The General Transcription Machinery and Preinitiation Complex Formation

Samuel Y. Hou and Cheng-Ming Chiang

Department of Biochemistry, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106-4935

Key Words: RNA polymerase II, general transcription factors, TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, preinitiation complex


Transcription of protein-coding genes in eukaryotes is regulated by RNA polymerase II (pol II). By itself, pol II is unable to direct site-specific initiation of transcription and requires a host of accessory proteins, termed general transcription factors (GTFs), to commence basal level transcription dictated by the core promoter elements. The GTFs for pol II-mediated transcription include TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. The transcription cycle, whether basal or regulated transcription, can be divided into multiple stages: preinitiation complex (PIC) assembly, initiation, promoter clearance, elongation, termination, and reinitiation. Before initiation, TFIID binding to the core promoter marks the beginning for PIC assembly. This is followed by the entry of TFIIA, TFIIB, pol II/TFIIF, TFIIE, and TFIIH, either in a stepwise fashion or as a preassembled pol II holoenzyme complex, to form a stable PIC which is ready to make RNA when ribonucleoside triphosphates and an energy source are provided. Formation of the PIC is a critical and often rate-limiting step in transcriptional regulation. Here we discuss the properties of pol II and each GTF in relation to PIC assembly, which occurs prior to the formation of the first phosphodiester bond in transcription initiation.


A: Discovery of RNA Polymerase Activity

The foundation of molecular biology, where information flows from DNA to RNA to protein, known as the Central Dogma, was first proposed by Francis Crick in 1958 (Crick, 1958). The discovery of RNA viruses and prions provided an expanded view of the flow of genetic information. With slight modifications, the Central Dogma as proposed by Francis Crick still holds true (Crick, 1970). The initial step of this process, the transfer of genetic information from DNA to RNA, is termed transcription. The enzymatic activity of RNA polymerase, which governs this genetic transfer, was first isolated by Weiss and Gladstone in 1959 from rat liver nuclei (Weiss and Gladstone, 1959). This enzyme could synthesize RNA in a DNA-dependent manner, as evidenced by the observation that upon the addition of DNase, incorporation of [a-32P]CTP into RNA was severely diminished (Weiss and Gladstone, 1959). While the isolation of a single bacterial RNA polymerase from Escherichia coli was achieved over the next few years (Chamberlin and Berg, 1962), the biochemical purification of this eukaryotic enzyme remained elusive for an entire decade until purification of this enzyme was accomplished in 1969 by Roeder and Rutter from sea urchin embryo nuclei (Roeder and Rutter, 1969).

Surprisingly, Roeder and Rutter isolated not just one, but three different RNA polymerase activities, which were named RNA polymerase I, II, and III (or A, B, and C respectively), based upon their chromatographic fractionation on a DEAE-Sephadex column. RNA

Corresponding Author: Cheng-Ming Chiang, Tel: (216) 368-8550, Fax: (216) 368-3419, E-mail: [email protected]

polymerase I eluted first at the lowest salt concentration, while RNA polymerase III came off the column at the highest salt concentration (Roeder and Rutter, 1969). Although the existence of three eukaryotic RNA polymerases were reported in 1969, their functions remained undefined until the specific activities of each RNA polymerase were resolved based upon their differential sensitivities to a-amanitin (Weinmann and Roeder, 1974; Weinmann et al., 1974), a drug isolated from the death cap fungus, Amanita phalloides, that inhibits the activity of RNA polymerase II at low concentrations and that of RNA polymerase III at high concentrations. Using a-amanitin sensitivity assay with endogenous RNA polymerases in isolated nuclei, Roeder and colleagues discovered that RNA polymerase I is primarily involved in transcribing 18S and 28S ribosomal RNAs, while RNA polymerase II transcribes mRNAs, and RNA polymerase III is responsible for synthesis of cellular 5S rRNA, tRNAs, and adenovirus VA RNAs. These results were consistent with the finding that RNA polymerase I is localized within nucleoli, the sites for rRNA gene transcription, whereas RNA polymerase II and III are normally present in the nucleoplasm (Roeder and Rutter, 1970).

