INSERTIONAL MUTAGENESIS OF A PLASMID-BORNE ESCHERICHIA-COLI RPOB GENE REVEALS ALTERATIONS THAT INHIBIT BETA-SUBUNIT ASSEMBLY INTO RNA-POLYMERASE

A plasmid was constructed that overproduces the Escherichia coli RNA polymerase β subunit from a lac promoter-rpoB fusion. The overproduced, plasmid-encoded β subunit assembled into functional RNA polymerase that supplied greater than 90% of the transcriptional capacity of the cells. Excess β subunit segregated into insoluble bodies and was not deleterious to cell growth. By insertion of a XhoI linker sequence (CTCGAG) and accompanying deletion of variable amounts of rpoB sequences, 13 structural alterations were isolated in the first and last thirds of the plasmid-borne rpoB gene. Twelve of these alterations appeared to reduce or prevent assembly of plasmid-encoded β subunit into RNA polymerase. One alteration had no discernible effect on assembly or function of the β subunit; eight others appeared to inhibit assembly but still produced detectable transcriptional activity. Three of these nine alterations produced β-subunit polypeptides that inhibited cell growth at 32°C, even though they were present in less than 50% of the cell RNA polymerase. When assembled into RNA polymerase, these three altered β subunits apparently affected essential RNA polymerase functions. Four of the recovered alterations appeared to inhibits completely or almost completely assembly of the β subunit into RNA polymerase. The results are consistent with a hypothesis that sequences in the first third of the β-subunit polypeptide are especially important for proper folding and assembly of the β subunit.