User:Chloe Paul/Sandbox 1 - Revision history

Chloe Paul at 08:26, 20 May 2011

Chloe Paul — Fri, 20 May 2011 08:26:58 GMT

←Older revision		Revision as of 08:26, 20 May 2011
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	== The Structure of RTP ==		== The Structure of RTP ==
-	<StructureSection <applet load='1bm9' size='500' side='right' caption='Structure of RTP (PDB entry [[1bm9]])' scene=''>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 (α1, α2, α3, α4), one β strand (β1) and two β ribbons(β2 and β3). Int also contains a distorted 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 conformation <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='500' side='right' caption='Structure of RTP (PDB entry [[1bm9]])' scene='Chloe_Paul/Sandbox_1/Basicrtp/1'>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 (α1, α2, α3, α4), one β strand (β1) and two β ribbons(β2 and β3). Int also contains a distorted 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 conformation <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 ==		== RTP binding to DNA ==

Chloe Paul at 07:47, 20 May 2011

Chloe Paul — Fri, 20 May 2011 07:47:35 GMT

←Older revision		Revision as of 07:47, 20 May 2011
Line 3:		Line 3:

	== The Structure of RTP ==		== The Structure of RTP ==
-	<StructureSection <applet load='1bm9' size='~~400~~' side='right' caption='Structure of RTP (PDB entry [[1bm9]])' scene=''>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 (α1, α2, α3, α4), one β strand (β1) and two β ribbons(β2 and β3). Int also contains a distorted 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 conformation <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='500' side='right' caption='Structure of RTP (PDB entry [[1bm9]])' scene=''>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 (α1, α2, α3, α4), one β strand (β1) and two β ribbons(β2 and β3). Int also contains a distorted 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 conformation <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 ==		== RTP binding to DNA ==
-	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 load='1f4k' size='500' 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>

-	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 pseudosummetric palendromic 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.	+	== The Asymetric binding ==
		+	<StructureSection load='2efw' size='500' 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 pseudosummetric palendromic 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 ==		== Termination Mechanism ==

Chloe Paul at 01:57, 19 May 2011

Chloe Paul — Thu, 19 May 2011 01:57:51 GMT

←Older revision		Revision as of 01:57, 19 May 2011
Line 1:		Line 1:
	== Introduction ==		== Introduction ==
-	Replication Terminator Protein (RTP) from Bacillus Subtilis is a protein of current scientific investigation in terms of its ability to bind DNA its symmetric and asymmetric nature, and the mechanism upon which it terminates DNA replication. Belonging to a group of Replication Terminator Proteins that are commonly found in prokaryotes (in particular within the Bacillaceae family)<ref> R.D. Finn, J. Mistry, J. Tate, P. Coggill, A. Heger, J.E. Pollington, O.L. Gavin, P. Gunesekaran, G. Ceric, K. Forslund, L. Holm, E.L. Sonnhammer, S.R. Eddy, A. Bateman The Pfam protein families database [http://pfam.sanger.ac.uk/family/PF02334#tabview=tab0] Nucleic Acids Research (2010) Database Issue 38:D211-222</ref>; RTP is often compared to another protein with similar intracellular function, Termination Utilisation Sequence (Tus) from E. coli. RTP has been shown to exist in both symmetric (in solution and when bound to palindromic DNA sequences) and asymmetric states (when bound to native DNA). ~~Its structural properties have proven~~ to be integral to ~~its function; as~~ it must be able to bind DNA and ~~have polarity~~ (despite it being a homomeric dimer) in order to specifically block DNA replication from one direction.	+	Replication Terminator Protein (RTP) from Bacillus Subtilis is a protein of current scientific investigation in terms of its ability to bind DNA, its symmetric and asymmetric nature, and the mechanism upon which it terminates DNA replication. Belonging to a group of Replication Terminator Proteins that are commonly found in prokaryotes (in particular within the Bacillaceae family)<ref> R.D. Finn, J. Mistry, J. Tate, P. Coggill, A. Heger, J.E. Pollington, O.L. Gavin, P. Gunesekaran, G. Ceric, K. Forslund, L. Holm, E.L. Sonnhammer, S.R. Eddy, A. Bateman The Pfam protein families database [http://pfam.sanger.ac.uk/family/PF02334#tabview=tab0] Nucleic Acids Research (2010) Database Issue 38:D211-222</ref>, RTP is often compared to another protein with similar intracellular function, Termination Utilisation Sequence (Tus) from E. coli. RTP has been shown to exist in both symmetric (in solution and when bound to palindromic DNA sequences) and asymmetric states (when bound to native DNA). The structure of RTP has been shown to be integral to it's function. RTP must be able to bind DNA (and therefore must be positively charged) and bind asymetrically (despite it being a homomeric dimer) in order to specifically block DNA replication from one direction.

