Sandbox20: Difference between revisions

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>.