User:Chloe Paul/Replication Terminator Protein: Difference between revisions

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== The Structure of RTP ==

<StructureSection <applet load='1bm9' size='300' side='right' caption='Structure of RTP (PDB entry [[1bm9]])' scene='User:Chloe_Paul/Replication_Terminator_Protein/Rtpmonobasic/2'>RTP has been found to exist as a symmetric α+β protein in solution and a homomeric dimer through crystal structure determination <ref name="bussiere">PMID: 7867072</ref>.These identical monomers each contain four α helices (<scene name='User:Chloe_Paul/Replication_Terminator_Protein/Rtpmonobasica1/1'>α1</scene>, α2, α3, α4), one β strand (β1) and two β ribbons(β2 and β3). It also contains a distordered N-terminal region. When the two monomers come together and the two α4 helices bind, forming a dimer with an overall rectangle shape of 66Å x 35 Å x 30 Å <ref name="bussiere" />. The long C-terminal helices (α4) facilitate binding by establishing a hydrophobic core between the monomers (residues 93-103.) The helices form an antiparallel coiled-coil structure and additionally contribute an amino acid to the hydrophobic core of the other monomer (residue 122)<ref name="bussiere" />. Both monomers still remain structurally similar when they form the dimer in solution. It should be noted that when in solution the flexible loop between β2 and β3 are able to assume different conformations <ref name="bussiere" />. This later gives rise to the "wing-up, wing-down" conformation when bound to native DNA<ref name="vivian">PMID:17521668</ref>.</StructureSection>

<StructureSection <applet load='1bm9' size='300' side='right' caption='Structure of RTP (PDB entry [[1bm9]])' scene='User:Chloe_Paul/Replication_Terminator_Protein/Rtpmonobasic/4'>RTP has been found to exist as a symmetric α+β protein in solution and a homomeric dimer through crystal structure determination <ref name="bussiere">PMID: 7867072</ref>.These identical monomers each contain four α helices (<scene name='User:Chloe_Paul/Replication_Terminator_Protein/Rtpmonobasica1/3'>α1</scene>, <scene name='User:Chloe_Paul/Replication_Terminator_Protein/Rtpmonobasica2/1'>α2</scene>, <scene name='User:Chloe_Paul/Replication_Terminator_Protein/Rtpmonobasica3/2'>α3</scene>, <scene name='User:Chloe_Paul/Replication_Terminator_Protein/Rtpmonobasica4/2'>α4</scene>), one β strand (<scene name='User:Chloe_Paul/Replication_Terminator_Protein/Rtpmonobasicb1/1'>β1</scene>) and two β ribbons (<scene name='User:Chloe_Paul/Replication_Terminator_Protein/Rtpmonobasicb2/2'>β2</scene> and <scene name='User:Chloe_Paul/Replication_Terminator_Protein/Rtpmonobasicb3/1'>β3</scene>) forming a <scene name='User:Chloe_Paul/Replication_Terminator_Protein/Rtpmonobasicbsheet/1'>β-sheet</scene>. It also contains a distordered <scene name='User:Chloe_Paul/Replication_Terminator_Protein/Rtpmonobasicn/1'>N-terminal region</scene>. When the two monomers come together and the two α4 helices bind, forming a dimer with an overall rectangle shape of 66Å x 35 Å x 30 Å <ref name="bussiere" />. The long C-terminal helices (α4) facilitate binding by establishing a hydrophobic core between the monomers (residues 93-103.) The helices form an antiparallel coiled-coil structure and additionally contribute an amino acid to the hydrophobic core of the other monomer (residue 122)<ref name="bussiere" />. Both monomers still remain structurally similar when they form the dimer in solution. It should be noted that when in solution the flexible loop between β2 and β3 are able to assume different conformations <ref name="bussiere" />. This later gives rise to the "wing-up, wing-down" conformation when bound to native DNA<ref name="vivian">PMID:17521668</ref>.</StructureSection>