	== The Structure of RTP ==		== The Structure of RTP ==
-	<StructureSection <applet load='1bm9' size='400' side='right' caption='Structure of RTP (PDB entry [[1bm9]])' scene=''>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 ~~subunits/~~monomers each contain four α helices (α1, α2, α3, α4) ~~and three~~ β ~~strands~~ (β1, β2, β3) ~~along with~~ a ~~disordered~~ N-terminal region. When the two ~~subunits~~ come together and the two α4 helices ~~align~~, ~~they form~~ a dimer with an overall rectangle shape of 66Å x 35 Å x 30 Å <ref name="bussiere" />. The long C-terminal helices (α4) ~~are involved in three interactions: contributing to the~~ hydrophobic core (residues 93-103), an antiparallel coiled-coil structure ~~(the two α4 helices coming together)~~ and ~~contributing~~ 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 conformation <ref name="bussiere" />. ~~Later giving~~ 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='400' side='right' caption='Structure of RTP (PDB entry [[1bm9]])' scene=''>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 (α1, α2, α3, α4), one β strand (β1) and two β ribbons(β2 and β3). Int also contains a distorted 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 conformation <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 ==		== RTP binding to DNA ==
-	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 ~~(comprising of~~ 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 ~~not involved in pairing~~. Structurally RTP interacts with DNA through ~~its alpha~~ helices in the major grooves, its anti-parallel β-~~strands~~ in the minor grooves ~~and the~~ flexible N-terminal regions ~~wrapping~~ with non-specific ionic interactions around the DNA <ref name=“wilce”>PMID:11224562</ref>.	+	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>.

-	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 ~~palindromic/~~symmetric DNA (sDNA) which ~~was demonstrated to maintain the symmetry of~~ RTP. However in nature, RTP was found to have a polar mechanism leading to further investigations of how RTP bound to DNA. It was later shown that when RTP bound to native/non-symmetric DNA (nDNA) ~~that~~ it induced an asymmetric "wing-up, wing-down" form of RTP with a two faces. One face is described as ~~being~~ the “permissive” face as it allows the replication fork to proceed ~~through when it approaches this face first~~. ~~Whilst~~ the ~~other face, known as~~ the ~~“blocking” face demonstrates~~ the ~~action~~ of ~~terminating~~ the ~~approaching replication fork~~. It is the concept of these two faces that give rise to the polar mechanism of RTP.	+	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 pseudosummetric palendromic 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.

-	==~~The Fork Arrest~~ Mechanism==	+	== 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". ~~This determines the polarity~~ of the ~~mechanism~~. 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 which arrests the replication fork when it approaches from the appropriate direction <ref name=“kaplan”>PMID:19298368</ref>.	+	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.

-	== ~~Comparison to Tus~~ ==	+	== 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. Investigations in to 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.		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. Investigations in to 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.

