Temperature-sensitive Escherichia coli mutant producing a temperature-sensitive sigma subunit of DNA-dependent RNA polymerase.

Abstract

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...

Amino-terminal amino acids modulate sigma-factor DNA-binding activity.

“…The ability of the fusion proteins to rescue ~ mutant phenotypes was assessed by transforming the pGEX derivatives of ~7o into P90A5c (Calendar et al 1988), which carries the temperature-sensitive rpoD285 allele (Harris et al 1978). rpoD285 is identical to rpoD800, as determined by sequence analysis.…”

Regions of the Escherichia coli primary sigma factor σ70 that are involved in interaction with RNA polymerase core enzyme

“…The wild-type j 70 and the tagged derivative were equally functional in vitro (data not shown), and the tagged derivative suppressed the growth defect of the rpoD800 temperature sensitive mutant (Harris et al 1978;Hu & Gross 1983) at a nonpermissive temperature. The j 70 has a naturally occurring HMK recognition site (serine 366 is to be phosphorylated) between the conserved regions 1 and 2.…”

Genetic and physiological studies of Bacillus subtilis sigma A mutants defective in promoter melting

“…For example, growth of cells containing a temperature-sensitive allele of Escherichia coli a0 at the nonpermissive temperature results in an approximately 10-fold drop in the rate of total RNA synthesis (11,13). In contrast, mutants defective in many of the alternative a factors are viable, though often conditionally lethal, and bulk RNA synthesis rates are not grossly altered.…”