== RTP binding to DNA ==

<StructureSection load='1f4k' size='300' side='right' caption='RTP bound to symmetric DNA (PDB entry [[1f4k]])' scene=''>RTP has been of interest in terms of its specific binding to DNA because it doesn’t use the common DNA structural motifs such as a basic leucine zipper, zinc finger or helix-turn-helix motif. It has been established that RTP, like Tus, is sequence specific, as it binds as the Ter sites. These comprise two sequences that are imperfect inverted repeats <ref name="bussiere" /><ref name="vivian" />. This means that RTP needs to be able to recognise specific bases in the helical DNA structure by reading the exposed edges of the bases located in the major and minor grooves of DNA. Structurally RTP interacts with DNA through the α3 helices in the major grooves, its anti-parallel β-sheets (β2 and β3) in the minor grooves. The flexible N-terminal regions wrap with non-specific ionic interactions around the DNA <ref name=~~“wilce”~~>PMID:11224562</ref>. </StructureSection>

<StructureSection load='1f4k' size='300' side='right' caption='RTP bound to symmetric DNA (PDB entry [[1f4k]])' scene='User:Chloe_Paul/Replication_Terminator_Protein/Rtpsym/1'>RTP has been of interest in terms of its specific binding to DNA because it doesn’t use the common DNA structural motifs such as a basic leucine zipper, zinc finger or helix-turn-helix motif. It has been established that RTP, like Tus, is sequence specific, as it binds as the Ter sites. These comprise two sequences that are imperfect inverted repeats <ref name="bussiere" /><ref name="vivian" />. This means that RTP needs to be able to recognise specific bases in the helical DNA structure by reading the exposed edges of the bases located in the major and minor grooves of DNA. Structurally RTP interacts with DNA through the α3 helices in the major grooves, its anti-parallel β-sheets (β2 and β3) in the minor grooves. The flexible N-terminal regions wrap with non-specific ionic interactions around the DNA <ref name="wilce">PMID:11224562</ref>. </StructureSection>

== ~~The~~ Asymmetric ~~binding~~ ==

== Symmetric and Asymmetric Conformations ==

<StructureSection load='2efw' size='300' side='right' caption='RTP bound to native DNA (PDB entry [[2efw]])' scene=''>The RTP:DNA interaction has been shown to be able to induce two different conformations of RTP depending upon the nature of the DNA. Early experiments used to determine how they interacted, used symmetric DNA (sDNA) which resulted in RTP binding symmetrically. However in nature, RTP was found to have a polar mechanism which implied ~~asymetric~~ binding, leading to further investigations of how RTP bound to DNA. It was later shown that when RTP bound to native or non-symmetric DNA (nDNA) it induced an asymmetric "wing-up, wing-down" form of RTP ~~with a~~ two faces. One face, known as the “blocking” face acts to terminate the approaching replication fork. The other face is described as the “permissive” face as it allows the replication fork to proceed along the DNA. These faces correspond to the A site and B site of the Ter sequence of DNA respectively. These DNA sites are the two halves of the pseudosymmetric palindromic sequence. The conformation and thus function of the RTP monomer depends on which site the RTP monomer binds to. It is the concept of these two faces that give rise to the polar mechanism of RTP.</StructureSection>

<StructureSection load='2efw' size='300' side='right' caption='RTP bound to native DNA (PDB entry [[2efw]])' scene='User:Chloe_Paul/Replication_Terminator_Protein/Rtpasym/3'>The RTP:DNA interaction has been shown to be able to induce two different conformations of RTP depending upon the nature of the DNA. Early experiments used to determine how they interacted, used symmetric DNA (sDNA) which resulted in RTP binding symmetrically<ref name="wilce" />. However in nature, RTP was found to have a polar mechanism which implied asymmetric binding, leading to further investigations of how RTP bound to DNA. It was later shown that when RTP bound to native or non-symmetric DNA (nDNA) it induced an asymmetric "wing-up, wing-down" form of RTP<ref name="vivian" />.

