Sandbox20: Difference between revisions

In B. subtilis, Ter sites are 30 bp in length with two imperfect inverted 16 bp repeats overlapping at a TAT motif.<ref> PMID: 17521668</ref>  The upstream portion of the Ter site is called TerA; and the downstream portion, TerB. The sequence differences between these cause the bound RTP dimers to bind with different affinity and generate an assymetric complex capable of halting the progression of the replication fork only if the B site is encountered first.<ref> PMID: 17521668</ref> The mechanism by which this is achieved is discussed below in relation to the structure of the <scene name='Sandbox20/2efw/3'>RTP complex</scene>.

The structure of an RTP monomer bears greatest similarity to the "''classic winged-helix''" motif, in which 'wings' project from the loop between the final two β sheets of their compact αβααββ structure.<ref>PMID: 10679470</ref> The two major variations from this theme are the absence of a β1 sheet (the corresponding region is instead termed the β1 loop), and the presence of a fourth elongate α-helix at the C-terminus, which facilitates dimerisation.<ref>PMID: 11224562</ref> Each of these secondary structural elements are indicated in this <scene name='Sandbox20/2efw/8'>model</scene>.

The association of α4 helices into an antiparellel coiled coil leads to dimerisation (right). The conformation is further stabilised by interhelical salt bridges outside this region, as well as an aromatic network on the inner surface which form a hydrophobic core. <ref>PMID: 17521668</ref> The phenylalanine and tryptophan residues that form part of this network are shown in the image (below). The crystal structure of [[1bm9| RTP in its unbound state]] was determined in 1995. A [[1j0r| C110S mutant]] was then generated to prevent the aggregation of RTP through cysteine oxidation. With a very similar structure and almost no change in dimerisation and DNA-binding capacities, the mutant was the protein of choice for later studies.<ref>PMID: 14559228 </ref>

When an RTP dimer binds to a TerA or TerB site, the basic residues of the α3 helix are positioned in the major groove, and the β-ribbon rests within the minor groove.<ref>PMID: 7867072</ref> Non-specific ionic interactions between the N-terminus and the DNA backbone stabilise the complex.

The dissociation constant of the RTP-TerA complex is greater than that of RTP-TerB, indicating an inherently a lower binding affinity.<ref>PMID: 17521668</ref>  However, following the binding of RTP to TerA, a positive cooperative effect facilitates the binding of RTP to TerA.<ref>PMID: 7867072</ref> It has been proposed that RTP bends the DNA at the TerB site in a manner that favours RTP binding at TerA. The RTP dimer at TerB may also present a surface for stabilising interactions with the dimer at TerA through its β1 loop and β3 strand.

The asymmetric arrangement of the RTP dimer means that certain regions of the protein are accessible from one face only. In particular, Tyr33 makes contact with the replication fork in the wing-down monomer only, as shown in this <scene name='Sandbox20/2efw/20'>model</scene>. Interestingly, residues in proximity to Tyr33 carry similarity to those of the leading face of DnaB helicase, suggesting some direct interaction. This may contribute to the suppression of helicase activity <ref>pdb: 7867072</ref>. For unknown reasons, both the TerA and TerB sites must be occupied for full replication termination activity. <ref>pdb: 17521668</ref>