Interested individuals must follow Steps 1, 2 AND 3 at the link on how to apply
Supervisors: Dr Laura Spagnolo (University of Edinburgh) [email protected]
and Dr Atlanta Cook (University of Edinburgh) [email protected]
in collaboration with Robert Galletto (Washington University in St Louis).
Rap1 is a conserved, multifunctional, sequence-specific, DNA-binding protein involved in transcriptional activation and silencing. It is an essential factor for telomere length regulation and maintenance. Besides its prominence in ageing, its deletion in mice leads to obesity and insulin resistance (1).
Rap1 binds multiple sites in duplex telomere DNA, forming the telomeric core. Fine detail on dissected Rap1 domains is available (2). Full-length Rap1 from S. cerevisiae interacts with DNA over 16 successive binding sites, leading to local DNA stiffening (3). There isn’t three-dimensional information on the full-length protein, nor on its complex with telomeric DNA.
This project will be carried out in close collaboration with Dr Roberto Galletto, Department of Biochemistry and Molecular Biophysics, University of Washington, US. Galletto will provide full-length ScRap1, as well as mutants, and telomeric DNA.
Our collaborator’s preliminary work shows that recombinant ScRap1 can be purified to homogeneity, binds telomeric DNA sequences as well as its interacting partners. The aim of this project is to achieve a three-dimensional reconstruction of ScRap1 in isolation, as well as of its multimeric complex with telomeric DNA. We will use transmission electron microscopy techniques coupled to single particle image processing. Initially, we will set out to analyse the samples with negative staining techniques (Year 1). We will then perform cryo-electron microscopy experiments, to achieve finer detail for the two structures (Years 2 and 3). We will perform 2D classification of negatively stained sample to visualize structural differences between the wild type protein and phenotype-specific mutants in yeast. This structural information will be integrated with biophysical work carried out in our collaborator’s laboratory (Isothermal Titration Calorimetry, Surface Plasmon Resonance), to provide a mechanistic model of how Rap1 forms the core of a telomere.
The student will be involved in all aspects of the electron microscopy work, from sample preparation, to data acquisition and image processing. This is a unique opportunity to develop a well-rounded profile in structural electron microscopy, a true single molecule technique, in which there is currently a shortage of experienced personnel.
(1) Duong and Sahin “RAP1: Protector of Telomeres, Defender against Obesity”, Cell Reports 2013
(2) Lewis and Wuttke “Telomerase and telomere-associated proteins: structural insights into mechanism and evolution”, Structure 2012
(3) LeBihan et al., “Effect of Rap1 binding on DNA distortion and potassium permanganate hypersensitivity”, Acta Cryst D 2013.