Mechanism of assembly of the RNA polymerase II preinitiation complex. Transcription factors delta and epsilon promote stable binding of the transcription apparatus to the initiator element.

J.W Conaway,J.N Bradsher,R.C Conaway

doi:10.1016/s0021-9258(19)50211-3

Abstract

Assembly of RNA polymerase II with the core region of TATA box-containing promoters requires the action of the TATA factor and four transcription factors designated alpha, beta gamma, delta, and epsilon, which have each been purified to near homogeneity from rat liver. Evidence from previous studies argues that alpha and beta gamma play a crucial role in delivering RNA polymerase II to the promoter (Conaway, R. C., Garrett, K. P., Hanley, J. P., and Conaway, J. W. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6205-6209). Here we describe the interaction of transcription factor delta with preinitiation intermediates assembled in the presence of either recombinant yeast TFIID or the high molecular mass, endogenous TATA factor tau from rat liver (Conaway, J. W., Hanley, J. P., Garrett, K. P., and Conaway, R. C. (1991) J. Biol. Chem. 266, 7804-7811). Results of template challenge experiments argue that delta enters the preinitiation complex through interactions with multiple components of the transcription apparatus. We observe that, in the presence of recombinant TFIID, delta interacts stably with the preinitiation complex only in the presence of RNA polymerase II, alpha, and beta gamma, whereas, in the presence of tau, delta is capable of interacting stably with the Initial Complex independently of RNA polymerase II. Results of restriction site protection experiments reveal that delta and epsilon promote binding of the transcription apparatus to the Initiator element and support the model that RNA polymerase II assembles at the core promoter in at least two discrete steps, first "touching down" near the TATA element and finally extending its interaction downstream to encompass the cap site.

Highlights

Previous findings indicate that 6 and t function directly and stoichiometrically in formation of the complete preinitiation complex and, in concert with a,Br, and the TATA factor, promote formation of stable protein-DNA contacts that anchor the transcription apparatus at the core promoter [32, 34].' We have extended these observations and, in this report, describe the interaction of 6 with preinitiation intermediates assembled in the presence of either recombinant yeast TFIID or thehigh molecular mass, endogenous TATA factor T from rat liver
We showed that 6 co-purifies closely with DNA-dependent ATPase(dATPase) activity when analyzed by hydrophobic interaction and cation and anion exchange HPLC [31]
Of RthNeA Polymerase I I PreinitiaCtiomn plex modification of the previous purification procedure, we have have recently reported purification of a HeLa cell transcripnow confirmedthe associationof these polypeptides with bothtion factor, designated BTF2, which is associated with multranscription and ATPase(dATPase) activitiesm; oreover, we tiple polypeptides rangingin size from 90 to 35 kDa; the have shown that DNA-dependent ATPase activity co-sedi- possibility thatthisfactorhas associated DNA-dependent mentswiththetranscriptionactivity of 6 duringsucrose ATPase activity has nobteen addressed

Summary

EXPERIMENTAL PROCEDURES

Inc. [u-~'P]CTP(>800 Ci/mmol) and [cY-~'P]~A(T>8P00 Ci/mmol) were from ICN. Polyvinyl alcohol, type 11, and heparin were obtained from Sigma. Assay of Runoff Transcription-Except as indicated in the figure legends, assays were performed as described [29] with -200 ng of NdeI-digested pDN-AdML [40] or pN4 [41] and approximately 2 ng of a (fraction V), 10 ng of /3r (fraction V), 40 ng of 6 (fraction VI), 20 ng of e (fraction V), 60 ng of T (fraction V) or 50 ng of recombinant yeast TFIID (AcA44 fraction), and 0.003 unit of RNA polymerase 11. Assay of dATPase-Reaction mixtures (20 pl) contained 40 mM Tris-HC1, pH 7.9, 7 mM MgC12,50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mg/ml bovine serum albumin, 2% (v/v) glycerol, 5 p~ dATP, 1 pCi of [a-32P]dATP, and250 ng of a 60-base pair doublestranded oligonucleotide,designated Ad(-50 to +lo),which contains the core region of the AdML promoter [40]. Fractions (2 drops) were collected from the bottom of tubes through a20-gauge needle

RESULTS

TSK SP S-PW

DISCUSSION