Inhibition of DNA gyrase activity in an in vitro transcription-translation system stimulates gyrA expression in a DNA concentration dependent manner. Evidence for the involvement of factors which may be titrated.
Stimulation of gyrA expression in an in vitro transcription-translation system by novobiocin, a DNA gyrase inhibitor, depends on the DNA concentration in the extract. At low DNA concentrations (less than 20 micrograms/ml) we note significant stimulation (2 to 25 x) upon the addition of novobiocin; at high DNA concentrations, stimulation is minimal. This observation is not due to the limited capacity of the system to transcribe or relax DNA. Using an extract prepared from a novobiocin-resistant strain of Escherichia coli, we were able to show that DNA gyrase mediates the novobiocin-enhanced expression. In an experiment with a fixed level of a gyrA-lac template, we found that the addition of a second non-Lac template increased expression from the gyrA-lac template, while concomitantly decreasing the extent of novobiocin stimulation. These observations are consistent with an inhibitory factor that can be titrated by increasing the DNA concentration and whose effects are minimized when the DNA template is in a relaxed conformation. The results of a mixed-extract experiment using two extracts that differ in their activities and degrees of novobiocin stimulation are also consistent with an inhibitory factor that mediates the relaxation-induced stimulation of transcription.[1]References
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