B: The General Transcription Machinery

The biochemical identification of all three eukaryotic RNA polymerases was not achieved until 1975. Analysis and comparison of these eukaryotic RNA polymerases revealed that RNA polymerase I, II and III contain multiple subunits, some of which appear to be common among all three polymerases (Sklar et al., 1975). In this chapter, we concentrate on the formation of an initiation-competent RNA polymerase II (hereafter referred to as pol II) transcription complex, since pol II is responsible for transcription of all protein-coding genes in eukaryotes. Although pol II was eventually isolated and found to contain 12 subunits (Young, 1991), purified pol II was not able to recognize specific promoters and accurately initiate transcription. Site-specific initiation was only observed in whole cell lysates or nuclear extracts. This led to the hypothesis that other essential or accessory factors were required for accurate initiation of gene-specific transcription.

Biochemical evidence for necessary accessory factors became evident when crude subcellular fractions supplemented with purified pol II were able to accurately transcribe natural adenovirus DNA template in vitro (Weil et al., 1979). This was followed by the chromatographic purification of subcellular fractions to identify the accessory factors, later termed general transcription factors (GTFs). Roeder and colleagues purified these subcellular fractions over a Whatman Pll phosphocellulose ion exchange column to isolate A, B, C, and D fractions, which correspond to the nuclear proteins sequentially eluted by 0.1, 0.3, 0.5 (or 0.6), and 0.85 (or 1.0) M KCl-containing buffer, and then proved that the A, C, and D components were necessary for accurate initiation of transcription by pol II (Matsui et al., 1980). The protein factors present in the A and D fractions necessary for pol II-mediated transcription were named TFIIA and TFIID, respectively. The C fraction was later fractionated into accessory factors TFIIB, TFIIE, TFIIF, and TFIIH (Sawadogo and Roeder, 1985a; Reinberg and Roeder, 1987; Flores et al., 1989; Flores et al., 1992; Ge et al., 1996). These accessory factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) were collectively defined as GTFs. The nomenclature for pol II GTFs soon became TFII(letter). TF represents Transcription Factor, the Roman numeral II specifies pol II transcription, and the "letter" indicates which purification fraction the specific GTF was isolated from (see Fig.2.1). Three components of the general transcription machinery are multisubunit complexes: pol II is composed of 12 subunits, TFIID is comprised of 14 subunits, and TFIIH has 9 subunits (Wu etal., 1998).

C: The Sequential Assembly Pathway

Although accessory factors for accurate initiation of pol II transcription were being identified at a rapid pace, little was known about how these GTFs are assembled at the promoter region where transcription initiates. Phil Sharp and colleagues were the first to identify the hierarchical nature of GTF assembly at the promoter region using native gel electrophoresis, together with DNase I footprinting, to establish the order of addition and the relative positions of GTFs in relation to the promoter, thus suggesting a model for transcription initiation that proceeds in a stepwise manner (Buratowski et al., 1989). Specifically, TFIID first recognizes the promoter, followed by TFIIA, then TFIIB, later pol II, and finally TFIIE (TFIIF and TFIIH had yet to be identified; Buratowski et al., 1989). After all the GTFs were identified and purified to near homogeneity, this stepwise manner of GTF assembly was further defined as: TFIID recognition of the promoter as the first step, followed by TFIIA and TFIIB stabilizing promoter-bound TFIID, and recruiting pol II/TFIIF to the promoter. After formation of a stable TFIID-TFIIA-TFIIB-pol II/TFIIF-promoter complex, TFIIE is then recruited and followed by TFIIH (Fig. 2.2A). This stepwise manner of assembly became known as the sequential assembly pathway for the formation of the preinitiation complex (PIC).

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