A gene affecting the or subunit of DNA-dependent RNA polymerase is tightly linked to dnaG at 66 min on the Escherichia coli chromosome. In order to create an easily selectable marker in this region, we inserted transposon-10, which carries a gene determining resistance to tetracycline (tet) near 66 minm and the order toC-dnaG--tet was determined. We used frequency of cotransduction with tet as a criterion to screen a collection of spontaneous temperature-sensitive Escherichia coli mutants that might affect the a subunit. One such mutant was found to map at the or locus. The a subunit isolated from this mutant is unstable at 460C in vitro and has an altered electrophoretic mobility. The temperature sensitivity of RNA synthesis in this mutant indicates that most transcription in E. coli is cr dependent.The a subunit of Escherichia coli DNA-dependent RNA polymerase is responsible for much of the specificity of in vitro transcription (1, 2). For example, RNA polymerase core enzyme (lacking a subunit) binds weakly to many sites on T7 phage DNA in vitro, whereas RNA polymerase holoenzyme (containing a subunit) binds strongly and only to those sites at which in vivo transcription begins (3).Elucidation of the role of the a protein in the specificity of gene expression has been delayed, however, by the lack of a subunit mutants. Previous genetic studies on the electrophoretic differences among the a proteins of enteric bacteria suggested that the structural gene for a maps at about 66 min on the E. coli genome (4, 5). In order to rapidly screen potential a mutants in this region of the genome, we have inserted the translocatable tetracycline-resistance (tet) element TnlO near the a gene. By using this readily selectable marker for rapid mapping of mutants, we have identified a spontaneous temperature-sensitive (ts) mutant that produces an altered a protein and is deficient in the synthesis of RNA at nonpermissive temperatures.MATERIALS AND METHODS Media, Buffers, and Chromatographic Materials. LB broth (6) supplemented with 0.4% glucose was used; the NaCl concentration was adjusted to 0.1 M. L plates consist of LB broth and 2% Difco agar. LC plates contain, in addition, 5 mM CaCd2 but 1.5% agar. Davis minimal medium is described by Calendar and Lindahl (7). Buffer P50 is described by Gonzalez et al. (8) and TGED buffer is described by Burgess and Jendrisak (9). TPG-CAA medium (10) enriched with 0.8% glucose, 0.01% arginine, 0.5 mM CaCI2, and 20 amino acids and trace metals each at 1 ttg/ml was used for radioactive labeling. Bio-Gel A-1.5m used for gel filtration and the ion-exchange material Bio-Rex 70 were purchased from Bio-Rad. (11), selecting for tetracycline-resistance and the ability to grow at 42'C (dnaG +). This procedure yielded transductants with a tet gene that is highly cotransducible with dnaG. C-2359 is one of these.Purification of RNA Polymerase. RNA polymerase holoenzyme was purified according to the procedure of Burgess and Jendrisak (9). After the enzyme was eluted from the A-1.5m column, i...

The RNA polymerase sigma subunits of Escherichia coli K, E. coli C, and Salmonella typhimurium can be resolved by electrophoresis. Using this technique, we have analyzed Salmonella strains carrying F' plasmids from E. coli K in order to map the gene for the sigma factor. Partial diploid analyses show the location of the sigma gene at 62-66 min on the E. coli genetic map. This gene is cotransducible with toWC and dnaG, at 66 min. The properties of homologous components of the transcription and translation apparatuses often vary between strains of enteric bacteria. Such natural differences provide a convenient tool for genetic mapping of these components when mutants are not available. In order to make use of such differences, investigators individually transfer small regions of the chromosome from one bacterial strain to another strain. The recipients are then screened for the desired phenotype, by using an appropriate assay. With this technique, it has been possible to map some of the genes coding for ribosomal proteins (1) as well as the gene coding for transcription termination factor rho (2). Until now, however, no genetic mapping has been reported for the RNA polymerase (RNA nucleotidyltransferase, nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) sigma subunit, which determines the selectivity of transcription in vitro (3). We have observed electrophoretic differences between the RNA polymerase sigma subunits of Escherichia coli strain K-12 (K), E. coli strain C, and Salmonella typhimurium, and have used these differences to map an E. coli K gene which causes synthesis of the sigma subunit characteristic of E. coli strain K.MATERIALS AND METHODS Chemicals and Buffers. Buffers and DNA-cellulose were prepared as described by Burgess and Jendrisak (4 Bacterial Strains. The bacterial strains used are described in Table 1. New merodiploid strains were constructed by transferring F' plasmids from E. coli K merodiploid strains into various Salmonella typhimurium and E. coli C strains, each containing nutritional mutations such that the presence of the F' plasmid would allow growth on minimal glucose medium. Transfer was accomplished by either of two methods. Method 1: a minimal glucose plate was spread with 0.1 ml (107-108 colony-forming units) of an overnight culture of the donor strain and 0.05 ml of the recipient strain, followed by incubation at 370 for 48 hr (13). Method 2: 0.5 ml samples of each overnight culture were inoculated into flasks containing 10 ml of minimal glucose medium supplemented with the nutritional requirements of each strain. These cultures were shaken at 370 until the optical density of the suspension was doubled. One milliliter of the recipient strain, 2 ml of the donor strain and 3 ml of LB broth were then added to a 250 ml Erlenmeyer flask and shaken very slowly at 370 for 60-90 min. The mating mixtures were centrifuged, concentrated to 10% of the initial volume, and 0.2 ml aliquots were spread on minimal glucose plates, followed by incubation at 370 for 48 hr. C...