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Protein structure, function and dynamics: Interactions and structures of the DNA-partitioning proteins KorB and IncC from the plasmid RK2.

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  • Full or part time
    Dr E I Hyde
    Dr S A White
  • Application Deadline
    Applications accepted all year round
  • Self-Funded PhD Students Only
    Self-Funded PhD Students Only

Project Description

This project is a collaboration between several groups of scientists (including Scott White and Chris Thomas; both in Biosciences, Birmingham; Melanie Grant, Dentistry, Birmingham; and David Scott in Nottingham and Harwell Research complex).
The aims are to
i) understand the molecular basis of DNA partitioning in bacteria by determining the structures and interactions of the proteins and DNA involved.
ii) determine how co-operativity between DNA-binding proteins can be achieved over a range of different DNA operator separations
iii) determine the energetic and structural role of regions of intrinsic disorder in proteins in these processes and hence in a wide range of key proteins.

Background : The low copy number, broad host range plasmid RK2 has been studied extensively as a small model genome, as an expression vector and as a carrier of multiple antibiotic resistance genes between bacterial species (1). In order to be stably maintained, RK2 encodes a system for active DNA partitioning to ensure that, when the cell divides, each daughter cell receives a copy of the DNA. This includes KorB, a DNA-binding protein and IncC, an ATPase, that are homologous to the ParB and ParA family partitioning proteins found on most bacterial chromosomes. KorB protein is structurally one of the best characterised of the ParB family. It contains two regions for which the 3D structures have been determined, and two regions that we have shown to be intrinsically unstructured in the free protein (2). IncC, uniquely, is transcribed in two forms, a shorter form, homologous to chromosomal ParA proteins, and a longer form which we have recently shown has an additional, intrinsically disordered, N-terminal 100 amino-acid region that binds DNA. Such disordered regions are found in a number of ‘hub’ proteins, in all kingdoms, that interact with multiple partners, including in cell signalling and transcription. These systems present a challenge to molecular analyses, as the proteins exist in a dynamic ensemble of interconverting conformations, and novel approaches have to be developed to study them. Our multipronged, biophysical studies are aimed towards determining the interactions of KorB with its partners, including IncC and DNA, and how these interactions affect its conformational range. KorB is also a weak transcription repressor that interacts co-operatively with other repressors encoded by the plasmid, such as KorA, which we have studied extensively (2, 3). We are also studying the KorB/KorA/DNA complex to determine how co-operativity between the two proteins is achieved over a range of different DNA operator separations to stringently regulate gene expression. Understanding DNA partitioning and gene regulation at a molecular level is a key first step to developing new targets for antibiotics to stop the spread of this group of plasmids, as well as targeting bacterial partitioning in general. The methods developed and the conclusions from this study will also be directly applicable to numerous proteins with disordered regions, which are involved in key functions such as cell signalling, regulation and cancer biology.

Methods: The student will be trained in a variety of biophysical methods, as well as protein purification and cloning. Full-length KorB, IncC, and KorA proteins, and some individual domains, have already been overexpressed and purified. The contacts within and between these proteins and any changes to their interactions in the presence of DNA, will be examined by chemical crosslinking followed by mass spectrometry. The interactions of the proteins will be confirmed by bacterial-2 hybrid methods and tested by mutagenesis. The energetics of binding, with and without the intrinsically disordered regions, will be measured using isothermal calorimetry or fluorescence. The structures of the proteins, both free and in complex, will be determined using NMR spectroscopy or X-ray crystallography, where possible, or modelled on structural homologues and the interactions observed used to dock the structures in the complexes. The conformational ranges of the proteins and complexes will be studied by small angle scattering (2), while their dynamics will be examined by NMR and MD simulations.

Conclusion: By the end of the study, we hope to have
(i) model structures, and conformational ranges for the partitioning proteins and their complexes
(ii) developed better understanding of the functional role of intrinsically disordered regions in these key proteins, including thermodynamics
(iii) developed better models for DNA partitioning and co-operative gene regulation in this system.

Funding Notes

All applicants should indicate in their applications how they intend to fund their studies. We have a thriving community of international PhD students and encourage applications at any time from students able to find their own funding or who wish to apply for their own funding (e.g. Commonwealth Scholarship, Islamic Development Bank).

The postgraduate funding database provides further information on funding opportunities available and further information is also available on the School of Biosciences website


1. Kolatka, K. et al, (2010) Plasmid, 64, 119-134. Replication and partitioning of the broad-host-range plasmid, pRK2.
2. Rajasekar, K. V. et al; (2010) J. Biol. Chem. 285, 15440-15449. Order and disorder in the domain organisation of the plasmid partition protein KorB
3. Bingle, L.E.H. et al (2008) Mol. Micro 70, 1502-151, A single aromatic residue in transcriptional repressor KorA is critical for co-operativity with its co-regulator KorB.

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