The molecular organization and functional characteristics of the PAD1 replicon-encoded par stability determinant were examined. par encodes two convergently transcribed RNAS of approximately 210 and 65 nucleotides designated RNA I and RNA II, respectively. The sequence of RNA II is largely complementary to RNA I, suggesting that RNA II could regulate RNA I function as an anti-sense RNA. Results of functional studies are consistent with a role for par as a post-segregational killing system, the first to be identified in Gram-positive bacteria, with RNA I encoding the toxin and RNA II the antidote. These results include: (i) destabilization of par-containing replicons in the presence of a second complete par or the RNA II coding sequence in the same cell; (ii) par-dependent stabilization of a highly unstable vector at the expense of host-cell growth rate; and (iii) protection of cells from the toxic effects of overexpression of RNA I by RNA II supplied in trans.

A plasmid was constructed that overproduces the Escherichia coli RNA polymerase 0i subunit from a lac promoter-rpoB fusion. The overproduced, plasmid-encoded , subunit assembled into functional RNA polymerase that supplied greater than 90% of the transcriptional capacity of the cells. Excess ,B 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 , subunit into RNA polymerase. One alteration had no discernible effect on assembly or function of the 0 subunit; eight others appeared to inhibit assembly but still produced detectable transcriptional activity. Three of these nine alterations produced ,B-subunit polypeptides that inhibited cell growth at 32C, even though they were present in less than 50% of the cell RNA polymerase. When assembled into RNA polymerase, these three altered ,B subunits apparently affected essential RNA polymerase functions. Four of the recovered alterations appeared to inhibit completely or almost completely assembly of the ,B subunit into RNA polymerase. The results are consistent with a hypothesis that sequences in the first third of the 1-subunit polypeptide are especially important for proper folding and assembly of the 13 subunit.The 1 subunit of Escherichia coli RNA polymerase is an attractive target for systematic analysis of structure-function relationships in the transcription complex. The 1 (Mr, 151,000), 1' (Mr, 155,000), and two a (Mr, 37,000) subunits constitute core RNA polymerase, an arrangement that is conserved among eubacteria (12, 72). Promoter specificity is conferred on core by a family of or subunits (58); in E. coli, a70 (Mr,70,000) is the major a factor. 13 and 13' are encoded by the rpoB and rpoC genes, which are located contiguously at 90 min in a polycistronic operon that includes genes for four ribosomal proteins (14,33,37,49,68). a and U70 are encoded by the unlinked rpoA (73 min) and rpoD (67 min) genes, respectively, which also are in operons encoding ribosomal components (22,27,42). All four genes have been cloned, sequenced, and verified by conditional lethal mutations to be essential for RNA polymerase function in vivo (2,5,11,32,(54)(55)(56)59).The 13 subunit is intimately involved in most of the known activities of procaryotic RNA polymerase and contains significant sequence similarities to the second-largest subunit of yeast and insect RNA polymerase II (13, 64). Mutations in rpoB (predominantly between codons 500 and 575) confer resistance to the transcription inhibitors rifampin and streptolydigin (28,36, 57). Jin and Gross have determined the genetic alterations of 17 different rifampin-resistant (Rif) mutants (28); many of them either increase or decrease RNA hairpin-dependent transcription pausing (15), rho-independ...