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