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1. The two polypeptide chains that comprise aspartate carbamoyltransferase in Escherichia coli are encoded by adjacent cistrons expressed in the order, promoter-leader-catalytic cistron-regulatory cistron (p-leader-pyrBI). 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 repression controls and thus possess the intact control regions. 2. By deleting an internal fragment with specific restriction endonucleases, it was possible to construct shortened fragments which no longer produced the regulatory polypeptide. 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 transcriptional control, the regulatory polypeptide was not required for expression or control of the catalytic cistron. As expected, 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 c3 trimer purified from the native holoenzyme by neohydrin dissociation. 3. Insertion of Mu d1(lac Apr) 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 adjacent to the 5'-terminus of the regulatory cistron and separated by 10-20 bp. 4. DNA sequence analysis of the 5'-proximal regions of pyrBI revealed that an extensive leader sequence separated the promoter and first structural gene pyrB. This leader 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.