Transcription initiated by RNA polymerase II and purified transcription factors from liver. Cooperative action of transcription factors tau and epsilon in initial complex formation

Abstract

Synthesis of accurately initiated transcripts has been reconstituted with RNA polymerase II and a set of five transcription factors purified from rat liver. In addition to three previously identified factors alpha, beta gamma, and delta (Conaway, R. C., and Conaway, J. W. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7356-7360), transcription in the reconstituted liver system requires two novel factors designated tau and epsilon. These five transcription factors comprise two functional classes: (i) promoter recognition factors (tau and epsilon), which interact with template DNA to facilitate formation of a stable initial complex that is subsequently recognized and bound by RNA polymerase II, and (ii) RNA chain initiation factors (alpha, beta gamma, and delta), which do not participate in formation of the initial complex, but which are essential for transcription initiation.

Fig. 1. Resolution and purification of transcription factors from rat liver

P-cell, phosphocellulose; CM-Seph, carboxymethyl-Sephadex; HAP, hydroxylapatite; φ, phenyl.

Fig. 2. TSK 4000SW size exclusion HPLC of τ

Runoff transcription reactions with pDN-AdML as template and chromatography were performed as described under “Experimental Procedures.” In this and subsequent figures, AdML Runoff Transcript refers to the relative synthesis, per transcription reaction, of the 260-nucleotide runoff transcript synthesized from the AdML promoter in pDN-AdML (expressed in arbitrary units determined by densitometry of autoradiograms). Standards for size exclusion chromatography were bacteriophage λ DNA (V₀); thyroglobulin (Thyro), 670 kDa; ferritin (Fer), 440 kDa; catalase (Cat), 232 kDa; aldolase (Aldo), 158 kDa.

Fig. 3. TSK 4000SW size exclusion HPLC of ε

Runoff transcription reactions with pDN-AdML as template and chromatography were performed as described for τ under “Experimental Procedures.” Standards for size exclusion chromatography were bacteriophage λ DNA (V₀), thyroglobulin (Thyro), 670 kDa; ferritin (Fer), 440 kDa; catalase (Cat), 232 kDa; aldolase (Aldo), 158 kDa; chymotrypsinogen A (Chymo), 25 kDa.

Fig. 4. Transcription depends on RNA polymerase II and transcription factors α, βγ, δ, ε; and τ

Runoff transcription reactions with pDN-AdML as template were performed as described under “Experimental Procedures,” except that RNA polymerase II and transcription factors were omitted from reaction mixtures as indicated, pol II, polymerase II.

Fig. 5. Resolution of two stages in runoff transcription

Runoff transcription reactions with pDN-AdML as template were performed as described under “Experimental Procedures,” except that RNA polymerase II, transcription factors, and template were all added to reaction mixtures together, transcription was initiated by addition of all four ribonucleoside triphosphates, and heparin was not added to the reactions. The concentration of KCl was varied as indicated in the figure. When the concentration of KCl was varied during the Preincubation stage, the concentration of KCl in the Initiation and Elongation stage was held constant at 70 mM (– – –). When the concentration of KCl was varied during the Initiation and Elongation stage, the concentration of KCl in the Preincubation stage was held constant at 150 mM (——). pol II, RNA polymerase II,

Fig. 6. Time course of the high salt preincubation

Runoff transcription reactions with pDN-AdML as template were performed as described under “Experimental Procedures,” except that RNA polymerase II and transcription factors were all added at the beginning of the high salt preincubation. The length of the high salt preincubation was varied as indicated.

Fig. 7. τ and ε function in the high salt preincubation stage

Runoff transcription reactions were performed as described under “Experimental Procedures” and in Table II. pol II, RNA polymerase II.

Fig. 8. τ and ε are both required for template committment

Runoff transcription reactions were performed as described under “Experimental Procedures.” Incubations were as described in the figure, pol, RNA polymerase II; pDN, pDN-AdML; hep, heparin.

Fig. 9. RNA polymerase II associates stably with DNA templates containing a preassembled initial complex

Runoff transcription reactions were performed as described under “Experimental Procedures.” Incubations were as described in the figure, pol, RNA polymerase II; pDN, pDN-AdML.