Abstract
The delta protein is a dispensable subunit of Bacillus subtilis RNA polymerase (RNAP) that has major effects on the biochemical properties of the purified enzyme. In the presence of delta, RNAP displays an increased specificity of transcription, a decreased affinity for nucleic acids, and an increased efficiency of RNA synthesis because of enhanced recycling. Despite these profound effects, a strain containing a deletion of the delta gene (rpoE) is viable and shows no major alterations in gene expression. Quantitative immunoblotting experiments demonstrate that delta is present in molar excess relative to RNAP in both vegetative cells and spores. Expression of rpoE initiates from a single, sigmaA-dependent promoter and is maximal in transition phase. A rpoE mutant strain has an altered morphology and is delayed in the exit from stationary phase. For biochemical analyses we have created derivatives of delta and sigmaA that can be radiolabeled with protein kinase A. Using electrophoretic mobility shift assays, we demonstrate that delta binds core RNAP with an apparent affinity of 2.5 x 10(6) M-1, but we are unable to demonstrate the formation of a ternary complex containing core enzyme, delta, and sigmaA.
Highlights
RNA polymerase (RNAP)1 from bacteria consists of a multisubunit enzyme complex that is the main target for the regulation of gene expression
Primer extension analysis (Fig. 1B) with RNA purified from vegetatively growing cells and from in vitro transcription assays using B. subtilis RNA polymerase containing A shows that transcription starts with approximately equal frequency at either of two purine residues located 84 and 86 base pairs from the translation start site
Other strains in which the fusion was placed at the SP locus, either in wild type or in an rpoE mutant, produced essentially identical results
Summary
RNA polymerase (RNAP) from bacteria consists of a multisubunit enzyme complex that is the main target for the regulation of gene expression. A vast array of other proteins makes contacts with RNAP at one or more steps of the transcription cycle and modifies its activities in various ways [1] Such ancillary polypeptides modify RNAP during the promoter binding, initiation, elongation, and termination steps to effect changes in gene expression. The carboxyl-terminal region is highly acidic, consisting mostly of aspartate (37%), glutamate (34%), and hydrophobic residues (25%) and is essential for displacing RNA bound to RNA polymerase [14] This RNA displacement activity may explain our previous observation that ␦ enhances RNAP recycling in multiple cycle transcription reactions, stimulating overall transcription [13]. We describe the expression of the rpoE locus, the phenotype of an rpoE deletion mutant, and preliminary biochemical analysis of the interactions between ␦ and RNAP
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