These two wings form the two faces of RTP.One face, known as the “blocking” face acts to terminate the approaching replication fork. The other face is described as the “permissive” face as it allows the replication fork to proceed along the DNA. These faces correspond to the A site and B site of the Ter sequence of DNA respectively. These DNA sites are the two halves of the pseudosymmetric palindromic sequence. The conformation and thus function of the RTP monomer depends on which site the RTP monomer binds to. It is the concept of these two faces that give rise to the polar mechanism of RTP.</StructureSection>

== Termination Mechanism ==

As previously noted the role of RTP is to terminate replication of the bacterial chromosome. It was originally assumed that the role of RTP was simply to arrest the replication fork allowing the DNA to cleanly separate <ref name=“wake”>PMID: 9271849</ref>. The proposed mechanism noted that the replication fork is only able to disrupt the RTP/Ter interaction when approaching the A-site/"blocking face". The directionality of the Ter sites (ie. the orientation of A site vs B site) will determine from which direction replication will be arrested. However recent research has indicated a more complex mechanism involving interactions between bound RTP and the replication fork helicase. The results of this research have confirmed a RTP/DnaB interaction in vivo, further suggesting this interaction plays an important role in replication fork arrest <ref name=“gautam”>PMID:11124956</ref>. This has lead to the development of a new helicase-specific model involving protein-protein interactions between the replication fork helicase and RTP protein which arrests the replication fork when it approaches from the appropriate direction <ref name=“kaplan”>PMID:19298368</ref>. This evidence ~~alows~~ us to move from a simple "fork arrest model" to a more complex understanding of termination.

As previously noted the role of RTP is to terminate replication of the bacterial chromosome. It was originally assumed that the role of RTP was simply to arrest the replication fork allowing the DNA to cleanly separate <ref name=“wake”>PMID: 9271849</ref>. The proposed mechanism noted that the replication fork is only able to disrupt the RTP/Ter interaction when approaching the A-site/"blocking face". The directionality of the Ter sites (ie. the orientation of A site vs B site) will determine from which direction replication will be arrested. However recent research has indicated a more complex mechanism involving interactions between bound RTP and the replication fork helicase. The results of this research have confirmed a RTP/DnaB interaction in vivo, further suggesting this interaction plays an important role in replication fork arrest<ref name=“gautam”>PMID:11124956</ref>. This has lead to the development of a new helicase-specific model involving protein-protein interactions between the replication fork helicase and RTP protein which arrests the replication fork when it approaches from the appropriate direction<ref name=“kaplan”>PMID:19298368</ref>. This evidence allows us to move from a simple "fork arrest model" to a more complex understanding of termination.

== Further Directions ==

RTP is frequently compared to Termination Utilisation Sequence (Tus) from E. coli. These two proteins display similar intracellular function with binding to Ter sites resulting in replication termination, despite the significant lack of identity and similarity between them (22% identity, 44% similarity) ~~(Ref)~~. Structurally these proteins differ as Tus has been demonstrated to be a monomer and an additional 300kbp larger than RTP (Ref). Investigations into their comparative function have shown that the substitution of RTP for Tus in the E.coli system, will demonstrate no phenotypic difference, and hence share the same function as replication terminators. ~~However the~~ question still remains to be answered how can two structurally different proteins give rise to the same intracellular function. Hopefully, further investigations will be able to shed more light as to how RTP and Tus, from B. subtilis and E. coli respecively, arrest the replication fork mechanism.

Further directions for research in relation to RTP and its function include: investigations into the helicase model and how it works, comparisons with similar proteins with a similar function and comparisons with initiator proteins (such as DnaA)<ref name="krause">PMID:9405146</ref>.

RTP is frequently compared to Termination Utilisation Sequence (Tus) from E. coli. These two proteins display similar intracellular function with binding to Ter sites resulting in replication termination, despite the significant lack of identity and similarity between them (22% identity, 44% similarity)<ref name="bussiere" />. Structurally these proteins differ as Tus has been demonstrated to be a monomer and an additional 300kbp larger than RTP<ref name="bussiere" />. The question still remains to be answered how can two structurally different proteins give rise to the same intracellular function. Hopefully, further investigations will be able to shed more light as to how RTP and Tus, from B. subtilis and E. coli respecively, arrest the replication fork mechanism.