	==References==		==References==
	<references />		<references />

Chloe Paul at 05:27, 18 May 2011

Chloe Paul — Wed, 18 May 2011 05:27:47 GMT

←Older revision		Revision as of 05:27, 18 May 2011
Line 1:		Line 1:
	== Introduction ==		== Introduction ==
	Replication Terminator Protein (RTP) from Bacillus Subtilis is a protein of current scientific investigation in terms of its ability to bind DNA its symmetric and asymmetric nature, and the mechanism upon which it terminates DNA replication. Belonging to a group of Replication Terminator Proteins that are commonly found in prokaryotes (in particular within the Bacillaceae family)<ref> R.D. Finn, J. Mistry, J. Tate, P. Coggill, A. Heger, J.E. Pollington, O.L. Gavin, P. Gunesekaran, G. Ceric, K. Forslund, L. Holm, E.L. Sonnhammer, S.R. Eddy, A. Bateman The Pfam protein families database [http://pfam.sanger.ac.uk/family/PF02334#tabview=tab0] Nucleic Acids Research (2010) Database Issue 38:D211-222</ref>; RTP is often compared to another protein with similar intracellular function, Termination Utilisation Sequence (Tus) from E. coli. RTP has been shown to exist in both symmetric (in solution and when bound to palindromic DNA sequences) and asymmetric states (when bound to native DNA). Its structural properties have proven to be integral to its function; as it must be able to bind DNA and have polarity (despite it being a homomeric dimer) in order to specifically block DNA replication from one direction.		Replication Terminator Protein (RTP) from Bacillus Subtilis is a protein of current scientific investigation in terms of its ability to bind DNA its symmetric and asymmetric nature, and the mechanism upon which it terminates DNA replication. Belonging to a group of Replication Terminator Proteins that are commonly found in prokaryotes (in particular within the Bacillaceae family)<ref> R.D. Finn, J. Mistry, J. Tate, P. Coggill, A. Heger, J.E. Pollington, O.L. Gavin, P. Gunesekaran, G. Ceric, K. Forslund, L. Holm, E.L. Sonnhammer, S.R. Eddy, A. Bateman The Pfam protein families database [http://pfam.sanger.ac.uk/family/PF02334#tabview=tab0] Nucleic Acids Research (2010) Database Issue 38:D211-222</ref>; RTP is often compared to another protein with similar intracellular function, Termination Utilisation Sequence (Tus) from E. coli. RTP has been shown to exist in both symmetric (in solution and when bound to palindromic DNA sequences) and asymmetric states (when bound to native DNA). Its structural properties have proven to be integral to its function; as it must be able to bind DNA and have polarity (despite it being a homomeric dimer) in order to specifically block DNA replication from one direction.
		+
		+	== The Structure of RTP ==
		+	<StructureSection <applet load='1bm9' size='400' side='right' caption='Structure of RTP (PDB entry [[1bm9]])' scene=''>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 subunits/monomers each contain four α helices (α1, α2, α3, α4) and three β strands (β1, β2, β3) along with a disordered N-terminal region. When the two subunits come together and the two α4 helices align, they form a dimer with an overall rectangle shape of 66Å x 35 Å x 30 Å <ref name="bussiere" />. The long C-terminal helices (α4) are involved in three interactions: contributing to the hydrophobic core (residues 93-103), an antiparallel coiled-coil structure (the two α4 helices coming together) and contributing 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 conformation <ref name="bussiere" />. Later giving rise to the "wing-up, wing-down" conformation when bound to native DNA<ref name="vivian">PMID:17521668</ref>.</StructureSection>

-	== The Structure of RTP ==
-	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 subunits/monomers each contain four α helices (α1, α2, α3, α4) and three β strands (β1, β2, β3) along with a disordered N-terminal region. When the two subunits come together and the two α4 helices align, they form a dimer with an overall rectangle shape of 66Å x 35 Å x 30 Å <ref name="bussiere" />. The long C-terminal helices (α4) are involved in three interactions: contributing to the hydrophobic core (residues 93-103), an antiparallel coiled-coil structure (the two α4 helices coming together) and contributing 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 conformation <ref name="bussiere" />. Later giving rise to the "wing-up, wing-down" conformation when bound to native DNA<ref name="vivian">PMID:17521668</ref>.
-
	== RTP binding to DNA ==		== RTP binding to DNA ==
	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 (comprising of 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 not involved in pairing. Structurally RTP interacts with DNA through its alpha helices in the major grooves, its anti-parallel β-strands in the minor grooves and the flexible N-terminal regions wrapping with non-specific ionic interactions around the DNA <ref name=“wilce”>PMID:11224562</ref>.		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 (comprising of 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 not involved in pairing. Structurally RTP interacts with DNA through its alpha helices in the major grooves, its anti-parallel β-strands in the minor grooves and the flexible N-terminal regions wrapping with non-specific ionic interactions around the DNA <ref name=“wilce”>PMID:11224562</ref>.
-

	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 palindromic/symmetric DNA (sDNA) which was demonstrated to maintain the symmetry of RTP. However in nature, RTP was found to have a polar mechanism leading to further investigations of how RTP bound to DNA. It was later shown that when RTP bound to native/non-symmetric DNA (nDNA) that it induced an asymmetric "wing-up, wing-down" form of RTP with a two faces. One face is described as being the “permissive” face as it allows the replication fork to proceed through when it approaches this face first. Whilst the other face, known as the “blocking” face demonstrates the action of terminating the approaching replication fork. It is the concept of these two faces that give rise to the polar mechanism of RTP.		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 palindromic/symmetric DNA (sDNA) which was demonstrated to maintain the symmetry of RTP. However in nature, RTP was found to have a polar mechanism leading to further investigations of how RTP bound to DNA. It was later shown that when RTP bound to native/non-symmetric DNA (nDNA) that it induced an asymmetric "wing-up, wing-down" form of RTP with a two faces. One face is described as being the “permissive” face as it allows the replication fork to proceed through when it approaches this face first. Whilst the other face, known as the “blocking” face demonstrates the action of terminating the approaching replication fork. It is the concept of these two faces that give rise to the polar mechanism of RTP.

