Chapter Ol

Transcription: The Never Ending Story

James A. Goodrich1 and Robert Tjian2

1 Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309 2Molecular and Cell Biology Department, University of California, Berkeley, Berkeley, CA 94720

Key Words: transcription, promoter, activator, coactivator, general factors, chromatin, RNA polymerase II


After more than 30 years of intense and sustained activity, the field of transcriptional control in eukaryotes continues to deliver unexpected and revealing montages of the remarkably complex yet elegant consequences of evolution. Transcription research started from humble beginnings with the isolation of 3 distinct RNA polymerases. This was followed by a rich period of mapping promoters, enhancers and the isolation of the first sequence specific DNA binding regulatory factors. These studies in turn led to the unraveling of the multi-subunit pre-initiation apparatus culminating with the modern era of co-activators and chromatin remodeling complexes. Throughout this opus of biochemical discovery we have witnessed a beautiful convergence of in vitro biochemical tour-de-force combined with the power of molecular genetics and cell biology. In this short preamble, we offer a brief and very likely incomplete history of the maturing of eukaryotic transcription and its prospects for the future.

Fumbling in the Dark: Hoping for Simplicity

Emboldened by the inspiring successes of pioneering work in the biochemistry of DNA replication and bacterial phage transcription, early workers struggling with animal and human gene regulation followed suit by isolating not one but three distinct enzymes: RNA polymerase I, II and III each dedicated to the synthesis of rRNA, mRNA, and tRNA/5sRNA

respectively (Krebs and Chambon, 1976; Sklar et al., 1975). However, due to the lack of promoter specific DNA templates or the ability to obtain sufficient quantities of "cloned" DNA, the ability of these 3 distinct enzymes to discriminate between the different classes of genes remained obscure. Nevertheless, the chromatographic separation and in vitro biochemical assays for detecting the RNA polymerases opened the first doors to the future development of high fidelity promoter specific and eventually activator regulated transcription in cell free systems.

Because, eukaryotic RNA polymerases behaved in a rather promiscuous and DNA template independent fashion in vitro, there was a brief period, (after the discovery of heterogeneous nuclear RNA) in which it was popular to posit that, unlike bacterial transcription which is temporally regulated by cascades of cr-factors, eukaryotic transcription may be "unregulated". Instead, one imagined that post transcriptional RNA processing (i.e. splicing, poly A addition, capping, etc.) would largely determine the population of mRNA's destined for gene product expression. Although this "random transcription" model fit with some early data regarding the apparent lack of promoter DNA selectivity in vitro of eukaryotic RNA polymerases, it soon became clear from studies of mammalian viruses (SV40, Adeno 2) that at the very least, specific DNA sequences that lie near transcription start sites (i.e. TATA elements and GC boxes) played some role in determining elements of the eukaryotic "promoter" (Fig. 1.1) (Myers et al, 1981; Rio et al., 1980; Tjian, 1978).

As is often the case with biology in general but especially in the study of eukaryotic transcriptional regulation, we invariably opted for simplicity and hoped that a well defined -3 5/-10 like element such as the

Corresponding Author: Robert Tjian, Tel: (510) 642-0884, FAX: (510) 643-9547, E-mail:[email protected]

popular TATA box of Ad2 would suffice to designate the necessary cis-regulatory information of a promoter (Corden et al., 1980; Hu and Manley, 1981). This rather minimalist view was, however, decisively toppled when both in vitro and cell based assays were developed that revealed the existence of important upstream distal as well as proximal DNA sequences in eukaryotic promoters (Baneiji et al., 1981; Benoist and Chambon, 1981; Fromm and Berg, 1983; Gidoni et al., 1985; Myers and Tjian, 1980; Picard and Schaffher, 1984). With the emergence of cloned promoter sequences and DNA template dependent in vitro transcription reactions measured by run-off and primer-extension assays the combinatorial nature of multiple cis-control elements of eukaryotic gene regulatory units became firmly embedded (Mitchell and Tjian, 1989). Add to these in vitro assays the advent of transient transfection assays and microinjection in animal cells that revealed the existence of "orientation and distance independent" enhancer elements and we began, for the first time, to get a glimpse of the complex regulatory network of gene transcription that would follow in succeeding decades (McKnight and Tjian, 1986; McKnight, 1982; Picard and Schaffiier, 1984; Treisman et al., 1983). To this day, the precise mechanisms mediating "long distance" enhancer or silencer functions remain largely obscure despite many plausible models including DNA looping, scanning etc.

Mappig cis-control dements; Proximal promoter elememts and enhancers


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