==References==

@@ Line 2: / Line 2: @@
 == The Structure of RTP ==
-<StructureSection <applet load='1bm9' size='300' side='right' caption='Structure of RTP (PDB entry [[1bm9]])' scene='User:Chloe_Paul/Replication_Terminator_Protein/Rtpmonobasic/2'>RTP has been found to exist as a symmetric α+β protein in solution and a homomeric dimer through crystal structure determination <ref name="bussiere">PMID: 7867072</ref>.These identical monomers each contain four α helices (<scene name='User:Chloe_Paul/Replication_Terminator_Protein/Rtpmonobasica1/1'>α1</scene>, α2, α3, α4), one β strand (β1) and two β ribbons(β2 and β3). It also contains a distordered N-terminal region. When the two monomers come together and the two α4 helices bind, forming a dimer with an overall rectangle shape of 66Å x 35 Å x 30 Å <ref name="bussiere" />. The long C-terminal helices (α4) facilitate binding by establishing a hydrophobic core between the monomers (residues 93-103.) The helices form an antiparallel coiled-coil structure and additionally contribute an amino acid to the hydrophobic core of the other monomer (residue 122)<ref name="bussiere" />. Both monomers still remain structurally similar when they form the dimer in solution. It should be noted that when in solution the flexible loop between β2 and β3 are able to assume different conformations <ref name="bussiere" />. This later gives rise to the "wing-up, wing-down" conformation when bound to native DNA<ref name="vivian">PMID:17521668</ref>.</StructureSection>
+<StructureSection <applet load='1bm9' size='300' side='right' caption='Structure of RTP (PDB entry [[1bm9]])' scene='User:Chloe_Paul/Replication_Terminator_Protein/Rtpmonobasic/4'>RTP has been found to exist as a symmetric α+β protein in solution and a homomeric dimer through crystal structure determination <ref name="bussiere">PMID: 7867072</ref>.These identical monomers each contain four α helices (<scene name='User:Chloe_Paul/Replication_Terminator_Protein/Rtpmonobasica1/3'>α1</scene>, <scene name='User:Chloe_Paul/Replication_Terminator_Protein/Rtpmonobasica2/1'>α2</scene>, <scene name='User:Chloe_Paul/Replication_Terminator_Protein/Rtpmonobasica3/2'>α3</scene>, <scene name='User:Chloe_Paul/Replication_Terminator_Protein/Rtpmonobasica4/2'>α4</scene>), one β strand (<scene name='User:Chloe_Paul/Replication_Terminator_Protein/Rtpmonobasicb1/1'>β1</scene>) and two β ribbons (<scene name='User:Chloe_Paul/Replication_Terminator_Protein/Rtpmonobasicb2/2'>β2</scene> and <scene name='User:Chloe_Paul/Replication_Terminator_Protein/Rtpmonobasicb3/1'>β3</scene>) forming a <scene name='User:Chloe_Paul/Replication_Terminator_Protein/Rtpmonobasicbsheet/1'>β-sheet</scene>. It also contains a distordered <scene name='User:Chloe_Paul/Replication_Terminator_Protein/Rtpmonobasicn/1'>N-terminal region</scene>. When the two monomers come together and the two α4 helices bind, forming a dimer with an overall rectangle shape of 66Å x 35 Å x 30 Å <ref name="bussiere" />. The long C-terminal helices (α4) facilitate binding by establishing a hydrophobic core between the monomers (residues 93-103.) The helices form an antiparallel coiled-coil structure and additionally contribute an amino acid to the hydrophobic core of the other monomer (residue 122)<ref name="bussiere" />. Both monomers still remain structurally similar when they form the dimer in solution. It should be noted that when in solution the flexible loop between β2 and β3 are able to assume different conformations <ref name="bussiere" />. This later gives rise to the "wing-up, wing-down" conformation when bound to native DNA<ref name="vivian">PMID:17521668</ref>.</StructureSection>
 == RTP binding to DNA ==
-<StructureSection load='1f4k' size='300' side='right' caption='RTP bound to symmetric DNA (PDB entry [[1f4k]])' scene=''>RTP has been of interest in terms of its specific binding to DNA because it doesn’t use the common DNA structural motifs such as a basic leucine zipper, zinc finger or helix-turn-helix motif. It has been established that RTP, like Tus, is sequence specific, as it binds as the Ter sites. These comprise two sequences that are imperfect inverted repeats <ref name="bussiere" /><ref name="vivian" />. This means that RTP needs to be able to recognise specific bases in the helical DNA structure by reading the exposed edges of the bases located in the major and minor grooves of DNA. Structurally RTP interacts with DNA through the α3 helices in the major grooves, its anti-parallel β-sheets (β2 and β3) in the minor grooves. The flexible N-terminal regions wrap with non-specific ionic interactions around the DNA <ref name=“wilce”>PMID:11224562</ref>. </StructureSection>