Chloe Paul at 23:52, 15 May 2011

Chloe Paul — Sun, 15 May 2011 23:52:17 GMT

←Older revision		Revision as of 23:52, 15 May 2011
Line 1:		Line 1:
	== Introduction ==		== Introduction ==
-	Replication Terminator Protein (RTP) from Bacillus Subtilis is a protein of current scientific investigation in terms of its symmetric and asymmetric nature, ~~its ability to bind DNA~~ and the mechanism upon which it terminates DNA replication. Belonging to a group of Replication Terminator Proteins that are commonly found in prokaryotes (in particular within the Bacillaceae family)<ref> R.D. Finn, J. Mistry, J. Tate, P. Coggill, A. Heger, J.E. Pollington, O.L. Gavin, P. Gunesekaran, G. Ceric, K. Forslund, L. Holm, E.L. Sonnhammer, S.R. Eddy, A. Bateman The Pfam protein families database [http://pfam.sanger.ac.uk/family/PF02334#tabview=tab0] Nucleic Acids Research (2010) Database Issue 38:D211-222</ref>; RTP is often compared to another protein with a similar intracellular function, Termination Utilisation Sequence (Tus) from E. coli. RTP has been shown to exist in both symmetric (in solution and when bound to palindromic DNA sequences) and asymmetric states (when bound to native DNA). Its structural properties have proven to be integral to its function; as it must be able to bind DNA and have polarity (despite it being a homomeric dimer) in order to specifically block DNA replication from one direction ~~and yet be “permissive” at the other end~~.	+	Replication Terminator Protein (RTP) from Bacillus Subtilis is a protein of current scientific investigation in terms of its ability to bind DNA its symmetric and asymmetric nature, and the mechanism upon which it terminates DNA replication. Belonging to a group of Replication Terminator Proteins that are commonly found in prokaryotes (in particular within the Bacillaceae family)<ref> R.D. Finn, J. Mistry, J. Tate, P. Coggill, A. Heger, J.E. Pollington, O.L. Gavin, P. Gunesekaran, G. Ceric, K. Forslund, L. Holm, E.L. Sonnhammer, S.R. Eddy, A. Bateman The Pfam protein families database [http://pfam.sanger.ac.uk/family/PF02334#tabview=tab0] Nucleic Acids Research (2010) Database Issue 38:D211-222</ref>; RTP is often compared to another protein with similar intracellular function, Termination Utilisation Sequence (Tus) from E. coli. RTP has been shown to exist in both symmetric (in solution and when bound to palindromic DNA sequences) and asymmetric states (when bound to native DNA). Its structural properties have proven to be integral to its function; as it must be able to bind DNA and have polarity (despite it being a homomeric dimer) in order to specifically block DNA replication from one direction.

	== The Structure of RTP ==		== The Structure of RTP ==
-	RTP has been found to exist as a symmetric protein in solution as a homomeric dimer through crystal structure determination <ref name="bussiere">PMID: 7867072</ref>.These identical subunits/monomers each contain four α helices (α1, α2, α3, α4) and three β strands (β1, β2, β3) along with a disordered N-terminal region. When the two subunits come together and the two α4 helices align, they form ~~the~~ dimer with an overall rectangle shape of 66Å x 35 Å x 30 Å <ref name="bussiere" />. The long C-terminal helices (α4) are involved three interactions: contributing to the hydrophobic core (residues 93-103), an antiparallel coiled-coil structure (the two α4 helices coming together) and ~~the~~ contributing 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 conformation <ref name="bussiere" />.	+	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 subunits/monomers each contain four α helices (α1, α2, α3, α4) and three β strands (β1, β2, β3) along with a disordered N-terminal region. When the two subunits come together and the two α4 helices align, they form a dimer with an overall rectangle shape of 66Å x 35 Å x 30 Å <ref name="bussiere" />. The long C-terminal helices (α4) are involved in three interactions: contributing to the hydrophobic core (residues 93-103), an antiparallel coiled-coil structure (the two α4 helices coming together) and contributing 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 conformation <ref name="bussiere" />. Later giving rise to the "wing-up, wing-down" conformation when bound to native DNA<ref name="vivian">PMID:17521668</ref>.

