Sandbox20
This sandbox is in use for Usyd BCHM3981 until June, 2011. Please do not edit this page.
IntroductionIntroduction
Replication Termination in E. coli and B. subtilisReplication Termination in E. coli and B. subtilis
Replication forks proceed from the origin of the circular chromosome in opposite directions, 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.
Space
Space
Space
Space
Space
Space
Space
Space
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 .
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.
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 .
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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Structural OverviewStructural Overview
Original picture is .
DNA BindingDNA Binding
Tus binds by an interaction between beta sheets (shown in green) and the major groove of DNA. This is .
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.