Eucaryotic transcription complexes are specifically associated in large sedimentable structures: rapid isolation of polymerase I, II, and III transcription factors.

Abstract

RNA synthesis in eucaryotes takes place on template molecules that are activated by stably associating with limiting transcription factors. In this paper we demonstrate that such stable transcription complexes can be specifically sedimented from in vitro transcription reaction mixtures by mild centrifugation. This occurs with stable complexes of genes transcribed by all three classes of eucaryotic RNA polymerase and with S-100 as well as whole-cell extracts. However, the transcriptional capacity of the isolated complex differs for the three polymerase classes. The pelleted ribosomal DNA (polymerase I) complex contains all the factors necessary for transcription, each purified 25-to 50-fold, whereas the pelleted adenovirus major late promoter (polymerase II) complex lacks a factor that remains in the supernatant. In the case of 5S DNA (polymerase III), a necessary factor associates slowly with the sedimentable complex. Notably, the interactions responsible for this rapid sedimentation are specific for DNA molecules in stable complexes, suggesting that the in vitro sedimentable complex mirrors the in vivo structural organization of active genes.Eucaryotic transcription factors stably associate with the DNA molecules that serve as templates for RNA synthesis. Formation of such stable transcription complexes has been demonstrated in vitro with genes transcribed by all three classes of polymerase (1, 3, 5-7, 9, 11, 22, 24, 25, 27) and thus appears to be the general means of maintaining the activated state of eucaryotic genes. The constituents of these stable complexes and their functional roles are currently under considerable investigation. The DNA sequences involved in stably binding polymerase I, II, and III transcription factors have been delineated for genes that encode 45S rRNA (B. Sollner-Webb, J. Tower, V. Culotta, and J. Windle, in J. Setlow and A. Hollaender, ed., Genetic Engineering, in press), the adenovirus major late transcript (7), and 5S RNA (21), respectively. In all cases, stable complex formation utilizes the same template region that was previously identified as the promoter. Considerable progress has also been made in isolating and characterizing the proteins involved in the transcription complex. In many cases, however, these studies have been hindered by the difficulty in purifying these factors without effecting significant losses of activity; despite much effort, most eucaryotic transcription factors have only been purified to a limited extent.As an alternative to traditional chromatographic methods, we have utilized a biological fractionation as the first step in purifying transcription factors, taking advantage of their specific binding to template molecules. We show that stable complexes and the associated transcription factors can be quantitatively and selectively sedimented from in vitro transcription reaction-inixtures, whereas the bulk of the extract protein remains soluble. This procedure is effective in isolating transcription complexes of all three classes of eucaryotic polymerase. ...

Factor C*, the specific initiation component of the mouse RNA polymerase I holoenzyme, is inactivated early in the transcription process.

“…released in an active form by treating the recovered complex with micrococcal nuclease or with high salt (14,60). The factor C* from the pelleted complex, from the supernatant, and from the combined pellet plus supernatant are then assayed by addition to cycloheximide-treated cell extract along with a second rDNA template.…”

“…6) or 40-jil (in Fig. 3 to 5) reaction mixtures containing 4 Rl of extract depleted of NTPs (see above) plus 3 jig of template per ml were incubated at 30'C for 60 min and then separated from any residual unbound factors by pelleting at 10,000 x g for 5 min at 40C and resuspension in 25 Rl of transcription buffer lacking NTPs (14). For the time course reactions, resuspended preinitiation complexes formed on the 450-nt runoff template were supplemented with 500 jiM each ATP, GTP, and UTP and 50 jiM CTP (in Fig.…”

“…To assay remaining C* activity, the unlabeled reactions prepared as described in the previous paragraph were put on ice and the transcription complex was pelleted by centrifugation at 10,000 X g for 5 min at 40C (14). After careful removal of the supernatants, transcription factors bound to the template were released by micrococcal nuclease digestion in a 4-RI reaction mixture (60) (in Fig.…”

Factors and nucleotide sequences that direct ribosomal DNA transcription and their relationship to the stable transcription complex.

“…We have recently found that these stable transcription complexes can be quantitatively pelleted upon centrifugation of an in vitro transcription reaction mixture (8). The activated rDNA template that is recovered in the pellet fraction is freed from the vast majority of the extract protein, polymerase I, and transcriptionally inactive rDNA molecules, yet the pellet retains the full transcriptional activity of the parent reaction.…”

“…S-100 extracts were prepared from logarithmically growing mouse tissue culture cells (line L1210) and HeLa cells as previously decribed (8,24). In vitro transcriptions were performed and analyzed basically as described previously (7).…”