	== RTP binding to DNA ==		== RTP binding to DNA ==
-	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 (comprising of two sequences that are imperfect inverted repeats <ref name="bussiere" /><ref name=~~”vivian”>PMID:17521668<~~/~~ref~~>). 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 not involved in pairing. Structurally RTP interacts with DNA through its alpha helices in the major grooves, its anti-parallel β-strands in the minor grooves and the flexible N-terminal regions wrapping with non-specific ionic interactions around the DNA <ref name=“wilce”>PMID:11224562</ref>.	+	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 (comprising of 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 not involved in pairing. Structurally RTP interacts with DNA through its alpha helices in the major grooves, its anti-parallel β-strands in the minor grooves and the flexible N-terminal regions wrapping with non-specific ionic interactions around the DNA <ref name=“wilce”>PMID:11224562</ref>.


-	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 palindromic/symmetric DNA (sDNA) which was demonstrated to maintain the symmetry of RTP. However in nature, RTP was found to have a polar mechanism leading to further investigations of how RTP bound to DNA. It was later shown that when RTP bound to native/non-symmetric DNA (nDNA) that it induced an asymmetric form of RTP with a two faces. One face is described as being the “permissive” face as it allows the replication fork when it approaches this face first ~~to proceed through~~. Whilst the other face, known as the “blocking” face demonstrates the action of terminating the approaching replication fork. It is the concept of these two faces that give rise to the polar mechanism of RTP.	+	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 palindromic/symmetric DNA (sDNA) which was demonstrated to maintain the symmetry of RTP. However in nature, RTP was found to have a polar mechanism leading to further investigations of how RTP bound to DNA. It was later shown that when RTP bound to native/non-symmetric DNA (nDNA) that it induced an asymmetric "wing-up, wing-down" form of RTP with a two faces. One face is described as being the “permissive” face as it allows the replication fork to proceed through when it approaches this face first. Whilst the other face, known as the “blocking” face demonstrates the action of terminating the approaching replication fork. It is the concept of these two faces that give rise to the polar mechanism of RTP.

	==The Fork Arrest Mechanism==		==The Fork Arrest 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. This ~~explains~~ the polarity of the mechanism. However recent research has indicated a more complex mechanism involving interactions between ~~the~~ 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 which arrests the replication fork when it approaches from the appropriate direction <ref name=“kaplan”>PMID:19298368</ref>.	+	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". This determines the polarity of the mechanism. 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 which arrests the replication fork when it approaches from the appropriate direction <ref name=“kaplan”>PMID:19298368</ref>.
		+
		+	== Comparison to Tus ==
		+	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. Investigations in to 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.

	==References==		==References==
	<references />		<references />

Chloe Paul at 11:54, 14 May 2011

Chloe Paul — Sat, 14 May 2011 11:54:53 GMT

←Older revision		Revision as of 11:54, 14 May 2011
Line 6:		Line 6:

	== RTP binding to DNA ==		== RTP binding to DNA ==
-	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 (comprising of two sequences that are imperfect inverted repeats ~~(Vivian et al, 2007~~ <ref name="bussiere" />). 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 not involved in pairing. Structurally RTP interacts with DNA through its alpha helices in the major grooves, its anti-parallel β-strands in the minor grooves and the flexible N-terminal regions wrapping with non-specific ionic interactions around the DNA ~~(Wilce et al, 2001)~~.	+	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 (comprising of two sequences that are imperfect inverted repeats <ref name="bussiere" /><ref name=”vivian”>PMID:17521668</ref>). 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 not involved in pairing. Structurally RTP interacts with DNA through its alpha helices in the major grooves, its anti-parallel β-strands in the minor grooves and the flexible N-terminal regions wrapping with non-specific ionic interactions around the DNA <ref name=“wilce”>PMID:11224562</ref>.


Line 12:		Line 12:

	==The Fork Arrest Mechanism==		==The Fork Arrest 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 ~~(Wake 1997~~.) The proposed mechanism noted that the replication fork is only able to disrupt the RTP/ter interaction when approaching the A-site. This explains the polarity of the mechanism. However recent research has indicated a more complex mechanism involving interactions between the 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 ~~(Gautam 2001~~.) This has lead to the development of a new helicase-specific model involving protein-protein interactions between the replication fork helicase and RTP which arrests the replication fork when it approaches from the appropriate direction ~~(Kaplan 2009~~.)	+	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. This explains the polarity of the mechanism. However recent research has indicated a more complex mechanism involving interactions between the 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 which arrests the replication fork when it approaches from the appropriate direction <ref name=“kaplan”>PMID:19298368</ref>.

