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>

Tus is the 36 kDa protein responsible for termination of replication in ''Escherichia coli''.<ref>PMID: 8051142</ref> The ''E. coli'' chromosome contains a set of six polar Ter DNA sequences arranged such that three with the same directionality are located on either half of the chromosome to the form the fork trap.<ref>PMID: 16148308</ref> The Ter sites contain a 20 bp consensus element to which a monomer of Termination Utilisation Substance (Tus) binds. The unidirectional blocking of the replication fork occurs by preventing the helicase activity of DnaB. However, whereas RTP most likely achieves this through direct protein-protein interactions, Tus causes the displacement of a cytosine residue to disrupt the normal DNA conformation required for helicase activity.<ref>PMID: 16814717</ref>

===Structure of the Tus-Ter Complex===

The structure of the Tus protein in complex with TerA revealed a previously undescribed backbone conformation (<scene name='Sandbox20/Tus/2'>original model</scene>).<ref>pdb:  8857533</ref> It can be divided into the major amino and carboxy domains, in which the α-helical regions of each are spanned by a central β-sandwich which makes contact with the DNA duplex. Three helices within the amino domain (αI αII, αIII) form an antiparallel bundle aligned parallel to the DNA (blue, left). Another two helices (αIV, αV) clamp the DNA phosphate backbone at the non-permissive end. This generates the cytosine-specific pocket with the crucial residues for anti-helicase activity.<ref>PMID: 16814717</ref> The main DNA-binding domain, however, is the exposed side of the double β sheet layer which provides several base-specific interactions. This lies within the major groove and causes a conformational change in the DNA involving a deepening of the major groove, and an expansion of the minor one (green, left).<ref>PMID: 8857533</ref>

Tus is among the most stable monomeric, sequence-specific, double-stranded DNA-binding proteins.<ref>PMID: 16814717</ref> This is due to a combination of three major sets of interactions; base-specific polar interactions within the major groove, non-polar contacts with the carboxy domain, and a phosphate clamp within the amino domain. The three β-sheets which span the major groove of DNA make both base-specific and base non-specific bonds, as shown in this <scene name='Sandbox20/Tus/19'>model</scene>. In particular, three glutamine residues on the βJ strand form bidentate hydrogen bonds to bases at the permissive end of the complex (below centre).<ref>PMID: 8857533</ref> Interspersed with these residues on the same strand, residues such as isoleucine make Van der Waals and hydrophobic interactions to sugar and base moieties (below right). The distribution of bonds to each strand of the duplex is highly asymmetric, and one strand is largely exposed to the solvent. This contributes to allowing the passage of the fork from one side only. <ref>PMID: 16814717</ref>

The ''phosphate clamp'' is located at the end of the aIV and aV helices, closest to where the replication fork is stalled. <ref>pdb:  8857533</ref> It ensures the protein does not come loose at the critical end and allow helicase activity to occur. It involves five, mostly van der Waals, contacts with the sugar-phosphate backbone.<ref>PMID: 8857533</ref>

The displacement of a <scene name='Sandbox20/Tus/13'>single conserved cytosine</scene> nucleotide from the double helix determines the polarity of fork arrest.<ref>PMID: 16814717</ref> This is located at the edge of the non-permissive face, and defines the point at which helicase activity is halted. Upon interaction with Tus the cyotsine is no longer base-paired, and is instead associated with residues within a well-defined recognition pocket.<ref>PMID: 16814717</ref> The specific interactions which stabilise this are shown in this <scene name='Sandbox20/Tus/9'>model</scene>.  

Before the structure of the Tus-Ter complex was determined, mutation of Glu49 was shown to eliminate anti-helicase activity without affecting DNA binding.<ref>PMID: 11493686</ref> This could not be explained by the [[1ecr|original crystal structure]] as it is not located close enough to make direct contact with the conserved cytosine.<ref>PMID: 11493686</ref> However, the [[2ewj|more recent structure]] revealed that this is due the water-mediated hydrogen bond formed between it and the adenine residue adjacent to cytosine.<ref>PMID: 16814717</ref> It is therefore likely, that the interaction is necessary to compensate for the disrupted H-bonding in the nucleotide adjacent to the displaced cytosine, as shown in this <scene name='Sandbox20/Tus/18'>model</scene>.