RNA Polymerases as Molecular Motors

By Henri Buc, Terence Strick

The Royal Society of Chemistry

Copyright © 2009 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-0-85404-134-3

Contents

There and Back Again: A Structural Atlas of RNAP Seth Darst, 1,
Part I From Promoter Recognition to Promoter Escape,
Chapter 1 Where it all Begins: An Overview of Promoter Recognition and Open Complex Formation Stephen Busby, Annie Kolb and Henri Buc,
Chapter 2 Opening the DNA at the Promoter; The Energetic Challenge Bianca Sclavi,
Chapter 3 Intrinsic In vivo Modulators: Negative Supercoiling and the Constituents of the Bacterial Nucleoid Georgi Muskhelishvili and Andrew Travers,
Chapter 4 Transcription by RNA Polymerases: From Initiation to Elongation, Translocation and Strand Separation Thomas A Steitz,
Chapter 5 Single-molecule FRET Analysis of the Path from Transcription Initiation to Elongation Achillefs N. Kapanidis and Shimon Weiss,
Chapter 6 Real-time Detection of DNA Unwinding by Escherichia coli RNAP: From Transcription Initiation to Termination Terence R. Strick and Andrey Revyakin,
Part II Transcription Elongation and Termination,
Interlude The Engine and the Brake Henri Buc and Terence Strick,
Chapter 7 Substrate Loading, Nucleotide Addition, and Translocation by RNA Polymerase Jinwei Zhang and Robert Landick,
Chapter 8 Regulation of RNA Polymerase through its Active Center Sergei Nechaev, Nikolay Zenkin and Konstantin Severinov,
Chapter 9 Kinetic Modeling of Transcription Elongation Lu Bai, Alla Shundrovsky and Michelle D. Wang,
Chapter 10 Mechanics of Transcription Termination Evgeny Nudler,
Conclusion Past, Present, and Future of Single-molecule Studies of Transcription Carlos Bustamante and Jeffrey R. Moffitt,
Subject Index, 315,

CHAPTER 1

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 assembled on the two α subunit N-terminal domains, with the β and β’ subunits forming a “crab claw” to accommodate DNA, with the catalytic centre of the enzyme right at the heart of the claw (Figure 1.1; full details are in Chapter 2). This organization is echoed in the structures of yeast RNA polymerase II that emerged from Roger Kornberg’s laboratory at the same time, underlining its importance at all levels of life.

Another major landmark in the study of bacterial RNA polymerases has been the realization that most bacteria contain multiple σ factors. This idea first emerged from studies by Rich Losick and others of the genes needed for spore formation by Bacillus subtilis. The products of some of these genes showed striking sequence similarities to already discovered σ factors. Since it was clear that σ factors were needed for both promoter specificity and open complex formation, Losick’s proposal that the sporulation pathway was driven by the synthesis of new σ factors, which switch on new sets of genes, was soon accepted. In fact, most bacteria contain one dominant σ factor and between 0 and 64 alternatives. The dominant σ, known as the “housekeeping σ” is an essential protein that is responsible for most transcription initiation. In E. coli, the predominant σ is σ with a molecular size of 70 kDa (613 amino acids) and it is this σ factor, encoded by the rpoD gene, that is found in most RNA polymerase preparations. The E. coli genome encodes six alternative σ factors (encoded by the rpoS, rpoH, rpoE, rpoF, rpoN and fecI genes), which are concerned with the management of different stresses. An increase in the intracellular level of an alternative σ factor (e.g., in response to a specific stress) results in the formation of a subpopulation of RNA polymerase holoenzyme molecules dedicated to initiate transcription at a particular subset of promoters. As more bacterial genomes have been studied, the belief that alternative σ factors have evolved to drive programs of microbial adaptation and differentiation has been reinforced.

DNA sequencing then allowed comparison of the primary structure of various s factors. Carol Gross and colleagues established that many of them contain four conserved regions of sequence that appear to fall in four protein domains. With the notable exception of the RpoN (σ) family of σ factors, this organization, or close variants, applies to most of the hundreds of σ factors that have now been characterized. In fact, biophysical studies have now confirmed the existence of four independent domains. In the RNA polymerase holoenzyme, Domains 2, 3 and 4 are arrayed on the surface, while Domain 1 slots in the crab claw DNA binding channel. The formation of a transcriptionally competent open complex involves recognition of different promoter elements by σ domain 2, and/or domain 3 and/or domain 4, expulsion of domain 1 from the crab claw channel, and entry of DNA surrounding the transcription startpoint (full details are in Chapter 2).

1.3 Promoters and Core Promoter Elements

As soon as it became possible to measure the synthesis of individual RNAs in E. coli, it became clear that there was enormous selectivity in gene expression. Some genes are highly transcribed whilst others are rarely transcribed. The idea that individual transcripts start and finish at specific points, and that much of the selectivity was due to differences in rates of initiation was embraced with enthusiasm. The rapid adoption of these concepts was probably due more to their simplicity than to the weight of experimental evidence, and, since they provided a convenient framework, pragmatism had the upper hand. What was incontrovertible was that specific mutations, located in front of the three genes that constitute the E. coli lactose (lac) operon, could reduce their simultaneous expression to near zero. The simplest and, as it turned out, the correct explanation was that the mutations were removing something that was channeling RNA polymerase to make a specific transcript. This something became known as a promoter, and as it became possible directly to determine transcript start points, and base by base mutational analysis could be achieved, detailed understanding of the architecture of the lac promoter, and other promoters, could be built up.