	==References==		==References==
	<references />		<references />

Chloe Paul at 11:32, 14 May 2011

Chloe Paul — Sat, 14 May 2011 11:32:34 GMT

←Older revision		Revision as of 11:32, 14 May 2011
Line 1:		Line 1:
	== Introduction ==		== Introduction ==
-	Replication Terminator Protein (RTP) from Bacillus Subtilis is a protein of current scientific investigation in terms of its symmetric and asymmetric nature, its ability to bind DNA and the mechanism upon which it terminates DNA replication. Belonging to a group of Replication Terminator Proteins that are commonly found in prokaryotes (in particular within the Bacillaceae family) (Pfam); RTP is often compared to another protein with a similar intracellular function, Termination Utilisation Sequence (Tus) from E. coli. RTP has been shown to exist in both symmetric (in solution and when bound to palindromic DNA sequences) and asymmetric states (when bound to native DNA). Its structural properties have proven to be integral to its function; as it must be able to bind DNA and have polarity (despite it being a homomeric dimer) in order to specifically block DNA replication from one direction and yet be “permissive” at the other end.	+	Replication Terminator Protein (RTP) from Bacillus Subtilis is a protein of current scientific investigation in terms of its symmetric and asymmetric nature, its ability to bind DNA and the mechanism upon which it terminates DNA replication. Belonging to a group of Replication Terminator Proteins that are commonly found in prokaryotes (in particular within the Bacillaceae family)<ref> R.D. Finn, J. Mistry, J. Tate, P. Coggill, A. Heger, J.E. Pollington, O.L. Gavin, P. Gunesekaran, G. Ceric, K. Forslund, L. Holm, E.L. Sonnhammer, S.R. Eddy, A. Bateman The Pfam protein families database [http://pfam.sanger.ac.uk/family/PF02334#tabview=tab0] Nucleic Acids Research (2010) Database Issue 38:D211-222</ref>; RTP is often compared to another protein with a similar intracellular function, Termination Utilisation Sequence (Tus) from E. coli. RTP has been shown to exist in both symmetric (in solution and when bound to palindromic DNA sequences) and asymmetric states (when bound to native DNA). Its structural properties have proven to be integral to its function; as it must be able to bind DNA and have polarity (despite it being a homomeric dimer) in order to specifically block DNA replication from one direction and yet be “permissive” at the other end.

	== The Structure of RTP ==		== The Structure of RTP ==
-	RTP has been found to exist as a symmetric protein in solution as a homomeric dimer through crystal structure determination ~~(Bussiere, 1995)~~. These identical subunits/monomers each contain four α helices (α1, α2, α3, α4) and three β strands (β1, β2, β3) along with a disordered N-terminal region. When the two subunits come together and the two α4 helices align, they form the dimer with an overall rectangle shape of 66Å x 35 Å x 30 Å. The long C-terminal helices (α4) are involved three interactions: contributing to the hydrophobic core (residues 93-103), an antiparallel coiled-coil structure (the two α4 helices coming together) and the contributing to the hydrophobic core of the other monomer (residue 122). 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~~. ~~(Bussiere, 1995)~~	+	RTP has been found to exist as a symmetric protein in solution as a homomeric dimer through crystal structure determination <ref name="bussiere">PMID: 7867072</ref>.These identical subunits/monomers each contain four α helices (α1, α2, α3, α4) and three β strands (β1, β2, β3) along with a disordered N-terminal region. When the two subunits come together and the two α4 helices align, they form the dimer with an overall rectangle shape of 66Å x 35 Å x 30 Å <ref name="bussiere" />. The long C-terminal helices (α4) are involved three interactions: contributing to the hydrophobic core (residues 93-103), an antiparallel coiled-coil structure (the two α4 helices coming together) and the contributing 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 conformation <ref name="bussiere" />.
-		+
	== RTP binding to DNA ==		== RTP binding to DNA ==
-	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 (comprising of two sequences that are imperfect inverted repeats (Vivian et al, 2007 ~~and Bussiere 1995)~~). 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 not involved in pairing. Structurally RTP interacts with DNA through its alpha helices in the major grooves, its anti-parallel β-strands in the minor grooves and the flexible N-terminal regions wrapping with non-specific ionic interactions around the DNA (Wilce et al, 2001).	+	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 (comprising of two sequences that are imperfect inverted repeats (Vivian et al, 2007 <ref name="bussiere" />). 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 not involved in pairing. Structurally RTP interacts with DNA through its alpha helices in the major grooves, its anti-parallel β-strands in the minor grooves and the flexible N-terminal regions wrapping with non-specific ionic interactions around the DNA (Wilce et al, 2001).


