Prof Peter Stockley

Peter Stockley is Professor of Biological Chemistry in the Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds. He obtained his B.Sc. in Chemistry from Imperial College, London in 1976 and his Ph.D. in Biochemistry, University of Cambridge in 1980. He joined the academic staff of the University of Leeds in 1986 after postdoctoral training at Harvard University.

Research Areas: Self-assembling systems: Protein-nucleic acid regulatory complexes; molecular recognition; gene expression; bionanoscience

Research Interests.

Overview:
The key feature of all living organisms that distinguishes them from simple chemical reactions is their ability to self-assemble complex macromolecular machines, from ribosomes to viruses, and the ability of those machines to participate in regulated functions. All these properties depend on the process of molecular recognition between gene products. We are studying a number of important model systems that allow us to dissect the molecular basis of such recognition events and hence the chemical underpinning of biological function. Our goals are to discover fundamental principles about the assembly and function of macromolecular machines, especially those involving protein-nucleic acid recognition, and to exploit this knowledge to create novel tools for research, diagnostics and/or therapy.

Current research projects.

1. Transcriptional control:

We have been studying the structure and function of the E.coli methionine repressor in collaboration with Prof. Simon Phillips for many years. The protein is a member of the ribbon-helix-helix family (RHH) of DNA-binding proteins. Its affinity for its target DNA operators is modulated by a small molecular weight co-repressor, S-adenosyl methionine, that is believed to operate via an unprecedented long-range electrostatic effect. In vitro structure and function work continues using NMR spectrometry to investigate the dynamics of the protein and its complexes (Wellcome Trust), whilst a BBSRC-funded project is allowing use to use transcriptomics to investigate the phenotypes of metJ knockouts in vivo and mutants with defined properties in vitro. We are also collaborating with Prof. David van Vranken, UC Irvine, who is synthesising a series of peptide mimics of the RHH motif in order to understand the molecular basis of sequence-specific DNA recognition.

As well as the met repressor we (Baumberg) have been studying the Bacillus subtilis arginine repressor-activator, AhrC, and its E.coli homologue ArgR. Unlike ArgR, AhrC activates genes for arginine catabolism, probably by playing a role in bending the DNA so that upstream activator proteins can contact RNAP and promote initiation. Catabolic promoters are controlled by sigmaL RNAPs. SigmaL is the Bacillus homologue of the major variant sigma factor in E.coli, sigma54. We are collaborating with Prof. Martin Buck, Imperial College, to dissect the functions of sigma54 using single molecule spectroscopy/FRET (with Prof Smith).

2. Viral assembly/Translational control:

The RNA bacteriophages are classical objects that have been studied intensively as models for various molecular processes. We are studying the sequence-specific basis of the RNA-coat protein interaction that occurs at a defined point in the phage lifecycle. Formation of a coat protein dimer-RNA stem-loop (19 nt) complex simultaneously leads to translational repression of the downstream replicase cistron and self-assembly of the phage T=3 shell around the phage RNA. We are probing the RNA-protein recognition event using a combination of X-ray crystallography and RNA chemical synthesis. The dynamic properties of the complex that forms, together with characterisation of the early intermediates in capsid assembly are being studied by NMR (with Prof Homans), mass spectrometry (with Dr Ashcroft), native chemical ligation (with Dr Warriner and Prof Radford) and electron/atomic force microscopy ( and Thomson).

3. Ligand-binding RNA aptamers/RNA-binding ligands

RNA aptamers are isolated from degenerate sequence libraries by repeated cycles of binding to the ligand of choice, separation of the bound from unbound species followed by RT-PCR to regenerate a DNA pool with lowered sequence degeneracy. Usually ten cycles of such SELEX procedures yields families of RNA sequences with the desired ligand binding characteristics. Aptamers have been isolated that bind everything from divalent metal ions to large proteins with affinities that can be in the sub-nanomolar range. Aptamers have been compared to antibodies in the range of targets that they can interact with, although they are much easier to isolate and manipulate than these protein ligands. We have automated aptamer selection using a Biomek liquid handling robot as part of an MRC Co-operative in Enabling Technologies that allows 10 cycles of SELEX to be done within 3 days.

We are using the ready availability of our RNA aptamers to explore their potential to provide research tools, diagnostic and therapeutic reagents in a wide range of fields. These include as reagents that block amyloid formation (in collaboration with Prof Radford); species that block transcription factor DNA-binding; antibiotic mimics and binders; regulators of viral protein function and cyclins; and as the capture ligands in chromatin immuno-precipitation (ChIP) assays (Bonifer).

In a particularly exciting collaboration with synthetic chemistry (with Prof Nelson) we are investigating the use of aptamers directed against a stereo-differentiated library of aminoglycoside derivatives (ADGs) to regulate gene expression in vivo in response to addition of the AGD to the growth medium. We are also developing chemical libraries that should bind sequence-specifically to RNAs and thus have the potential to act as anti-virals or antibiotics.

4. Virus-like particles/ Bionanoscience

We have exploited our fundamental understanding of the formation of the RNA bacteriophage capsids to create Virus-like particles (VLPs) that can be used to present immunogenic epitopes to the immune system, i.e. act as synthetic vaccines, or target drug species to particular cell types in a directed drug-delivery system. The use of viral capsids as scaffolds for the crystallisation of 'difficult' target molecules has been demonstrated previously and further extensions of this concept are being tried with other viral species.

Recently, the University of Leeds has agreed to invest £4.5M into the rapidly expanding area known as bionanoscience in which knowledge of the structure and function of natural macromolecular complexes can be exploited medically or commercially. We are heavily involved in developing new concepts in the applications of VLPs for these bionanoscience goals.