User:Chloe Paul/Sandbox 1

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IntroductionIntroduction

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)[1]; 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 RTPThe 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 [2].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 [2]. 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)[2]. 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 [2]. Later giving rise to the "wing-up, wing-down" conformation when bound to native DNA[3].

Structure of RTP (PDB entry 1bm9)

Drag the structure with the mouse to rotate

RTP binding to DNARTP 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 [2][3]). 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 [4].

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 MechanismThe 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 [5]. 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 [6]. 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 [7].

Comparison to TusComparison 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.

ReferencesReferences

  1. ↑ 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 [1] Nucleic Acids Research (2010) Database Issue 38:D211-222
  2. ↑ 2.0 2.1 2.2 2.3 2.4 Bussiere DE, Bastia D, White SW. Crystal structure of the replication terminator protein from B. subtilis at 2.6 A. Cell. 1995 Feb 24;80(4):651-60. PMID:7867072
  3. ↑ 3.0 3.1 Vivian JP, Porter CJ, Wilce JA, Wilce MC. An asymmetric structure of the Bacillus subtilis replication terminator protein in complex with DNA. J Mol Biol. 2007 Jul 13;370(3):481-91. Epub 2007 Mar 2. PMID:17521668 doi:S0022-2836(07)00259-8
  4. ↑ Wilce JA, Vivian JP, Hastings AF, Otting G, Folmer RH, Duggin IG, Wake RG, Wilce MC. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Biol. 2001 Mar;8(3):206-10. PMID:11224562 doi:10.1038/84934
  5. ↑ Wake RG. Replication fork arrest and termination of chromosome replication in Bacillus subtilis. FEMS Microbiol Lett. 1997 Aug 15;153(2):247-54. PMID:9271849
  6. ↑ Gautam A, Bastia D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. J Biol Chem. 2001 Mar 23;276(12):8771-7. Epub 2000 Dec 21. PMID:11124956 doi:10.1074/jbc.M009538200
  7. ↑ Kaplan DL, Bastia D. Mechanisms of polar arrest of a replication fork. Mol Microbiol. 2009 Apr;72(2):279-85. Epub 2009 Mar 4. PMID:19298368 doi:10.1111/j.1365-2958.2009.06656.x
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