Mechanism of a nucleotide dependent transcription activation process

The cells of every organism contain a chemical code within their DNA molecules that holds the information needed for making all the protein products required to make the cell grow and stay alive. The code is in the form of a linear sequence of nucleotide bases, the famous A,G, C & T. The order of these letters determines the subsequent linear order of amino acids of the cell's proteins, which ultimately determine its structure and function. Converting the nucleotide information into the encoded amino acid sequence is known as as gene expression. The first step in this process is one of the most important reactions inside cells and involves making an RNA copy - a transcript- of the DNA message. The enzymes involved in this process - RNA polymerases - must start the copying process at a defined point in DNA, known as the promoter. In cells, this process is tightly regulated and determines the pattern of genes being read and thus contributes to the precise execution of the cellular genetic program. We are studying the basic mechanisms that control the choice of start site and the controlled activation of the RNA polymerase from a passive DNA-binding protein into the active copying machine. In particular we are studying how the protein components of transcription machinery use the energy currency of the cell-a molecule called ATP-to function as small machines and to interconvert between different states. We would like to know the detailed features and the conformational changes in the transcription machinery during the initial steps - when the double stranded DNA is opened up in single strand forms and the template strand is delivered into the site of RNAP for synthesizing RNA.

Our overall goal is to develop a molecular level description of the action of a multi-subunit molecular machine controlling one of the most important processes in all biology, namely transcript initiation. This step is at the heart of regulating gene expression in all living organisms. The system chosen for these studies is the sigma54 transcriptional complex from E.coli in which initiation is dependent on a AAA+ ATP-hydrolysis driven activator protein, a mechanism resembling enhancer-dependent transcription initiation in eukaryotic cells. Such large multi-functional complexes require us to take an interdisciplinary approach encompassing structural, biophysical, genetic and protein biochemical techniques. We will build on the strengths of the research teams involved to interrogate the spatial relationships within the transcriptional complex as it undergoes isomerisation and adopts a state whereby the template DNA is delivered deep into the active site of the RNA polymerase. Our approach includes single particle cryoEM and site-specific labeling of subunits with fluorophores which are then interrogated by single molecule Förster Resonant Energy Transfer (smFRET). This will provide structure and kinetic information and distance constraints on large scale motions within the complex. In addition, we will apply hydrogen deuterium exchange (HDX) and mass spectrometry to map changes in subunit-subunit interfaces onto the amino acid sequences and known three-dimensional structures. This will provide short-range constraints. We will use time-resolved DNA footprinting to locate changes in DNA. The results will be correlated with parallel biochemical and site-directed mutagenesis experiments to identify key interactions that control subunit and domain movements. The data obtained will be integrated with the structural studies, yielding pseudo-atomic models of the activation pathway and one of the most detailed mechanistic descriptions of transcription initiation.

Many important scientific advances are only found to be useful many years after the original discovery. The work here focuses on the mechanism of a fundamental cellular process and therefore we expect any commercial impacts will occur in the longer term. Nevertheless, sigma54 and its activators are essential factors in the virulence of plant and animal pathogens. Therefore, understanding the detailed molecular mechanism of events involving sigma54 is essential if this pathway is ever to be exploited for antimicrobial development. Sigma54 and its activators are also involved in bioremediation by a number of bacteria. A detailed understanding of these gene regulation systems could be exploited to enhance bioremediation activities. Clearly an understanding of how sigma54 regulates gene transcription is of practical value in agriculture, medicine and biotechnology. Similarly understanding how directed motion and precise control in molecular machines arises from domain co-ordination is of major interest in considering how to exploit structures for nanotechnology and in designing inhibitors of such machines. The system we are studying mimics many of the functional features of the much more complicated RNAP Pol II system. Therefore, the insights obtained here will help us to understand aspects of the eukaryotic transcriptional machinery. The AAA+ protein family is one of the largest protein families known and its members are involved in numerous fundamental cellular processes, many of which are associated with human diseases. The advanced state of our knowledge in this system that relies on a AAA+ activator protein will have the added benefit of helping to understand the basic mechanisms underlying disease states which may then be exploited for novel drug development. The interdisciplinary approach of the collaborating groups in this proposal will greatly enhance training of the associated RAs, especially with respect to their ability to work within large interdisciplinary teams. They will also receive specific training in high-end modern techniques for studying large, complex and dynamic macromolecular systems. Such trained RAs (and associated PhD, masters and undergraduate students) are likely to benefit the biotechnology and pharmaceutical industries, as well as the academic base in the UK and abroad. Approaches to answering precise and penetrating questions of complex macromolecular systems will also feature in the training of staff associated with the project. We therefore anticipate medium term economic benefits arising from a well-trained UK and international research base, reflected in maintaining internationally competitive research intensive universities and associated industries.