@misc{oai:ir.soken.ac.jp:00000946, author = {寺社下, 美樹 and ジシャゲ, ミキ and JISHAGE, Miki}, month = {2016-02-17, 2016-02-17}, note = {Selection of a σ subunit of RNA polymerase is a powerful mechanism for switching transcription of a group of genes. For understanding the regulation of σ subunit utilization in Escherichia coli, I determined the intracellular levels of four members among 8 known σ subunits of E. coli strains, W3110 and MC4100, at various growth phases by a quantitative Western immunoblot analysis. The level of σ⁷⁰ is rather constant throughout the growth, and those of σ⁵⁴ and σ²⁸ are maintained at 10 and 50% the level of σ⁷⁰ in both strains growing at exponential and stationary phases, respectively. At the exponential growth phase σ³⁸ is undetectable but increases to up to 30% of σ⁷⁰ at the stationary phase, supporting the concept that σ³⁸ plays a key role in transcription of the stationary phase - expressed genes. Stress - coupled change in the intracellular level was observed for two σ subunits: an increase in σ³⁸ level and a decrease in σ²⁸ level upon exposure to heat shock at the exponential phase; and the increase in σ³⁸ level under a high osmolality condition at both the exponential and the stationary phases. I also found that the composition of σ subunits is different between bacterial strains, even under the same strain name. For instance, the compositions of σ²⁸ and σ³⁸ differ among three W3110 strains.
The growth transition from exponential to stationary phase is accompanied by the replacement of RNA polymerase - associated σ⁷⁰ subunit with σ³⁸. She found that the GST - fused σ⁷⁰ in stationary - phase cell extracts exists as a complex with a new protein, and it was designated Rsd (Regulator of Sigma D). A gene located at 90 min on the E. coli chromosome encodes Rsd, and is transcribed from upstream and downstream promoters with σ³⁸ and σ⁷⁰ holoenzymes, respectively. Over - expressed and purified Rsd protein can form a complex with σ70 in vitro but not with any other σ subunits; σ⁵⁴, σ³⁸, σ³², σ²⁸ and σ²⁴. A binding assay with proteolytic fragments of σ⁷⁰ suggested that Rsd interacts with the conserved region 4, the promoter - 35 recognition domain, and its downstream of σ⁷⁰. The isolated Rsd inhibited transcription in vitro to various extents depending on the promoters used. The intracellular level of Rsd starts to increase during the transition from growing to stationary phase.
Experiments using strains with disrupted rsd gene and those with overproduced Rsd indicated that Rsd is a negative factor for transcription directed by σ⁷⁰ and a positive factor for that by σ³⁸. The results indicate that Rsd is a stationary - phase E. coli protein regulating the activity of the σ⁷⁰ function, providing an evidence for the existence of the control of the activities as well as the levels of the σ subunits of RNA polymerase, contributing to a group switching of genes ofE. coli. Taking all the observations together, she discussed possible models for the switching mechanism., application/pdf, 総研大乙第64号}, title = {Control of the Synthesis and Functions of RNAPolymerase Sigma Subunits in Escherichia coli}, year = {} }