User:Bianca Varney/Bacterial Replication Termination: Difference between revisions
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====RTP Structure==== | ====RTP Structure==== | ||
Active RTP is a homodimer composed of 14.5 kDa subunits. The structure of the protein has been determined to a 2.6 A resolution using X-ray crystallography [3]. The RTP protein contains three major structural domains for its specific functionality; | Active RTP is a homodimer composed of 14.5 kDa subunits. The structure of the protein has been determined to a 2.6 A resolution using X-ray crystallography [3]. The RTP protein contains three major structural domains for its specific functionality; DNA-binding, DnaB interaction and dimer-dimer interaction domains [3]. The RTP is organized into a dimer by the association of their | ||
<scene name='User:Bianca_Varney/Bacterial_Replication_Termination/Dimer_interaction_domain/1'>long α helices</scene> within the C-terminus [3]. The ‘<scene name='User:Bianca_Varney/Bacterial_Replication_Termination/Dna-binding/1'>winged helix</scene>’ is believed to be involved as the major DNA-binding domain while two α helices, found central in the protein, also fit adjacently into the major groove, and DNA-binding is a result of a three helical bundle [3]. This binding interaction is vastly different from the Tus-''ter'' interactions. | |||
====RTP Mechanism of Action==== | ====RTP Mechanism of Action==== | ||
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An RTP dimer binds the core sequence and the complex formed allows a second dimer to cooperatively to bind to the auxiliary site. In the absence of a core site, the auxiliary site is unable to bind RTP. Furthermore, without the auxiliary site, RTP is unable to block the replication fork, as the interaction of both dimers has been suggested to provide enough DNA binding strength to displace the replication fork. This binding explains how the symmetrical RTP can block replication helicase machinery in an asymmetric manner. The blocking end occurs at the core site, while it is believed that the non-blocking auxiliary site may let replication through as there is less contact points of the dimer to the DNA and the replication machinery coming from this direction is predicted to displace the dimer that is weakly bound to the auxiliary site, which would then displace the dimer bound to the core [10]. Biochemical and mutational studies have identified particular residues that are vital for the functionality of RTP. Mutations within a hydrophobic region at residues Glu-30 and Tyr-33 causes the loss of contrahelicase ability [10]. These mutations do not affect dimer-dimer interactions or DNA binding activity and indicate that simple DNA binding is not able to block the replication fork. This provided evidence that RTP and the replication fork machinery interact specifically [10].</StructureSection> | An RTP dimer binds the core sequence and the complex formed allows a second dimer to cooperatively to bind to the auxiliary site. In the absence of a core site, the auxiliary site is unable to bind RTP. Furthermore, without the auxiliary site, RTP is unable to block the replication fork, as the interaction of both dimers has been suggested to provide enough DNA binding strength to displace the replication fork. This binding explains how the symmetrical RTP can block replication helicase machinery in an asymmetric manner. The blocking end occurs at the core site, while it is believed that the non-blocking auxiliary site may let replication through as there is less contact points of the dimer to the DNA and the replication machinery coming from this direction is predicted to displace the dimer that is weakly bound to the auxiliary site, which would then displace the dimer bound to the core [10]. Biochemical and mutational studies have identified particular residues that are vital for the functionality of RTP. Mutations within a hydrophobic region at residues Glu-30 and Tyr-33 causes the loss of contrahelicase ability [10]. These mutations do not affect dimer-dimer interactions or DNA binding activity and indicate that simple DNA binding is not able to block the replication fork. This provided evidence that RTP and the replication fork machinery interact specifically [10].</StructureSection> | ||
==The Terminus Utilization Substance ( | ==The Terminus Utilization Substance (''Escherichia coli'' )== | ||
<StructureSection load='2ewj' size='500' side='left' caption= 'Tus complexed to the ''E. coli'' ''ter'' site' scene=''>The ''E.coli'' protein that is responsible for termination is a 36kDa protein named Tus (Terminius Utilization Substance) that binds 23bp ''ter'' sites and arrests the replication helicase, DnaB, responsible for separating the two strands of DNA []. Unlike RTP termination sites, the ten ''E.coli'' ''ter'' sites do not contain inverted sequences or direct repeats and Tus binds as a monomer to a highly conserved core region of 13bp [8]. The tus-''ter'' complex is known to terminate replication by arresting the replication machinery in a in a polar manner however there is great discrepancy in evidence whether Tus specifically interacts or physically blocks the DnaB helicase to arrest its progression [1]. | <StructureSection load='2ewj' size='500' side='left' caption= 'Tus complexed to the ''E. coli'' ''ter'' site' scene=''>The ''E.coli'' protein that is responsible for termination is a 36kDa protein named Tus (Terminius Utilization Substance) that binds 23bp ''ter'' sites and arrests the replication helicase, DnaB, responsible for separating the two strands of DNA []. Unlike RTP termination sites, the ten ''E.coli'' ''ter'' sites do not contain inverted sequences or direct repeats and Tus binds as a monomer to a highly conserved core region of 13bp [8]. The tus-''ter'' complex is known to terminate replication by arresting the replication machinery in a in a polar manner however there is great discrepancy in evidence whether Tus specifically interacts or physically blocks the DnaB helicase to arrest its progression [1]. | ||
