Studies on the structure and expression of Escherichia coli pyrC, pyrD, and pyrF using the cloned genes

Abstract

The Escherichia coli pyrC, pyrD and pyrF genes were cloned on multicopy plasmids derived from pBR322 and analysed by means of restriction endonucleases. It was found that the pyrC gene is destroyed by cutting with the restriction endonuclease BamHI, that the entire pyrD gene can be isolated on a 1300-base pairs DNA fragment generated by EcoRI cleavage and that cutting with EcoRI removes the promotor and probably also the translational start site from the pyrF gene. More details on the restriction maps are presented. Further, it was found that the presence of a pyr gene in multiple copies on a plasmid does not significantly interfere with the activity of the chromosomal pyr genes. Using the 'minicell' technique, the polypeptides encoded by the three cloned pyr genes were identified. The relative molecular masses for the pyrC-encoded and pyrD-encoded polypeptides are 38 000-40 000 and 36 000-38 000, respectively. Thus in their native form, dihydroorotase and dihydroorotate oxidase appear to be dimeric proteins. The 'minicell' experiments positively identified a protein chain of Mr 23 000-24 000 as being a subunit of OMP decarboxylase encoded by pyrF. Moreover, the coding frame for this polypeptide seems to be expressed as the first gene in the operon with the coding frame for another protein chain of Mr 13 000-14 000. Since, however, the native OMP decarboxylase during sedimentation and gel filtration behaves as a protein of Mr 45 000 +/- 4000, this latter polypeptide (Mr 13 000-14 000) is hardly a component of the enzyme. Pyr-lac+ operon fusions were constructed by the Mu d1 procedure. By integrating an F'lac episome into the lac part of the fusions and determining the direction of chromosomal transfer from the resultant Hfr strains, the direction of pyrC transcription was found to be counter-clockwise, while pyrD and pyrF were found to be transcribed in a clockwise direction.