A TBP-containing multiprotein complex (TIF-IB) mediates transcription specificity of murine RNA polymerase I

4180-4186 Nucleic Acids Research, 1993, Vol. 21, No. 18 ©1993 Oxford University Press A TBP-containing multiprotein complex (TIF-IB) mediates transcription specificity of murine RNA polymerase I Dirk Eberhard, Laszlo Tora1, Jean-Marc Egly1 and Ingrid Grummt* Institute of Cell and Tumor Biology, German Cancer Research Center, D-69120 Heidelberg, Germany and 1lnstitut de Chimie Biologique, Faculte de Medecine, 11 rue Humann, 67085 Strasbourg cedex, France Received July 6, 1993; Revised and Accepted August 2, 1993 ABSTRACT INTRODUCTION Different molecular mechanisms are involved in the process of transcription initiation by the three classes of nuclear RNA polymerases. Class I, II and in RNA polymerase promoters differ not only in their DNA recognition sequences and their overall structural organization but also interact with a distinct set of basic transcription factors that are required for accurate transcription initiation (for review, see 1). These general factors serve in part to precisely position the RNA polymerase at the transcription start site. In addition to specific binding to promoter sequences, • To whom correspondence should be addressed they interact with each other and/or the polymerase to form a productive transcription initiation complex. To understand this process, the function of the factors necessary for initiation by the three classes of nuclear RNA polymerases must be determined. Transcription initiation of murine ribosomal RNA genes by RNA polymerase I (Pol I) has been shown to require in addition to Pol I four factors, designated TTF-IA, TTF-IB, TIF-IC, and UBF (2). Two of these factors, TIF-IB and UBF, are specific DNA binding proteins which function early in the initiation pathway. Promoter selectivity is conferred by TIF-IB, the basal factor which specifically interacts with the core region of the mouse rDNA promoter and nucleates transcription complex formation (3). Cooperative interaction between TTF-IB and the upstream binding factor UBF has been shown to increase binding of TIF-IB to its target sequence (4). Apparently, UBF recruits TTF-IB to the template which, in turn, allows for higher promoter activity. The association of TTF-IB with the rDNA promoter, in the presence or absence of UBF, creates a primary preinitiation complex that recruits Pol I, either alone or in association with the two auxiliary factors TIF-IA and TIF-IC to to the template to form a preinitiation complex (2). Much previous work has focussed on the functional differences which exist between the transcription apparatus of the different classes of RNA polymerases. Initially it was assumed that each class of polymerases would utilize a distinct set of accessory factors to transcribe its set of genes. However, recent experimental evidence suggest that the different polymerases use similar strategies, and even the same or closely related polypeptides, to transcribe their target genes. First, in yeast at least three subunits are shared between the Pol I, Pol n, and Pol III enzymes (for review, see 5). Second, at least one factor, the TATA-binding protein (TBP), is required for transcription by all three classes of RNA polymerases (6-12). Thus, TBP is a general transcription factor for all nuclear RNA polymerases. Presumably the choice for a given class of promoters is accomplished by association of TBP with other accessory proteins, termed TAFs (for TBP-associated factors). Recent studies utilizing biochemical and immunoaffinity approaches have resulted in the separation of distinct TBP complexes and the identification of specific TAFs TIF-IB is a transcription factor which interacts with the mouse ribosomal gene promoter and nucleates the formation of an initiation complex containing RNA polymerase I (Pol I). We have purified this factor to near homogeneity and demonstrate that TIF-IB is a large complex ( < 2 0 0 kDa) which contains several polypeptides. One of the subunits present In this protein complex Is the TATA-blnding protein (TBP) as revealed by copurlflcation of TIF-IB activity and TBP over different chromatographlc steps including Immunoafflnity purification. In addition to TBP, three tightly associated proteins (TAFs-l) with apparent molecular weights of 95, 68, and 48 kDa are contained In this multimeric complex. This subunit composition Is similar—but not Identical—to the analogous human factor SL1. Depletion of TBP from TIF-IB-containlng fractions by Immunoprecipltation eliminates TIF-IB activity. Neither TBP alone nor fractions containing other TBP complexes are capable of substituting for TIF-IB activity. Therefore, TIF-IB is a unique complex with Pol l-speciflc TAFs distinct from other TBPcontaining complexes. The Identification of TBP as an integral part of the murine rDNA promoter-specific transcription Initiation factor extends the previously noted similarity of transcriptional initiation by the three nuclear RNA polymerases and underscores the Importance of TAFs in determining promoter specificity. Nucleic Acids Research, 1993, Vol. 21, No. 18 4181 MATERIALS AND METHODS Partial purification of transcription factors and in vitro transcription assays 200 ml of a mixture of nuclear and cytoplasmic extracts were chromatographed on DEAE-Sepharose CL-6B, followed by Heparin-Ultrogel. Partial separation of individual factor activities was obtained by step-elution with buffer AM (20 mM Tris—HC1, pH 7.9, 0.1 mM EDTA, 20% glycerol, 5 mM MgCl2, 1 mM DTE, 0.5 mM PMSF) containing different salt concentrations. TIF-IA and TTF-IC eluted at 200 mM KC1, Pol I and UBF at 400 mM KC1, and TTF-IB at 600 mM KC1. The H-200 fraction was further purified by chromatography on Q-Sepharose (300 mM). In vitro transcription reactions (25 /tl) contained 10-50 ng of template DNA (pMrWT/Nde I) and a mixture of crude Pol I (H-400 fraction), partially purified TIF-IA and TTF-IC (Q-Sepharose fraction), and TIF-IB at different stages of purification. The cell-free transcription system and the analysis of the RNA has been described elsewhere (21, 22). Purification of TTF-IB A typical purification of TIF-IB started from about 6 x l 0 1 0 cultured Ehrlich ascites cells which were harvested in the exponential phase of growth. 600 ml of a mixture of nuclear and cytoplasmic extracts were purified by chromatography on DEAESepharose, Heparin-Ultrogel, CM-Sepharose, and Mono-S as described (3). TIF-IB was eluted from the Mono-S column with a salt gradient from 0.3 to 0.7 M KC1. Fractions containing TIFIB activity (eluting at 450 mM KC1) were pooled and used for affinity purification. Sedimentation velocity centrifugation TIF-IB (200 /tl of a Mono-S fraction) was layered onto a 3.5 ml linear (20-40%) glycerol gradient containing 3% sucrose in buffer AM-100 and centrifuged in a SW60 rotor at 55,000 rpm for 14 h at 4°C. 200 /tl fractions were collected and assayed in the reconstituted transcription system. 100 /J of each fraction were precipitated with 10% TCA and, after SDS-PAGE, TBP (...truncated)