Studies on the mechanism of glucocorticoid hormone induced alterations in rat thymic transcription—I. Evidence from reconstituted cross-over transcription assays that sequential increases and decreases in transcription are due to changes in the activity of rna polymerase ii rather than in the activity of chromatin template

Abstract

In experiments to determine the mechanism of glucocorticoid induced decreases in thymic transcription, adrenalectomized rats were injected with hydrocortisone (50 mg/kg) or vehicle. Thymic nuclei were used to prepare chromatins and soluble nuclear extracts containing RNA polymerase II for cross-over experiments. With calf thymus DNA or rat thymic chromatins as templates limiting RNA polymerase II from rats treated with hydrocortisone 3 h previously had 130% of the [ 3H]UMP incorporating activity of RNA polymerase II from control vehicle treated rats. In contrast, limiting RNA polymerase II from rats treated with hydrocortisone 12 h previously had 40–50% of the [ 3H]UMP incorporating activity of RNA polymerase II from controls. When limiting calf thymus DNA or rat thymic chromatins were used in 12 h cross-over experiments. Individual RNA polymerases II produced equal [ 3H]UMP incorporations, but RNA polymerase II activity from hydrocortisone treated rats was again only 50% of control values. Thus with template saturation, RNA polymerase II from hydrocortisone treated rats could not transcribe rat thymic chromatin templates to the level achieved by RNA polymerase II from control rats. This suggests that the activity, rather than the amount, of RNA polymerase II from hydrocortisone treated rats is reduced. Double reciprocal plots of [ 3H]UMP incorporation on rat chromatins with increasing concentrations of RNA polymerases II were made at 12 h. The apparent K m for RNA polymerase II from animals treated with hydrocortisone was identical to that of RNA polymerase II from controls, but the V max of RNA polymerase II from hydrocortisone treated animals was reduced. These data suggest the presence of an inhibitor of transcription or an RNA polymerase II defective in its capacity to initiate and/or elongate RNA transcripts. Further experiments demonstrated that these effects were not due to steroid induced changes in ribonuclease or protease activities.