino-acid leader peptide, and a sequence very similar to that of the attenuator of the trp operon. RNA transcripts from several bacterial strains were studied by Sl nuclease mapping. Under conditions leading to extensive enzyme synthesis there was a large production of transcript whose 5' end correlated with the putative promoter closer to the structural genes. At low levels of operon expression there was little transcript in the extracts and both promoters appeared to serve as initiation sites. The results are interpreted in terms of transcriptional control of the pyrB-pyrl operon according to an attenuation model that differs in novel ways from the mechanisms proposed for the regulation of amino acid biosynthesis. Pyrimidine synthesis in Escherichia coli and Salmonella typhimurium is regulated in part by the allosteric enzyme aspartate transcarbamoylase (ATCase; aspartate carbamoyltransferase; carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2), which catalyzes the first committed step in the biosynthetic pathway. Control is achieved in several ways. The enzyme itself regulates the formation of carbamoyl aspartate through the sigmoidal dependence of catalytic activity on the concentration of both substrates (1, 2). Also, ATCase is inhibited by CTP, the end product of the pyrimidine pathway, and activated by ATP (1). In addition to the regulation provided by the allosteric properties of ATCase, control is achieved at the level of protein synthesis. ATCase production is relatively low at high levels of UTP, a product of the pyrimidine biosynthetic pathway, whereas enzyme synthesis is increased as much as 150fold when the cellular concentration of UTP is very low (3). The mechanism of regulation of the synthesis of ATCase is not known. The catalytic and regulatory chains of E. coli ATCase are encoded by the pyrB and pyrI genes, respectively (4). Recent The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. ?1734 solely to indicate this fact. 120oylase in Escherichia coli: rB-pyrI operon versity of California, Berkeley, California 94720 studies of deletion mutants and molecular cloning experiments followed by nucleotide sequence analysis showed that the pyrB and pyri genes constitute an operon with the two contiguous genes separated by a 15-nucleotide, untranslated, intercistronic region (4). One pyrB deletion produced a normal amount of regulatory chains even though a substantial portion of the pyrB gene was removed. Another deletion, which shares a similar end point within pyrB, produced no regulatory polypeptide chain because of the removal of the promoter region (4). These results indicated that a single region adjacent to pyrB controls transcription of both pyrB and pyrI. Recently Roof et al. (5), on the basis of their nucleotide sequence determination of the promoter region, suggested that attenuation and other "overlapping" regulatory mechanisms were implicated in the control of the pyrB-pyrI operon. In an independent study, Turnbough et al. (6) determined the sequence of the 620 nucleotides preceding the pyrB structural gene. Also, they demonstrated by in vitro transcription experiments the presence of a UTP-dependent pause site and interpreted their results in terms of attenuation control (6). We have determined the nucleotide sequence of the promoter region from two genetically distinct sources of DNA and have characterized the in vivo transcripts from strains in which the intracellular levels of pyrimidine nucleotides were varied. The attenuator model presented here is similar to that of Turnbough et al. (6) and is analogous to those proposed for the regulation of operons involved in amino acid biosynthesis (7, 8). MATERIALS AND METHODS Bacterial Strains and Media. Both E. coli and S. typhimurium strains were used in these studies. The episome F393 argF lac proAB (P22 pyrB) was derived from specialized transduction of pyrB into F128 argF lac proAB of E. coli K-12 (9). F393 carries the intact pyrB-pyrI operon and its regulatory elements (4). Mutation pyrH700 encodes a partially defective UMP kinase. Strains carrying this mutation have decreased levels of UDP and UTP and as a result overproduce ATCase (10, 11). Mutation pyrB655 is a deletion that removes all of pyrB (12). Because pyrB655 and pyrH700 are available only in S. typhimurium, we have employed a hybrid organism in which the pyrB-pyrI operon from E. coli is present in S. typhimurium (4, 9, 13). The E. coli strain AT2535 carrying the pyrB59 allele (4) was used in the selection oftransformants maintaining pyrB+ plasmids. When uracil was used in the growth medium for the various bacterial strains, the concentration was 20 ,g/ml. Plasmid Construction. Conditions for restriction endonuclease digestions and ligation reactions, as well as plasmid purification and transformation techniques, were described earlier Abbreviations: ATCase, aspartate transcarbamoylase; kb, kilobase(s); bp, base pair(s). * To whom reprint requests should be addressed. This content downloaded from 157.55.39.78 on Mon, 20 Jun 2016 07:22:26 UTC All use subject to http://about.jstor.org/terms 1208 Biochemistry: Navre and Schachman