Sandbox20
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IntroductionIntroduction
Replication Termination in E. coli and B. subtilisReplication Termination in E. coli and B. subtilis
The replication of the chromosomal DNA in most bacterial species occurs through a bidirectional mechanism, in which two replication forks progress in opposite directions from the unique origin of replication. A [replisome]
creating a characteristic theta structure . Although the use of two active polymerase complexes is clearly faster than one, this mechanism generates difficulty when it comes to terminating replication. Among other factors, the presence of DNA-associated proteins causes the two replication complexes to proceed at different rates, and they therefore do not necessarily meet directly opposite the origin. However, the position of termination is not random, and is defined by the presence of termination sites which associate protein factors capable of halting the advance of the replication fork from only one direction. From their positions within the chromosome and their directionality, you can see that this occurs only once the complex has traversed more than half of the total DNA.
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The ProteinsThe Proteins
RTPRTP
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Two structurally identical RTP molecules form a dimeric complex upon binding to DNA. The Ter site is 30 bp in length, and contains an imperfect inverted 16 bp repeat overlapping at a highly conserved trinucleotide sequence (TAT). Differences in sequence distinguish the upstream Ter A domain from the downstream Ter B domain. These bind their respective RTP molecules in slightly different manners, creating an asymmetric complex which will only halt the progression of the replication fork if the B site is encountered first. The mechanism by which this is achieved is discussed below in relation to the structure of the .
Structural OverviewStructural Overview
The structure of an RTP monomer bears greatest similarity to the "classic winged-helix" motif, where 'wings' project from the loop between the final two β sheets of a compact αβααββ structure. The two major variations from this theme are the absence of a β1 sheet (the corresponding region is termed the β1 loop), and the presence of a fourth elongate α-helix, which facilitates dimerisation. Each of these secondary structural elements are indicated in the structure .
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DNA BindingDNA Binding
13-15 residues contribute to the attachment of an RTP molecule to the Ter DNA site. Most of this is contributed by the basic residues of the , which lies in the major groove of DNA.
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The asymmetry of the dimer is shown by the names 'wing up' and 'wing down'. It is measured by the angle between the a2 and a3 heices, as shown .
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Replication Termination ActivityReplication Termination Activity
The consequence of these different conformations is most prominent in the position of the B1 sheet. This is evident in the Tyr33 residue, Space, which contact DNA only in the wing-down conformation.
TusTus
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RoleRole
Tus is the 36 kDa protein responsible for termination of replication in Escherichia coli. The chromosome of E. coli 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. This generates what is termed a replication-fork trap, which prevents replication from occurring towards the origin. The Ter sites contain a 20 bp consensus element to which a monomer of Termination Utilisation Substance (Tus) binds to form a polar DNA-protein complex which halts the progression of the replicative machinery from one direction only. The fork arrest mechanism depends on the blocking of the helicase activity of DnaB, which is the first component of the replisome to encounter the Ter-Tus complex.
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Structural OverviewStructural Overview
The structure of the Tus protein was determined in complex with TerA by Kamada et al., and shown to be a previously undescribed backbone conformation (.). It is divided into two domains (amino and carboxy), in which α-helical regions of each are spanned by a central β-sandwich which contacts 13 bp of DNA duplex (#Indicate domains). Three helices within the amino domain (αI αII, αIII) form an antiparallel bundle aligned parallel to the DNA (#Helix bundle). Another two helices (αIV, αV) clamp the DNA phosphate backbone at the non-permissive end, and forms the cytosine-specific pocket containing the crucial residues for anti-helicase activity (#Phosphate clamp). 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 (#Sheet position).
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DNA BindingDNA Binding
Tus is among the most stable monomeric, sequence-specific, double-stranded DNA-binding proteins. Three major sets of interactions contribute to this; (1) the phosphate clamp within the amino domain, (2) base-specific polar interactions by the β-sheet within the major groove, and (3) non-polar contacts within the carboxy domain.
1. Interaction between beta sheets (shown in green) and the major groove of DNA ().
Replication Termination ActivityReplication Termination Activity
Tus binds to a conserved cytosine residue which is not base paired . The interactions between residues of the Tus protein and this unpaired cytosine nucleotide are shown in more detail .
Its ability to do this depends on the conserved glutamate residue E49. "The crystal structure of the Tus-Ter lock shows that Glu49 of Tus makes a water-mediated hydrogen bond with the 50- phosphate of the displaced A(7) nucleotide residue, and it would thus be expected to be partially defective in formation of the locked species." This is shown
E49K does not affect DNA binding but does affect anti-helicase activity in the ‘trapped’ complex E49 makes an indirect hydrogen bond to the phosphate of the ‘displaced’ nucleotide.
The HistoryThe History
Summary of progression of understanding.