Sandbox20: Difference between revisions

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~~Replication termination protein (RTP) binds to the Ter sites of~~ B. subtilis, ~~to halt progression of the replication fork. Two structurally identical RTP molecules form a dimeric complex upon binding to DNA. The~~ Ter ~~site is~~ 30 bp in length~~, and contains an~~ imperfect inverted 16 bp ~~repeat~~ overlapping at a ~~highly conserved trinucleotide sequence (~~TAT). ~~Differences in sequence distinguish~~ the ~~upstream ''~~Ter ~~A'' domain from~~ the downstream ~~''Ter B'' domain~~. ~~These~~ bind ~~their respective RTP molecules in slightly~~ different ~~manners, creating~~ an ~~asymmetric~~ complex ~~which will only halt~~ the progression of the replication fork if the B site is encountered first. The mechanism by which this is achieved is discussed below in relation to the structure of the <scene name='Sandbox20/2efw/3'>RTP complex</scene>.

In B. subtilis, Ter sites are 30 bp in length with two imperfect inverted 16 bp repeats overlapping at a TAT motif. The upstream portion of the Ter site is called TerA; and the downstream portion, TerB. The sequence differences between these cause the bound RTP dimers to bind with different affinity and generate an assymetric complex capable of halting the progression of the replication fork only if the B site is encountered first.The mechanism by which this is achieved is discussed below in relation to the structure of the <scene name='Sandbox20/2efw/3'>RTP complex</scene>.

===~~Structural Overview~~===

===The RTP Dimer===

The structure of an RTP monomer bears greatest similarity to the "''classic winged-helix''" motif, in which 'wings' project from the loop between the final two β sheets of a compact αβααββ structure. The two major variations from this theme are the absence of a β1 sheet (the corresponding region is instead termed the β1 loop), and the presence of a fourth elongate α-helix, which facilitates dimerisation. Each of these secondary structural elements are indicated in ~~the structure~~ <scene name='Sandbox20/2efw/8'>~~shown here~~</scene>.

The structure of an RTP monomer bears greatest similarity to the "''classic winged-helix''" motif, in which 'wings' project from the loop between the final two β sheets of a compact αβααββ structure. The two major variations from this theme are the absence of a β1 sheet (the corresponding region is instead termed the β1 loop), and the presence of a fourth elongate α-helix at the C-terminus, which facilitates dimerisation. Each of these secondary structural elements are indicated in <scene name='Sandbox20/2efw/8'>this structure</scene>.

Dimerisation is enabled by the association of α4 helices into an antiparellel coiled coil, and the conformation is subsequently stabilised by formation of interhelical salt bridges outside this region. An aromatic network forms on the inner surface to make a hydrophobic core, which confers additional stability. Residues that form part of this network are F11, F18, F45, F50 and Y22. The crystal structure of RTP in its unbound state was determined in 1995 (1BM9). A C110S mutant was then generated to prevent the aggregation of RTP through cysteine oxidation. With a very similar structure (1J0R) and almost no change in dimerisation and DNA-binding capacities, the mutant was the protein of choice for later studies.

The crystal structure of RTP was originally determined in 1995 <ref>PMID: 7867072</ref> in its unbound state, and submitted under the entry [[1bm9]]. Since then, three additional structures have been determined. The first of these, [[1f4k]], is of the RTP-DNA complex. The second, [[1j0r]] is of a single cysteine mutant. The most recent, [[2efw]], indicates the DNA-RTP complex in its native state and reveals an insight into the molecular mechanism of contrahelicase activity.

===DNA Binding===

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@@ Line 18: / Line 18: @@
 <Structure load='2EFW' size='300' frame='true' align='right' caption='RTP' scene='Sandbox20/2efw/3' />
-Replication termination protein (RTP) binds to the Ter sites of B. subtilis, to halt progression of the replication fork. Two structurally identical RTP molecules form a dimeric complex upon binding to DNA. The Ter site is 30 bp in length, and contains an imperfect inverted 16 bp repeat overlapping at a highly conserved trinucleotide sequence (TAT). Differences in sequence distinguish the upstream ''Ter A'' domain from the downstream ''Ter B'' domain. These bind their respective RTP molecules in slightly different manners, creating an asymmetric complex which will only halt the progression of the replication fork if the B site is encountered first. The mechanism by which this is achieved is discussed below in relation to the structure of the <scene name='Sandbox20/2efw/3'>RTP complex</scene>.
+In B. subtilis, Ter sites are 30 bp in length with two imperfect inverted 16 bp repeats overlapping at a TAT motif. The upstream portion of the Ter site is called TerA; and the downstream portion, TerB. The sequence differences between these cause the bound RTP dimers to bind with different affinity and generate an assymetric complex capable of halting the progression of the replication fork only if the  B site is encountered first.The mechanism by which this is achieved is discussed below in relation to the structure of the <scene name='Sandbox20/2efw/3'>RTP complex</scene>.
-===Structural Overview===
+===The RTP Dimer===
 [[Image:RTP 1 Symmetry.jpg | thumb | upright=1.3| left| The two subunits of the RTP dimer complex.]] [[Image:RTP Dimerisation.jpg | thumb | upright=1.2| right| Interactions between a4 helices facilitates dimerisation of RTP on the Ter DNA site.]]
-The structure of an RTP monomer bears greatest similarity to the "''classic winged-helix''" motif, in which 'wings' project from the loop between the final two β sheets of a compact αβααββ structure. The two major variations from this theme are the absence of a β1 sheet (the corresponding region is instead termed the β1 loop), and the presence of a fourth elongate α-helix, which facilitates dimerisation. Each of these secondary structural elements are indicated in the structure <scene name='Sandbox20/2efw/8'>shown here</scene>.
+The structure of an RTP monomer bears greatest similarity to the "''classic winged-helix''" motif, in which 'wings' project from the loop between the final two β sheets of a compact αβααββ structure. The two major variations from this theme are the absence of a β1 sheet (the corresponding region is instead termed the β1 loop), and the presence of a fourth elongate α-helix at the C-terminus, which facilitates dimerisation. Each of these secondary structural elements are indicated in <scene name='Sandbox20/2efw/8'>this structure</scene>.
+Dimerisation is enabled by the association of α4 helices into an antiparellel coiled coil, and the conformation is subsequently stabilised by formation of interhelical salt bridges outside this region. An aromatic network forms on the inner surface to make a hydrophobic core, which confers additional stability. Residues that form part of this network are F11, F18, F45, F50 and Y22. The crystal structure of RTP in its unbound state was determined in 1995 (1BM9). A C110S mutant was then generated to prevent the aggregation of RTP through cysteine oxidation. With a very similar structure (1J0R) and almost no change in dimerisation and DNA-binding capacities, the mutant was the protein of choice for later studies.
-The crystal structure of RTP was originally determined in 1995 <ref>PMID: 7867072</ref> in its unbound state, and submitted under the entry [[1bm9]]. Since then, three additional structures have been determined. The first of these, [[1f4k]], is of the RTP-DNA complex. The second, [[1j0r]] is of a single cysteine mutant. The most recent, [[2efw]], indicates the DNA-RTP complex in its native state and reveals an insight into the molecular mechanism of contrahelicase activity.
 ===DNA Binding===