Control of transcription at pause and termination sites is common in bacteria. Many transcriptional pause and termination events are thought to occur in response to formation of an RNA hairpin in the nascent transcript. Some mutations in the 13-subunit of Escherichia coli RNA polymerase that confer resistance to the transcription inhibitor rifampicin also alter the response to transcriptional pause and termination signals. Here, we report isolation of termination-altering mutations that do not confer rifampicin resistance and show that such mutations occur predominantly in limited regions of the 13-subunit polypeptide. One region is between amino acid residues 500 and 575, which encompasses the locations of almost all known rifampicin-resistance mutations. Many termination-altering mutations also occur in two other regions: between amino acid residues 740 and 840 and near the carboxyl terminus of the I~-subunit (amino acid residues 1225-1342). Amino acid sequences in these three regions of the 13-subunit are conserved between prokaryotic and eukaryotic 13-subunit homologs. Several mutations that alter transcription termination in vitro affect amino acid residues that are identical in prokaryotic and eukaryotic RNA polymerase 13-subunit homologs, suggesting that they alter an important function common to multisubunit RNA polymerases. We propose that these three regions of the 13-subunit may contact the nascent RNA transcript, the RNA-DNA heteroduplex, or the DNA template in the transcription complex and that mutations in these regions alter transcription pausing and termination by affecting these contacts.

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...

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