+<StructureSection load='1f4k' size='300' side='right' caption='RTP bound to symmetric DNA (PDB entry [[1f4k]])' scene='User:Chloe_Paul/Replication_Terminator_Protein/Rtpsym/1'>RTP has been of interest in terms of its specific binding to DNA because it doesn’t use the common DNA structural motifs such as a basic leucine zipper, zinc finger or helix-turn-helix motif. It has been established that RTP, like Tus, is sequence specific, as it binds as the Ter sites. These comprise two sequences that are imperfect inverted repeats <ref name="bussiere" /><ref name="vivian" />. This means that RTP needs to be able to recognise specific bases in the helical DNA structure by reading the exposed edges of the bases located in the major and minor grooves of DNA. Structurally RTP interacts with DNA through the α3 helices in the major grooves, its anti-parallel β-sheets (β2 and β3) in the minor grooves. The flexible N-terminal regions wrap with non-specific ionic interactions around the DNA <ref name="wilce">PMID:11224562</ref>. </StructureSection>
-== The Asymmetric binding ==
+== Symmetric and Asymmetric Conformations ==
-<StructureSection load='2efw' size='300' side='right' caption='RTP bound to native DNA (PDB entry [[2efw]])' scene=''>The RTP:DNA interaction has been shown to be able to induce two different conformations of RTP depending upon the nature of the DNA. Early experiments used to determine how they interacted, used symmetric DNA (sDNA) which resulted in RTP binding symmetrically. However in nature, RTP was found to have a polar mechanism which implied asymetric binding, leading to further investigations of how RTP bound to DNA. It was later shown that when RTP bound to native or non-symmetric DNA (nDNA) it induced an asymmetric "wing-up, wing-down" form of RTP with a two faces. One face, known as the “blocking” face acts to terminate the approaching replication fork. The other face is described as the “permissive” face as it allows the replication fork to proceed along the DNA.  These faces correspond to the A site and B site of the Ter sequence of DNA respectively. These DNA sites are the two halves of the pseudosymmetric palindromic sequence. The conformation and thus function of the RTP monomer depends on which site the RTP monomer binds to.  It is the concept of these two faces that give rise to the polar mechanism of RTP.</StructureSection>
+<StructureSection load='2efw' size='300' side='right' caption='RTP bound to native DNA (PDB entry [[2efw]])' scene='User:Chloe_Paul/Replication_Terminator_Protein/Rtpasym/3'>The RTP:DNA interaction has been shown to be able to induce two different conformations of RTP depending upon the nature of the DNA. Early experiments used to determine how they interacted, used symmetric DNA (sDNA) which resulted in RTP binding symmetrically<ref name="wilce" />. However in nature, RTP was found to have a polar mechanism which implied asymmetric binding, leading to further investigations of how RTP bound to DNA. It was later shown that when RTP bound to native or non-symmetric DNA (nDNA) it induced an asymmetric "wing-up, wing-down" form of RTP<ref name="vivian" />.
+These two wings form the two faces of RTP.One face, known as the “blocking” face acts to terminate the approaching replication fork. The other face is described as the “permissive” face as it allows the replication fork to proceed along the DNA.  These faces correspond to the A site and B site of the Ter sequence of DNA respectively. These DNA sites are the two halves of the pseudosymmetric palindromic sequence. The conformation and thus function of the RTP monomer depends on which site the RTP monomer binds to.  It is the concept of these two faces that give rise to the polar mechanism of RTP.</StructureSection>
 == Termination Mechanism ==
