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J. Biol. Chem., Vol. 265, Issue 27, 16244-16247, 09, 1990

Hormonal regulation of transcription of rDNA. The role of TFIC in formation of initiation complexes

PB Mahajan, PK Gokal and EA Thompson
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77550.

Glucocorticoids inhibit transcription of rDNA in P1798 lymphoma cells. This observation can be recapitulated in vitro in that extracts from hormone-treated cells are virtually incapable of transcribing from the cloned mouse rRNA promoter. However, such extracts can be reconstituted by addition of a RNA polymerase I transcription factor, called TFIC. TFIC has been purified to homogeneity. Assays have been developed which facilitate analysis of various aspects of initiation of transcription by RNA polymerase I in vitro. This paper describes a series of experiments designed to test two related hypotheses. It is proposed that TFIC is a bona fide initiation factor and that the inability of hormone-treated cells to synthesize rRNA is due to failure to form initiation complexes on rDNA. The data indicate that extracts from hormone-treated cells cannot form KCl or heparin-resistant initiated complexes upon the rRNA promoter. The ability to form such complexes is dependent upon the addition of TFIC. The lack of TFIC precludes formation of the first phosphodiester bond. At low concentrations of TFIC there is a more or less direct relationship between the amount of the factor and the number of initiated complexes formed. At higher concentrations, the system saturates and addition of TFIC beyond 80 pg/microliters (approximately 0.5 nM) has no effect upon initiation. Addition of TFIC to control extracts does not influence the formation of initiated complexes. This is consistent with the conclusion that control extracts contain excess TFIC, whereas hormone-treated extracts are depleted in this respect. The kinetics of reconstitution have been examined, and the results suggest that association of highly purified TFIC with the transcriptional apparatus is a relatively slow process, with a t1/2 of about 2 min. The data are consistent with the hypothesis that TFIC is an initiation factor and suggest that the active form of RNA polymerase I is associated with TFIC. It is proposed that in the absence of this association, initiation of transcription of rDNA does not occur.
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