Accurate initiation of rat ribosomal RNA gene transcription using a fractionated nuclear extract from normal liver and a hepatoma.

Abstract

Nuclear extracts obtained from normal rat liver and from Morris hepatoma 3924A were fractionated by DEAE-Sephadex chromatography. The fraction eluted with 175 mM (NH4)2SO4 (DE-B), which contains >90% of RNA polymerase I activity, supported accurate transcription of cloned rat rDNA. A similar fraction obtained from the cytosol had all of the factors required for rDNA transcription. However, its transcriptional activity was at most one-sixth that of the corresponding nuclear fraction, as determined by the amount of protein needed to produce a similar quantity of the transcript. Unfractionated nuclear or cytosol preparations did not yield an accurate transcript. Optimal KCI and magnesium concentrations for rDNA transcription were 60 mM and 5-7.5 mM, respectively. The extent of transcriptional activity was in the following order: hepatoma nuclear fraction DE-B > whole cell extract derived from rat mammary adenocarcinoma cells >> normal liver fraction DE-B. The hepatoma preparation produced at least 10 times the amount of transcript produced by the corresponding liver nuclear preparation. Transcriptional activity was proportional to the levels of RNA polymerase I and to the rate of rRNA synthesis in these tissues.The development of cell-free systems (1-3) has facilitated studies on the transcription of specific genes by class I, II, and III RNA polymerases (reviewed in refs. 4 and 5). These systems have provided the means to investigate the promoter regions for several genes and to identify the transcriptional regulatory factors. Either a whole-cell extract (3) or an S-100 fraction (2) has been used for these studies. A major disadvantage with cell extracts is the large number of cells needed to identify and characterize factors involved in the transcriptional process (6). This problem could be circumvented if tissue extracts could be prepared that would accurately transcribe purified genes by specific RNA polymerases. Methods that are used for preparing whole-cell extracts have not been extended to tissues probably because of the relatively higher ribonuclease levels in tissues.Since transcription occurs in the nucleus, most factors required for transcription must be found in this organelle. Consequently, a transcriptional system derived from the nuclei will have a distinct advantage over other systems. Over many years, the enzymology and subunit structure of RNA polymerase I and the modifications of its activity and/or amount in response to physiological and pathological stimuli have been studied in our laboratory (7-13). Recently, we have initiated studies on the identification of accessory factors required for transcription of rDNA by class I RNA polymerase. In the course of these studies, we prepared a nuclear extract from tissues that, upon a single chromatographic step, yields a fraction capable of accurate transcription of rDNA. The activity of the fractionated nuclear extract with respect to rDNA transcription was proportional to the rRNA synthetic activity of the tissue from which the initial nuclea...

Purification and characterization of a novel factor which stimulates rat ribosomal gene transcription in vitro by interacting with enhancer and core promoter elements.

“…It is of interest to determine whether E1BF has a potential regulatory role. Our laboratory has developed a cell-free system from adult rat liver and a rat hepatoma (18). The extract derived from the hepatoma was 10-fold more efficient than the liver extract in transcribing rat rDNA.…”