-As previously noted the role of RTP is to terminate replication of the bacterial chromosome. It was originally assumed that the role of RTP was simply to arrest the replication fork allowing the DNA to cleanly separate <ref name=“wake”>PMID: 9271849</ref>. The proposed mechanism noted that the replication fork is only able to disrupt the RTP/Ter interaction when approaching the A-site/"blocking face". The directionality of the Ter sites (ie. the orientation of A site vs B site) will determine from which direction replication will be arrested. However recent research has indicated a more complex mechanism involving interactions between bound RTP and the replication fork helicase. The results of this research have confirmed a RTP/DnaB interaction in vivo, further suggesting this interaction plays an important role in replication fork arrest <ref name=“gautam”>PMID:11124956</ref>. This has lead to the development of a new helicase-specific model involving protein-protein interactions between the replication fork helicase and RTP protein which arrests the replication fork when it approaches from the appropriate direction <ref name=“kaplan”>PMID:19298368</ref>. This evidence alows us to move from a simple "fork arrest model" to a more complex understanding of termination.
+As previously noted the role of RTP is to terminate replication of the bacterial chromosome. It was originally assumed that the role of RTP was simply to arrest the replication fork allowing the DNA to cleanly separate <ref name=“wake”>PMID: 9271849</ref>. The proposed mechanism noted that the replication fork is only able to disrupt the RTP/Ter interaction when approaching the A-site/"blocking face". The directionality of the Ter sites (ie. the orientation of A site vs B site) will determine from which direction replication will be arrested. However recent research has indicated a more complex mechanism involving interactions between bound RTP and the replication fork helicase. The results of this research have confirmed a RTP/DnaB interaction in vivo, further suggesting this interaction plays an important role in replication fork arrest<ref name=“gautam”>PMID:11124956</ref>. This has lead to the development of a new helicase-specific model involving protein-protein interactions between the replication fork helicase and RTP protein which arrests the replication fork when it approaches from the appropriate direction<ref name=“kaplan”>PMID:19298368</ref>. This evidence allows us to move from a simple "fork arrest model" to a more complex understanding of termination.
 == Further Directions ==
-RTP is frequently compared to Termination Utilisation Sequence (Tus) from E. coli. These two proteins display similar intracellular function with binding to Ter sites resulting in replication termination, despite the significant lack of identity and similarity between them (22% identity, 44% similarity) (Ref). Structurally these proteins differ as Tus has been demonstrated to be a monomer and an additional 300kbp larger than RTP (Ref). Investigations into their comparative function have shown that the substitution of RTP for Tus in the E.coli system, will demonstrate no phenotypic difference, and hence share the same function as replication terminators. However the question still remains to be answered how can two structurally different proteins give rise to the same intracellular function. Hopefully, further investigations will be able to shed more light as to how RTP and Tus, from B. subtilis and E. coli respecively, arrest the replication fork mechanism.
+Further directions for research in relation to RTP and its function include: investigations into the helicase model and how it works, comparisons with similar proteins with a similar function and comparisons with initiator proteins (such as DnaA)<ref name="krause">PMID:9405146</ref>.
+RTP is frequently compared to Termination Utilisation Sequence (Tus) from E. coli. These two proteins display similar intracellular function with binding to Ter sites resulting in replication termination, despite the significant lack of identity and similarity between them (22% identity, 44% similarity)<ref name="bussiere" />. Structurally these proteins differ as Tus has been demonstrated to be a monomer and an additional 300kbp larger than RTP<ref name="bussiere" />. The question still remains to be answered how can two structurally different proteins give rise to the same intracellular function. Hopefully, further investigations will be able to shed more light as to how RTP and Tus, from B. subtilis and E. coli respecively, arrest the replication fork mechanism.
 ==References==
 <references />