... understanding life in molecular detail

Prof Peter Stockley

Macromolecular Self-assembly; Virus assembly; Transcriptional control; Aptamers & Bionanotechnology


My laboratory is interested in understanding the molecular mechanisms regulating crucial cellular processes and macromolecular self-assembly, such as amyloid formation, as well as  (dis)assembly of virus particles. Our approach is largely based on in vitro biochemical experiments in amenable model systems, but emphasis is placed on being able to compare our in vitro data, wherever possible, with processes occurring in vivo.

Current major projects include:
  • Transcription initiation in the sigma54 class of bacterial promoters
  • Single molecule assays of virus assembly.
  • Evolution of virus assembly pathways
  • Nucleic aptamers as diagnostic, therapeutic and research tools

Assembly Mechanisms of Single-stranded RNA Viruses: Roles of RNA Genomes and Novel Drug Targets.

We have argued that single-stranded RNA viruses have genomes harbouring degenerate, multiple copy short sequences/secondary structures having affinity for their cognate coat proteins that we have termed Packaging Signals. The interactions that are this facilitated achieve a number of things that are beneficial for the virus and are this potential drug targets. Amongst these effects are: 1) compaction of the genome so that it will fit into the confines of the capsid; 2) binding energy to drive (1); 3) association of viral CPs with cognate genomes at low concentrations, reflecting the situation in vivo where initial CP concentrations are close to zero; 4) provide allosteric assistance to the CPs so that they form capsids of the correct shape and symmetry rapidly, thus preventing the occurrence of misformed or aggregated material, that would trigger host anti-viral defences; & 5) provide guidance throughout particle assembly that avoids the equivalent of Levinthal’s Paradox for protein folding.

The two-stage assembly mechanism for viral RNA. Our results suggest that there are three forms of RNA condensation/compaction in the context of virus assembly. (A) Non-specific condensation of viral RNA by multivalent ions (Mg2+, spermidine) inhibits assembly by coat proteins (middle). Only at very high CP concentrations (>5 μM) is this block overcome (right), showing that simple electrostatic condensation is not on pathway to capsid formation. (B) A two-stage mechanism of assembly  in which CPs first bind to cognate RNAs displaying multiple packaging sites (blue segments), distributed throughout the viral genome to facilitate protein-protein interactions, thus mediating a rapid RNA collapse (stage I). The collapse is followed by cooperative recruitment of additional CP subunits (stage II) even at low concentration (<1 μM) to complete capsid assembly. (C) Assembly with non-cognate (cellular) RNAs leads to weak interactions without observable initial RNA collapse. Dissociation of coat proteins from these complexes allows them to be captured by the cognate assembly pathway (red arrows, middle). A low yield alternative pathway occurs when non-specifically bound coat proteins nucleate assembly on cellular RNA. Since the coat protein binding sites are not correctly positioned these RNAs do not collapse and there can be multiple nucleation events leading to misassembled and multishell structures. Such pathways explain the assembly of non-cognate RNAs in vitro at relatively high coat protein concentrations.

The main features of PS-CP interactions had been established for the RNA bacteriophage MS2, and we had shown using single molecule fluorescence correlation spectroscopy (smFCS) that MS2 shared these properties with Satellite Tobacco Necrosis Virus (STNV). This smFCS work was reported in a PNAS paper [1] but failed to address a number of issues such as the nature and locations of the PSs in the MS2 genome and the nature of PS-CP interaction leading to RNA-dependent assembly of STNV. We have subsequently determined the locations of MS2, and a related phage GA, PSs and used them to predict that they share a conserved assembly pathway leading to a defined 3D structure for the encapsidated RNA [2]. This prediction has been validated by the first asymmetric cryo-electron tomographic reconstruction of a ssRNA virus that reveals a structure for the genome [3]. In addition, we used in vitro reassembly of STNV with its best PS site, B3, together with a crystal structure of a VLP assembled around B3, to show the molecular details of the PS effect on assembly [4]. The electrostatic component of this assembly mechanism has recently been confirmed by smFCS (unpublished).

Ablating PS-CP interactions would in our view be a beneficial anti-viral route and we have begun to exemplify this in both ensemble and sm reassembly experiments with STNV using clinically approved compounds such as antofine. Our goal was also to expand the range of viruses to include animal and human ones. To this end we have completed SELEX to identify putative PSs in Turnip Crinkle Virus, Cowpea Chlorotic Mosaic Virus, Brome Mosaic Virus, Human Parecho Virus, Polio & Hepatitis C. We find compelling evidence of similar PS-like sites in each case although we expect the molecular details and effects of the PS-CP interaction will be distinct in each case. For instance we find that PS sites in TCV seem to be clustered in pairs, encompassing the previously identified high affinity Minimal assembly Sequence or MAS. Dimer Mas fragments seem to be more efficient in ensemble reassembly experiments than single MASs. We are refining our in vitro assembly conditions before transferring them to smFCS assays.

