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Functional dissection of ParB homologue and global regulatory protein KorB of RK2

Muntaha, Sidra Tul (2010)
Ph.D. thesis, University of Birmingham.

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Abstract

RK2 is a low copy number plasmid responsible for spread and maintenance of important properties (including antibiotic resistance and degradation functions) among bacteria. Gene expression in RK2 is controlled by cooperativity among four repressors (i.e. KorA, KorB, KorC and TrbA) to tightly regulate replication, stable inheritance and conjugative transfer functions. KorB (358 aa) has dual roles as a global regulator and as an active partitioning protein. This study focuses on its role as a global regulatory protein and its interaction with DNA, RNAP and other repressor proteins (e.g KorA and TrbA) of RK2. It is shown for the first time that DNA binding by negatively charged protein KorB (-21) is modulated via a balance of charge in the internal region from aa 235 to 245. KorB binds O\(_B\) and silences the genes around, showing that KorB can spread. TrbA and KorA bound to DNA adjacent to KorB do not block gene silencing by KorB and indeed potentiate its repression, suggesting that KorB can spread past DNA binding proteins and thus that they do not act as road blocks. The fact that KorB E237A, which is defective in silencing, cannot repress at a distance when alone but can do so in presence of TrbA, provides strong evidence of looping. The fact that KorA and TrbA do not potentiate gene silencing by E237A, but do potentiate its repression, indicates strongly that gene silencing is because of spreading instead of looping. Full length KorB is required for distal repression. However, only the region 225-255 aa is critical for proximal repression by KorB. The results suggest a model in which KorB organises DNA loosely over a long region through a wrapping in a way that can accommodate other regulatory proteins. This nucleoprotein complex may also be critical for plasmid partitioning.

Type of Work:Ph.D. thesis.
Supervisor(s):Thomas, Chris M.
School/Faculty:Colleges (2008 onwards) > College of Life & Environmental Sciences
Department:School of Biosciences
Subjects:QH301 Biology
QH426 Genetics
Institution:University of Birmingham
ID Code:1381
This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder.
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