Aurintricarboxilic acid as a tool for investigating the template-bound and unbound forms of RNA polymerase I in permeabilized cells

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Abstract
The presence of template-bound and unbound RNA polymerase I in permeabilized cells was investigated. The two enzyme forms were defined on the basis of their different susceptibilities towards aurintricarboxilic acid (ATA). It was found that addition of ATA to permeabilized cells suppresses initiation of new RNA chains by RNA polymerase I but has no effect on the activity of the enzyme already engaged in transcription. This last activity is not affected even after washing the permeabilized cells for removal of the ATA. The RNA polymerase I activity solubilized from permeabilized cells pre-treated with ATA is 60–70% of that obtained from non-treated controls. The decrease of the solubilized enzyme activity was observed after purification of the enzymes by DEAE-Sephadex columns and cannot be attributed to the presence of inhibitory or activating factors in the enzyme preparations. The simplest interpretation of these findings is that two distinct RNA polymerase I fractions, showing different sensitivities towards ATA are present in permeabilized cells. These fractions should represent the template-bound and unbound RNA polymerase I. The results also show that the amount of ATA-insensitive activity is lower in nuclei than in permeabilized cells, suggesting that detachment of template-bound enzyme occurs during nuclei isolation.