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 beta subunit from a lac promoter-rpoB fusion. The overproduced, plasmid-encoded beta subunit assembled into functional RNA polymerase that supplied greater than 90% of the transcriptional capacity of the cells. Excess beta subunit segregated into insoluble inclusion 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 beta subunit into RNA polymerase. One alteration had no discernible effect on assembly or function of the beta subunit; eight others appeared to inhibit assembly but still produced detectable transcriptional activity. Three of these nine alterations produced beta-subunit polypeptides that inhibited cell growth at 32 degrees C, even though they were present in less than 50% of the cell RNA polymerase. When assembled into RNA polymerase, these three altered beta subunits apparently affected essential RNA polymerase functions. Four of the recovered alterations appeared to inhibit completely or almost completely assembly of the beta subunit into RNA polymerase. The results are consistent with a hypothesis that sequences in the first third of the beta-subunit polypeptide are especially important for proper folding and assembly of the beta subunit.
Landick
R
R
Department of Biology, Washington University, St. Louis, Missouri 63130.
Colwell
A
A
Stewart
J
J
D001483:Base Sequence; D004251:DNA Transposable Elements; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D002479:Inclusion Bodies / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation; D012293:Rifampin / Q000494:pharmacology; D013329:Structure-Activity Relationship
D004251:DNA Transposable Elements; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin