Where it all Begins: An Overview of Promoter Recognition and Open Complex Formation

STEPHEN BUSBY, ANNIE KOLB AND HENRI BUC

1.1 Gene Expression as a Driver of Life

The importance of transcription, the process by which information encoded in DNA is copied into RNA, cannot be overstated. As soon as the dogma that DNA makes RNA makes protein was established, the hunt was on for the machinery that orchestrates transcription. Thus, in the late 1950s and early 1960s, classical methods of protein fractionation were used to identify DNA-dependent RNA polymerase activity. Remarkably, in parallel, primarily using Escherichia coli genetics, Jacob, Monod and their colleagues were discovering gene regulatory proteins and establishing the paradigm that gene transcription was the key point at which gene regulation is effected. Thus, right from the start, Escherichia coli K-12 was established as the model system to use and, with the benefit of hindsight, it is easy to see now how 40 years of amazing progress was sparked by the fusion of two very different worlds, one populated by the biochemists and the other by the bacterial geneticists. Put very simply, the stories in this book expand on how the biochemistry explains the genetics and how the genetics gives reason to the biochemistry. The crucial discoveries that set the scene for these stories were made in the late 1960s: the characterization of the single multi-subunit RNA polymerase in E. coli, the discovery of promoters and terminators, and the realization that different genes are transcribed at widely differing frequencies. The pace accelerated with the arrival of cloning and DNA sequencing in the 1970s and in-depth studies of how different promoters are regulated exploiting increasingly sophisticated methodologies. The arrival of whole genome sequences in the late 1990s led to the complete catalogue of the different players and attempts to integrate our knowledge with systems biology approaches. And finally, the structural biologists have provided us with models of many of the major players, including the multi-subunit RNA polymerases, the principal topic of this book.

1.2 Escherichia coli RNA Polymerase

The view of bacterial RNA polymerase as a 500 kDa enzyme with subunit structure α2ββ'ωσ had a long and slow birth, emerging from heroic biochemistry in both the USA and in Germany. It is easy to overlook the difficulties encountered by the pioneers in this field of proving the integrity and function of such a large multi-subunit complex. DNA cloning technologies had not yet arrived and early efforts to demonstrate specific DNA-directed transcription mostly had to exploit viral templates, notably bacteriophages. Perhaps the most influential single observation was the chance discovery by Dick Burgess and colleagues in 1969 that passage of the preparation of E. coli RNA polymerase through phosphocellulose led to loss of its ability to initiate specific transcripts and that this loss was due to the loss of the σ factor. This led to the definition of two forms of RNA polymerase, the holo-enzyme with composition α2ββ'ωσ, and the core enzyme, α2ββ'ωσ, devoid of σ, and the notion of σ as the factor controlling transcript initiation. Another influential early finding came from Mike Chamberlin and colleagues, who showed that the transcriptionally competent complexes formed between the holoenzyme and DNA were resistant to heparin (see also ref. 4). In these complexes, which could form in the absence of any nucleotides, the heparin resistance arises from the template DNA strands being locally unwound around the transcription start site. These observations gave birth to the idea of a pathway to transcription initiation, with the kinetically competent or open complex being preceded by a heparin-sensitive closed complex in which the DNA strands are not open. Amazingly, the nature of closed complexes, the mechanics of the closed to open transition and the number of intermediates remain hot topics for study and debate today.

One of the early proofs that E. coli contained a single core RNA polymerase was that RNA synthesis could be completely inhibited by the drug, rifampicin, but a single point mutation can confer complete resistance and normal RNA synthesis. The location of these rifR mutations led to the identification of the co-transcribed rpoB and rpoC genes, which encode the RNA polymerase large β and β' subunits (1342 and 1407 amino acids respectively). Subsequently, the genes encoding the other RNA polymerase subunits were identified at different locations on the E. coli chromosome, and the pathway of subunit assembly was established. The first step is the formation of a dimer of two 329 amino acid α subunits, which acts as a scaffold for the addition of first β and then β'ω to give core enzyme. The holoenzyme is then formed by the addition of the σ subunit. This pathway was established by Akira Ishihama, who later showed that the C-terminal 100 amino acids of each α subunit are dispensable for RNA polymerase assembly. The reason for this is that the RNA polymerase α subunit consists of two domains, with the 230 amino acid N-terminal domain being essential for enzyme assembly, whilst the C-terminal contains a separate independently folding domain that plays a key role at certain promoters. We now have detailed structures for both the core and holo enzymes, due largely to the efforts of Seth Darst, Dmitry Vassylyev and their coworkers using RNA polymerases from thermophilic bacteria. The structures show the large β and β' subunits...