Line 13:		Line 13:
	==The Fork Arrest Mechanism==		==The Fork Arrest 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 (Wake 1997.) The proposed mechanism noted that the replication fork is only able to disrupt the RTP/ter interaction when approaching the A-site. This explains the polarity of the mechanism. However recent research has indicated a more complex mechanism involving interactions between the 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 (Gautam 2001.) This has lead to the development of a new helicase-specific model involving protein-protein interactions between the replication fork helicase and RTP which arrests the replication fork when it approaches from the appropriate direction (Kaplan 2009.)		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 (Wake 1997.) The proposed mechanism noted that the replication fork is only able to disrupt the RTP/ter interaction when approaching the A-site. This explains the polarity of the mechanism. However recent research has indicated a more complex mechanism involving interactions between the 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 (Gautam 2001.) This has lead to the development of a new helicase-specific model involving protein-protein interactions between the replication fork helicase and RTP which arrests the replication fork when it approaches from the appropriate direction (Kaplan 2009.)
		+
		+	==References==
		+	<references />

Chloe Paul at 08:22, 8 May 2011

Chloe Paul — Sun, 08 May 2011 08:22:47 GMT

←Older revision		Revision as of 08:22, 8 May 2011
Line 4:		Line 4:
	== The Structure of RTP ==		== The Structure of RTP ==
	RTP has been found to exist as a symmetric protein in solution as a homomeric dimer through crystal structure determination (Bussiere, 1995). These identical subunits/monomers each contain four α helices (α1, α2, α3, α4) and three β strands (β1, β2, β3) along with a disordered N-terminal region. When the two subunits come together and the two α4 helices align, they form the dimer with an overall rectangle shape of 66Å x 35 Å x 30 Å. The long C-terminal helices (α4) are involved three interactions: contributing to the hydrophobic core (residues 93-103), an antiparallel coiled-coil structure (the two α4 helices coming together) and the contributing to the hydrophobic core of the other monomer (residue 122). 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. (Bussiere, 1995)		RTP has been found to exist as a symmetric protein in solution as a homomeric dimer through crystal structure determination (Bussiere, 1995). These identical subunits/monomers each contain four α helices (α1, α2, α3, α4) and three β strands (β1, β2, β3) along with a disordered N-terminal region. When the two subunits come together and the two α4 helices align, they form the dimer with an overall rectangle shape of 66Å x 35 Å x 30 Å. The long C-terminal helices (α4) are involved three interactions: contributing to the hydrophobic core (residues 93-103), an antiparallel coiled-coil structure (the two α4 helices coming together) and the contributing to the hydrophobic core of the other monomer (residue 122). 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. (Bussiere, 1995)
		+
		+	== RTP binding to DNA ==
		+	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 (comprising of two sequences that are imperfect inverted repeats (Vivian et al, 2007 and Bussiere 1995)). 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 not involved in pairing. Structurally RTP interacts with DNA through its alpha helices in the major grooves, its anti-parallel β-strands in the minor grooves and the flexible N-terminal regions wrapping with non-specific ionic interactions around the DNA (Wilce et al, 2001).
		+
		+
		+	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 palindromic/symmetric DNA (sDNA) which was demonstrated to maintain the symmetry of RTP. However in nature, RTP was found to have a polar mechanism leading to further investigations of how RTP bound to DNA. It was later shown that when RTP bound to native/non-symmetric DNA (nDNA) that it induced an asymmetric form of RTP with a two faces. One face is described as being the “permissive” face as it allows the replication fork when it approaches this face first to proceed through. Whilst the other face, known as the “blocking” face demonstrates the action of terminating the approaching replication fork. It is the concept of these two faces that give rise to the polar mechanism of RTP.

	==The Fork Arrest Mechanism==		==The Fork Arrest 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 (Wake 1997.) The proposed mechanism noted that the replication fork is only able to disrupt the RTP/ter interaction when approaching the A-site. This explains the polarity of the mechanism. However recent research has indicated a more complex mechanism involving interactions between the 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 (Gautam 2001.) This has lead to the development of a new helicase-specific model involving protein-protein interactions between the replication fork helicase and RTP which arrests the replication fork when it approaches from the appropriate direction (Kaplan 2009.)		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 (Wake 1997.) The proposed mechanism noted that the replication fork is only able to disrupt the RTP/ter interaction when approaching the A-site. This explains the polarity of the mechanism. However recent research has indicated a more complex mechanism involving interactions between the 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 (Gautam 2001.) This has lead to the development of a new helicase-specific model involving protein-protein interactions between the replication fork helicase and RTP which arrests the replication fork when it approaches from the appropriate direction (Kaplan 2009.)

