Factors involved in specific transcription by mammalian RNA polymerase II. Identification and characterization of factor IIH.

O Flores,H Lu,D Reinberg

doi:10.1016/s0021-9258(18)45947-9

Abstract

Two new factors required for transcription of class II genes have been identified. These factors, TFIIH and TFIIJ, were required together with the previously described general factors (TFIIA, TFIIB, TFIID, TFIIE, and TFIIF) and RNA polymerase II for transcription of different class II genes. TFIIH was extensively purified, and the activity appeared to coelute with polypeptides of 33 and 95 kDa. The role of TFIIH and TFIIJ in preinitiation complex assembly was analyzed using mobility shift assays. It was found that TFIIH and TFIIJ association with the preinitiation complex was ordered and required the previous assembly of a preinitiation complex intermediate containing factors IID, IIB, IIF, IIE, and RNA polymerase II. A model for the ordered assembly of the general factors and RNA polymerase II is presented.

Highlights

From the Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854-5635
Valuable information on transcription initiation has been obtained from studies using partially purified transcription factors, a definitive underwas found that TFIIH and TFIIJ association with the standing requires the development of a system amenable to preinitiation complex was ordered and required the detailed biochemical analyses, namely a reconstituted tranprevious assembly of a preinitiation complex interme- scription system with homogeneous protein factors
Saltzman and Weinmann, 1989) and more recently from the isolation of the cDNA clones encoding the human general transcription factors BTF3 (Zheng et al, 1990),RAP 30 (Sopta et al, 1989), TFIID (Kao et al, Identification of the protein factors that govern transcrip- 1990, Peterson et al, 1990;Hoffmann et aL,1990)TFIIB (Ha tion initiation by RNA polymerase I1 has led to the delinea- et al, 1991),and TFIIE (Peterson et al, 1991). tion of two functional groups of transcription factors (TF).’

Summary

MATERIALS AND METHODS

Attention has been devoted toward the purification and char- Specific Transcription Assay and Transcription Factors-Reaction mixtures (40 pl) were incubated at 30 “Cfor 60 min and contained 20. The Mono S F/H protein fraction (25 mg)was dialyzed against buffer C,1.4 M (NH4)2SO4,loaded onto a phenylSuperose column (Pharmacia, HR 10/10), and eluted with an 80-ml linear gradient of ammonium sulfate (1.4-0 M).Aliquots of the column fractions were dialyzed against buffer C,0.1 M KC1 and assayed for TFIIF and TFIIHactivities as described in the text The first three fractionation steps(see Fig. 1) were essentially as described by Sumimoto et al (1990).The IIG SPSepharose fraction (100mg) wasdialyzed against buffer C, 0.1 M KCI, loaded onto an S-Sepharose column (5 mgof protein/ml of resin, Pharmacia) and eluted with a 200-ml linear gradient of KC1 (0.1-1.0 M). TFIIH andTFIIA activities coeluted on the S-Sepharose salt gradient (between 0.2 and 0.35 M KCl); one pool was made (50 mg) which was dialyzed against buffer C, 1.2 M (NH4),SO4 and loaded onto aphenyl-Superose column (Pharmacia, HR 10/ 10)equilibrated in the same buffer.

Phenylsuperose

RESULTS

DISCUSSION