Structure and expression of a Complex operon : pyrLBIX of Escherichia coli

Typescript (photocopy).Aspartate transcarbamoylase from Escherichia coli has been the subject of extensive analyses to determine the kinetic and physico-chemical characteristics of this hetero-multimeric enzyme which is known to be regulated by end product feedback inhibition and substrate cooperativity. The three dimensional structure with proper placement of protein secondary structures, as determined by x-ray crystallography, has been hampered by the lack of a complete amino acid sequence. Reported herein is the nucleotide sequence of the genes which encode the catalytic (pyrB) and regulatory (pyrI) polypeptides of ATCase, from which the deduced amino acid sequence has been used to confirm and correct a previously determined amino acid sequence obtained by conventional chemical modification and cleavage techniques on overlapping peptides. Also reported is the nucleotide sequence of a newly discovered gene, designated pyrX, found adjacent to the genes which encode ATCase. Including the sequence for the previously reported gene coding for a leader polypeptide, the structure of the operon is: promoter, leader, catalytic, regulatory, and the unknown (X) gene. Expression of this operon is regulated by attenuation in the leader coding sequence at a rho-independent terminator. Those features proposed to participate in coupling between RNA polymerase and the leading ribosome, whereby attenuation is overcome, have been examined by several methods, including deletional analysis and insertion of portions of the leader into lacZ. Three key factors are shown to control expression of the pyrBI operon. Firstly, the rate of transcription through the leader sequence prior to the attenuator is sensitive to the concentration of UTP. Secondly, the rate of translational initiation at an inefficient Shine-Dalgarno site delays polypeptide synthesis of the leader so that coupling occurs as a result of pausing by RNA polymerase. Thirdly, a ribosome stalled at two infrequently used arginine codons at the extreme end of the leader coding sequence prevents translational initiation at the pyrB Shine-Dalgarno, rendering a measure of control by the concentration of arginine, a product of the pathway which shares the substrate carbamoyl phosphate with ATCase