Expression of the pyrBI operon of Escherichia coli K-12, which encodes the subunits of the pyrimidine biosynthetic enzyme aspartate transcarbamylase, is negatively regulated by the intracellular levels of UTP. Previous experiments suggested a unique model for regulation of operon expression in which low UTP levels cause close coupling of transcription and translation of the pyrBI leader region. This close coupling suppresses transcriptional termination at an attenuator preceding the structural genes. In this study, we examined the regulatory role of translation and attenuation in operon expression. To determine whether the leader region is translated, we constructed a plasmid, designated pBHM17, in which the pyrBI promoter(s) and the first 11 codons for a putative 44-amino acid leader polypeptide are fused to codon 9 of lacZ. A transformant carrying this plasmid synthesized a beta-galactosidase fusion protein with the amino-terminal sequence of the leader polypeptide, demonstrating that the signals required for leader polypeptide synthesis function in vivo. Synthesis of the fusion protein was nearly insensitive to pyrimidine availability. In uracil-grown cells, the level of fusion protein synthesis encoded by plasmid pBHM17 was much greater than that encoded by a similar plasmid containing a pyrB::lacZ gene fusion, in which the pyrBI promoter-regulatory region is intact. These results indicate that the downstream leader sequence which includes the attenuator is required for regulation and functions as a transcriptional barrier. Oligonucleotide-directed mutagenesis was used to change the ATG leader polypeptide initiation codon of the intact pyrBI operon to ACG, which was shown to strongly inhibit translational initiation. This mutation greatly reduced operon expression and regulation as predicted by the attenuation control model.