Regulation of aspartate transcarbamoylase synthesis in Escherichia coli: analysis of deletion mutations in the promoter region of the pyrBI operon

Abstract

The catalytic and regulatory polypeptide chains of Escherichia coli aspartate transcarbamoylase are encoded by the pyrB and pyrI genes, respectively, which constitute a single transcriptional unit in the pyrBI operon. The DNA sequence immediately preceding the first structural gene, pyrB, contains a short open reading frame that could encode a 44-amino acid leader peptide and a (G+C)-rich region of dyad symmetry followed by eight thymidine residues. Synthesis of the enzyme is negatively controlled at the level of transcription depending on the cellular level of UTP, and an attenuation mechanism has been proposed to account for the 70-fold increase in pyrBI expression on pyrimidine starvation. The potential role of the dyad and eight thymidines as an attenuator was tested with a plasmid containing the promoter region of the pyrBI operon upstream of the galK coding sequence. When cells containing this plasmid, pPYRB10, were grown in a medium low in uracil, there was an 83-fold increase in galactokinase activity compared with the same cells grown at high uracil levels. This regulation is similar to that for aspartate transcarbamoylase synthesis in cells depleted of pyrimidines. Deletions constructed in the promoter region of pPYRB10 from the 3' side produced one plasmid that retained normal control of galK expression and five that exhibited greatly reduced regulation. Nucleotide sequence determination showed that the one deletion mutation that was functionally similar to the wild-type plasmid contained the entire region of dyad symmetry, including the eight thymidines. The plasmids with more extensive deletions lacked the region with dyad symmetry and the eight thymidines. One of the deletion mutants that exhibited very low levels of regulation lacks the entire sequence coding for the putative leader peptide up to the major promoter. The results demonstrating the crucial role of a 19-nucleotide sequence (from -33 to -15) support an attenuation model but indicate that other mechanisms also contribute to the regulation of the pyrBI operon.