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内容記述 |
RNA polymerase core enzyme with the subunit structure of α2ββ' (E) is functionally differentiated into different forms of holoenzyme by interaction with one of multiple molecular species of as subunit, the promoter recognition subunit. Up to now, six species of σ subunit have been identified in Escherichia coli. In order to understand the switching mechanism(s) of transcription by σ replacement in response to changes in growth conditions, such as those during growth phase change or under various stress responses, I compared the functional specificity between two sigma factors, σ70 (the major σ at exponentially growing phase) and σ38 (the essential as at stationary growth phase).<br /> At first, I compared the core enzyme-binding affinity and the promoter-binding activity between two σ subunits by get filtration column chromatography or by titration of the concentration of σ required for the maximum transcription in the presence of a fixed amount of core enzyme. The core enzyme-binding affinity of σ38 was found to be less than half the level of σ70. In addition, the holoenzyme concentration required for the maximum transcription of a fixed amount of templates was higher for σ38 than for σ70. Because the intracellular concentration of σ38 is not higher than that of σ70 even aftcr prolonged starvation in the stationary phase, these results suggest that the selective transcription of stationary-specific genes by Eσ38 holoenzyme may require either a specific reaction condition(s) or a specific factor(s) which enhances either σ38 binding to core enzyme or Eσ38 binding to promoters.<br /> Next, I carried out a systematic analysis of the effect of cellular factors, which vary depending on the cell growth conditions, including salt species, salt concentration, trehalose concentration, and DNA superhelicity, on the promoter recognition by Eσ70 and Eσ38 holoenzymes. The effects of potassium acetate and potassium glutamate, the natural solutes which accumulate in E. coli in response to the increased extracellular osmolarity, were examined in in vitro transcription directed by osmo-regulated promoters (osmB and osmY). The osmB and osmY transcription level increased gradually up to 300 to 400 mM of potassium glutamate but only when they were transcribed by Eσ38. In contrast, transcription at these promoters by Eσ70 decreased with increase in potassium glutamate concentration, illdicating that Eσ38 RNA polymerase itself monitors the intracellular salt concentration and changes its promoter<br />recognition properties.<br /> In the stationary growth-phase and under high osmolarity stress conditions, the intracellular concentration of trehalose increases. I then examined the effect of trehalose concentration on in vitro transcription by Eσ70 and Eσ38 holoenzymes. The optimum trehalose concentration for maximum transcription by Eσ38 was high, i.e., around 0.7 to 1.2 M. In contrast, the optimum trehalose concentration for maximum transcription by Eσ70 was lower, i.e., around 0.5 to 0.7 M. This enhancement of Eσ38 activity by high concentrations of trehalose was found to be due to the stimulation or<br />stabilization of Eσ38 holoenzyme formation.<br /> The superhelicity of chromosomal DNA in bacterial cells decreases in the stationary growth-phase and/or under the nutrient starvation conditions. I then examined the effect of DNA superhelicity on in vitro transcription by Eσ70 and Eσ38 holoenzymes. The optimum superhelical density for maximum transcription by Eσ38 was low, i.e., around 0 to -0.03, whereas Eσ70 required high levels of the DNA superhelicity. Moreover, the optimum superhelical density for maximum transcription in vitro by Eσ38 was almost the same as that of plasmids prepared from stationary-phase E. coli cells. The enhancing effects of high potassium glutamate concentration and low DNA superhelicity, and of high trehalose concentration and low DNA superhelicity on transcripthion by Eσ38 were additive, but those of potassium glutamate and trehalose were not additive.<br /> Taken all these results together, I propose the switching mechanism of RNA polymerase specificities as follows: i) transcription by Eσ38 is specifically enhanced under high concentrations of potassium glutamate or high concentrations of trehalose; ii) the low superhelicity of chromosomal DNA provides templates suitable for Eσ38. These specific conditions that enhance Eσ38 activities are in good agreement with the intracellular situation in E. coli cells growing under high osmolarity or starved conditions. |
要旨・審査要旨 / Abstract, Screening Result (297.0 kB)