References:

  1. Borodavka,A.,Tuma, R. & Stockley, P. G. (2012) Evidence that Viral RNAs have Evolved for Efficient, Two-stage Packaging. Proceedings Of The National Academy Of Sciences Of The United States Of America, 109, 15769-15774, doi/10.1073/pnas.1204357109.
  2. Eric C. Dykeman, Peter G. Stockley and R. Twarock. Identification of dispersed, cryptic packaging signals in two viral RNA genomes reveals a conserved assembly mechanism. JMB doi:pii: S0022-2836(13)00365-3. 10.1016/j.jmb.2013.06.005.
  3. Dent, K.C., Thompson, R., Barker, A.M., Barr, J.N., Hiscox, J.A., Stockley, P.G. & Ranson, N.A. (2013). The asymmetric structure of an icosahedral virus bound to its receptor suggests a mechanism for genome release. Structure. doi:pii: S0969-2126(13)00194-9. 10.1016/j.str.2013.05.012.
  4. Robert J. Ford, Amy M. Barker, Saskia E. Bakker, Robert H Coutts, Neil A. Ranson, Simon E.V.Phillips, Arwen R. Pearson & Peter G. Stockley. (2013)  Sequence-specific, RNA-protein interactions overcome electrostatic barriers preventing assembly of Satellite Tobacco Necrosis Virus coat protein. J Mol. Biol. 425, 1050-64. PMID:23318955
  5. Borodavka A, Tuma R, Stockley PG. (2013) A two-stage mechanism of viral RNA compaction revealed by single molecule fluorescence. RNA Biol. 10, 481-9.
  6. Stockley, P. G., Twarock, R.,, Bakker SE, Barker AM, Borodavka,A.,, Dykeman, E., Ford,R.J.,  Pearson AR, Phillips, S.E.V., Ranson NA& Tuma, R.. Packaging signals in single-stranded RNA viruses: Nature’s alternative to a purely electrostatic assembly mechanism. In press J Biological Physics. 39, 277-87. doi: 10.1007/s10867-013-9313-0. Epub 2013 Apr 12.
  7. Stockley, P. G., Ranson, N.A. & Twarock, R. (2013) A new paradigm for the roles of the genome in ssRNA viruses. Future Virology, 8, 531-543.

 Enhancer-dependent bacterial RNA polymerase initiation: insights from single molecule studies.

We have been using single molecule fluorescence spectroscopy to complement structural and mechanistic studies by our collaborators at Imperial College, London (Buck & Zhang) of the control of transcriptional initiation by the sigma54 class of bacterial transcription factors. Sigma54 holoenzyme does not undergo spontaneous isomerisation from the closed to the open transcriptional complex in contrast to the dominant bacteria, enzyme containing sigma70 subunits. This is a consequence of a physical barrier to the entry of the template into the active site posed by the Region I domain of the sigma factor. The physical location of this domain within the transcriptional machinery must be altered (remodelled) by a nucleotide hydrolysis event occurring on an adjacently bound activator protein. Using the activator PspF, a member of the AAA+ superfamily, we have been dissecting the pathway to open complex formation using single molecule FRET & FCS, in both solution and TIRF modes. We find that multiple ligand binding events including template and nucleotide-free activator binding lead to alterations in the preferred location of Region I. However, only during ATP hydrolysis is this sufficient to allow the template strand to enter the active site. These studies are providing novel insights into detailed molecular events in the complex machinery controlling transcription.

Experimental design and description of components for the smFRET assays. (A) Cartoon illustration of the pathway leading to open complex formation in σ54-dependent transcription machinery. (B) Schematic description of the domain architecture of showing the different domains of the molecule and the positions of dye-labelling. (C) Schematic description of nifH promoter used for smFRET assays. Positions of dye-labels used in TIRFM measurements are shown.

Detailed research programme                  Close ▲
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Professor of Biological Chemistry
BSc (Imperial) PhD (Cambridge)


Postdoctoral Research Fellow, (Harvard University) 1980-1983
Postdoctoral Research Assistant, (Harvard University) 1983-1985
Lecturer, Department of Genetics, (University of Leeds) 1986 -1992
Visiting Research Scholar, (The Scripps Research Institute, La Jolla)

Miall 10.29
School of Molecular and Cellular Biology
0113 343 3092
p.g.stockley@leeds.ac.uk

Selected Publications

  1. Dent, K.C., Thompson, R., Barker, A.M., Barr, J.N., Hiscox, J.A., Stockley, P.G. & Ranson, N.A. (2013). The asymmetric structure of an icosahedral virus bound to its receptor suggests a mechanism for genome release. Structure. doi:pii: S0969-2126(13)00194-9. 10.1016/j.str.2013.05.012.

  2. M WnÄ?k, M Ł Górzny,, M B Ward, C Wälti, A G Davies, R Brydson, S D Evans and P G Stockley  Fabrication and characterisation of gold nanowires templated on helical arrays of TMV coat proteins. (2013) Nanotechnology 24, 025605. doi: 10.1088/0957-4484/24/2/025605. Epub 2012 Dec 10.

  3. Galaway, F.A. & Stockley, P.G. (2012). MS2 Virus-Like Particles: A Robust, Semi- Synthetic Targeted Drug Delivery Platform. Molecular Pharmaceutics 10, 59-68. 

  4. Borodavka,A.,Tuma, R. & Stockley, P. G. (2012) Evidence that Viral RNAs have Evolved for Efficient, Two-stage Packaging. Proceedings Of The National Academy Of Sciences Of The United States Of America, 109, 15769-15774.