The picture that emerged of a typical promoter was that it contained two or more functionally important distinct sequence elements upstream of the transcription start site. The first such elements to be established were the -10 and -35 hexamers, and early studies attempted, with mixed success, to score promoter strength on the basis of the correspondence of the actual sequence to the -10 consensus 5′-TATAAT-3′, the -35 consensus 5′-TTGACA-3′ and the separation between the two elements. Crucially, most previously selected promoter “down” mutations fell in one or the other element and changed the sequence away from the consensus. Similarly, most known promoter “up” mutations also mapped to these elements and changed the promoter sequence towards the consensus. Subsequently, using suppression genetics, it was shown that the -10 and -35 elements were recognized by determinants in Domain 2 and Domain 4 respectively of the RNA polymerase σ subunit during promoter recognition, and details of the molecular basis of this recognition are now understood (Chapter 2). The simple model of the two principal promoter elements being recognized by two different domains of σ immediately provided an attractive explanation for how RNA polymerase containing alternative σ factors could recognize different promoters.

Although -10 and -35 elements play a major role in setting promoter activity, other elements that play important roles were soon discovered, and these can compensate for -10 and -35 elements that correspond poorly to the consensus. The base sequences immediately upstream of the -10 hexamer constitute the “extended -10” element that is recognized by Domain 3 of σ and, according to promoter context, can increase promoter strength by up to 20-fold. Similarly, the 20 base pair tract upstream of the -35 region provides a target for the two RNA polymerase a subunit C-terminal domains. This tract, known as the UP element, is found upstream of many strong promoters and functions by increasing the recruitment of RNA polymerase such that promoter strength can be increased up to 50-fold. Other determinants of promoter activity are the spacing between the -10 and -35 elements, which determines their juxtaposition as they are recognized by the RNA polymerase s subunit, and sequences downstream of the -10 hexamer, which determine the stability of the open complex and the facility with which RNA polymerase escapes from the open complex as the nascent RNA chain is elongated (Figure 1.2).

When it comes to bacterial promoters, the keyword is variety: just as many combinations of small coins can make up a dollar, different combinations of elements can make a promoter! However, it is here that one encounters a problem, because if the contributing elements, acting together, are too strong, the RNA polymerase has difficulty escaping, since the forces that recruited it cannot be undone. This is because the energy to move RNA polymerase out of the initiation complex has to come from the free energy associated with RNA formation. This explains why promoters have evolved to have non-consensus elements and why some of the strongest promoters, which recruit RNA polymerase most efficiently, are constructed so that the open complex is unstable, and hence RNA polymerase can be cleared rapidly. In fact, as demonstrated by Hermann Bujard, a sequence having all the consensus elements would be useless as a promoter, as RNA polymerase would bind to it, but then would be trapped.

Most of the research described in this book is concerned with RNA polymerase–DNA interactions, and focuses on simple promoters where RNA polymerase alone is sufficient for transcript initiation. However, in bacteria, the expression of most genes is regulated by the growth conditions and this involves different proteins that interact at promoters to activate or repress the process of transcript initiation. Thus, the activity of many promoters is set, not only by the different promoter elements but by these proteins. Some of these proteins control changes in transcription initiation in response to specific signals in the environment, e.g., the lac repressor responds to lactose, whilst others set the chromosomal context in which the promoter functions. Some transcription factors respond to global metabolic signals (e.g., carbon, nitrogen or oxygen supply) and interact at scores of different promoters. For example, initiation of transcription at the lac promoter, one of the first E. coli promoters to be studied in detail, is almost completely dependent on the cyclic AMP receptor protein (CRP), a global regulator that is often regarded as a paradigm for transcription factors. To understand fully the transactions that take place at bacterial promoters, an appreciation of how different proteins influence open complex formation is essential.

1.4 Biochemistry: It Works with RNA Polymerase!

It is fortunate that bacterial transcription can be reiterated in an in vitro system with purified DNA template and proteins. Landmark studies with bacteriophages DNA as templates clearly demonstrated that E. coli RNA polymerase was capable of specific initiation and termination of transcripts as well as elongation. These observations, made in the late 1960s, form the basis of the mechanistic studies described in this book. Of course, we now know of a host of proteins that interact with RNA polymerase to affect transcript initiation, elongation and termination, but the importance of the observation that the “simple” E. coli holoenzyme preparation was capable of reproducing the transcription cycle in a test tube cannot be overstated (Figure 1.2). Early experiments showed that the σ subunit was released from RNA polymerase as the nascent RNA chain grew. This led to the establishment of the σ cycle. At the time, it provided a rationale for the observation that E. coli cells contained substantially less σ than molar equivalents of core RNA polymerase (a finding that is not supported by a recent quantitative determination). Later, Sankar Adhya, Jack Greenblatt and colleagues showed that NusA was recruited to the core enzyme concomitant with the loss of σ and functioned as an elongation factor.

(Continues…)Excerpted from RNA Polymerases as Molecular Motors by Henri Buc, Terence Strick. Copyright © 2009 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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