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内容記述 |
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 σ<SUP>70</SUP> is rather constant throughout the growth, and those of σ<SUP>54</SUP> and σ<SUP>28</SUP> are maintained at 10 and 50% the level of σ<SUP>70</SUP> in both strains growing at exponential and stationary phases, respectively. At the exponential growth phase σ<SUP>38</SUP> is undetectable but increases to up to 30% of σ<SUP>70</SUP> at the stationary phase, supporting the concept that σ<SUP>38</SUP> 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 σ<SUP>38</SUP> level and a decrease in σ<SUP>28</SUP> level upon exposure to heat shock at the exponential phase; and the increase in σ<SUP>38</SUP> 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 σ<SUP>28</SUP> and σ<SUP>38</SUP> differ among three W3110 strains.<br /> The growth transition from exponential to stationary phase is accompanied by the replacement of RNA polymerase - associated σ<SUP>70</SUP> subunit with σ<SUP>38</SUP>. She found that the GST - fused σ<SUP>70</SUP> 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 σ<SUP>38</SUP> and σ<SUP>70</SUP> holoenzymes, respectively. Over - expressed and purified Rsd protein can form a complex with σ70 in vitro but not with any other σ subunits; σ<SUP>54</SUP>, σ<SUP>38</SUP>, σ<SUP>32</SUP>, σ<SUP>28</SUP> and σ<SUP>24</SUP>. A binding assay with proteolytic fragments of σ<SUP>70</SUP> suggested that Rsd interacts with the conserved region 4, the promoter - 35 recognition domain, and its downstream of σ<SUP>70</SUP>. 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.<br /> Experiments using strains with disrupted rsd gene and those with overproduced Rsd indicated that Rsd is a negative factor for transcription directed by σ<SUP>70</SUP> and a positive factor for that by σ<SUP>38</SUP>. The results indicate that Rsd is a stationary - phase E. coli protein regulating the activity of the σ<SUP>70</SUP> 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. |
要旨・審査要旨 / Abstract, Screening Result (259.9 kB)