Chloe Paul: adding fork arrest section

Chloe Paul — Wed, 04 May 2011 11:15:53 GMT

adding fork arrest section

←Older revision		Revision as of 11:15, 4 May 2011
Line 4:		Line 4:
	== The Structure of RTP ==		== The Structure of RTP ==
	RTP has been found to exist as a symmetric protein in solution as a homomeric dimer through crystal structure determination (Bussiere, 1995). These identical subunits/monomers each contain four α helices (α1, α2, α3, α4) and three β strands (β1, β2, β3) along with a disordered N-terminal region. When the two subunits come together and the two α4 helices align, they form the dimer with an overall rectangle shape of 66Å x 35 Å x 30 Å. The long C-terminal helices (α4) are involved three interactions: contributing to the hydrophobic core (residues 93-103), an antiparallel coiled-coil structure (the two α4 helices coming together) and the contributing to the hydrophobic core of the other monomer (residue 122). 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. (Bussiere, 1995)		RTP has been found to exist as a symmetric protein in solution as a homomeric dimer through crystal structure determination (Bussiere, 1995). These identical subunits/monomers each contain four α helices (α1, α2, α3, α4) and three β strands (β1, β2, β3) along with a disordered N-terminal region. When the two subunits come together and the two α4 helices align, they form the dimer with an overall rectangle shape of 66Å x 35 Å x 30 Å. The long C-terminal helices (α4) are involved three interactions: contributing to the hydrophobic core (residues 93-103), an antiparallel coiled-coil structure (the two α4 helices coming together) and the contributing to the hydrophobic core of the other monomer (residue 122). 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. (Bussiere, 1995)
		+
		+	==The Fork Arrest 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 (Wake 1997.) The proposed mechanism noted that the replication fork is only able to disrupt the RTP/ter interaction when approaching the A-site. This explains the polarity of the mechanism. However recent research has indicated a more complex mechanism involving interactions between the 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 (Gautam 2001.) This has lead to the development of a new helicase-specific model involving protein-protein interactions between the replication fork helicase and RTP which arrests the replication fork when it approaches from the appropriate direction (Kaplan 2009.)

Chloe Paul: New page: == Introduction == Replication Terminator Protein (RTP) from Bacillus Subtilis is a protein of current scientific investigation in terms of its symmetric and asymmetric nature, its ability...

Chloe Paul — Wed, 04 May 2011 07:22:35 GMT

New page: == Introduction == Replication Terminator Protein (RTP) from Bacillus Subtilis is a protein of current scientific investigation in terms of its symmetric and asymmetric nature, its ability...

New page

== Introduction ==
Replication Terminator Protein (RTP) from Bacillus Subtilis is a protein of current scientific investigation in terms of its symmetric and asymmetric nature, its ability to bind DNA and the mechanism upon which it terminates DNA replication. Belonging to a group of Replication Terminator Proteins that are commonly found in prokaryotes (in particular within the Bacillaceae family) (Pfam); RTP is often compared to another protein with a similar intracellular function, Termination Utilisation Sequence (Tus) from E. coli. RTP has been shown to exist in both symmetric (in solution and when bound to palindromic DNA sequences) and asymmetric states (when bound to native DNA). Its structural properties have proven to be integral to its function; as it must be able to bind DNA and have polarity (despite it being a homomeric dimer) in order to specifically block DNA replication from one direction and yet be “permissive” at the other end.

== The Structure of RTP ==
RTP has been found to exist as a symmetric protein in solution as a homomeric dimer through crystal structure determination (Bussiere, 1995). These identical subunits/monomers each contain four α helices (α1, α2, α3, α4) and three β strands (β1, β2, β3) along with a disordered N-terminal region. When the two subunits come together and the two α4 helices align, they form the dimer with an overall rectangle shape of 66Å x 35 Å x 30 Å. The long C-terminal helices (α4) are involved three interactions: contributing to the hydrophobic core (residues 93-103), an antiparallel coiled-coil structure (the two α4 helices coming together) and the contributing to the hydrophobic core of the other monomer (residue 122). 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. (Bussiere, 1995)