User:Nathan Harris/Tus
From Proteopedia
Tus is a DNA binding protein involved in the termination of bi-directional replication in Escherichia coli. Tus binds specifically to Ter sequences within the E. Coli genome forming a Tus- Ter complex which functions to trap replication forks. Tus binds to Ter sites as an asymmetric monomer creating a permissive and non-permissive face to allow for polar fork arrest [1].
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Biological role
Multiple Ter sites (TerA- TerJ) are located in regions destined for replication termination in E. coli. Tus binds specifically to these 23bp Ter sites forming a Tus-Ter complex . This complex allows for the blocking of an approaching replication fork in one direction, the non-permissive face, but not from the other direction, the permissive face. The ability to halt the replication machinery at the non-permissive face is thought to involve the inhibition of DnaB Helicase, preventing it from unwinding DNA. DnaB inhibition has been proposed to occur either through protein-protein interactions between Tus and DnaB, or by a physical block provided by Protein-DNA interactions i.e. the Tus-Ter complex [2]. Recent models suggest a potentially combination of these two mechanisms [3]. Evolution of this termination system has allowed for efficient replication by E. coli as it prevents any over expenditure of energy or time. Different replication proteins have been found in other model organisms, such as RTP in Bacillus subtilis. Despite similar biological roles of RTP and Tus they have significantly different structures [3].
Ter sites
The Tus protein binds as a monomer through several direct and indirect contacts to conserved Ter sites. Ter sites are signified by 23 bp of consensus sequences which maintain a highly conserved C6 and 13 bp core region that interacts with Tus. Additionally, Ter sites are arranged in groups of five located opposite to the origin of replication. Within each group the Ter sites have a coordinated polarity of termination [1].
Structure of Tus protein and binding interactions with TerA
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| Resources: | FirstGlance, OCA, PDBsum, RCSB | ||||||||
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Tus constitutes 308 amino acids and a mass of approximately 36 kDa. The structural components of Tus have been elucidated through crystal structures of Tus bound to [2]. Tus exhibits a unique binding motif to Ter sites previously undescribed from any known protein-DNA interactions.
Tus is divided into an and distinguished by two alpha helical regions and central β sheets combining to encompass a large central basic cleft. The consists of anti-parallel β strands and an which connect the amino and carboxy domains. Within this interdomain region, the , , and strands are responsible for specific and non-specific recognition of Ter.
The amino domain consists of three amphipathic alpha helices forming an anti-parallel bundle roughly parallel to Ter, a sandwich of anti-parallel β sheets and three loops. The major groove and minor groove are clamped by two alpha helices ( and ) which also contribute to the hydrophobic core of the protein. Within the β sandwich, contacts the alpha helical region, whereas is associated with DNA binding. Furthermore, the extended L4 loop is also involved in contacts to the minor groove.
The carboxy domain consists of a hydrophobic core stabilised by alpha helices and β strands (βGHNO). The is responsible for connecting helices and and also contacts the minor groove of DNA [1][2].
Confirmation changes induced on Ter sites
The Ter region in E.coli between bases T5 and A9 is significantly underwound upon binding with Tus. This region of DNA is altered from standard B form which is attributed to straddling of Ter by interdomain β strands (βF and βG) and the L4 connecting loop of Tus. Tus interacts with Ter in a previously undescribed manner with β strands of Tus inserting almost perpendicularly into the major groove to recognise Ter. Alteration of Ter is characterised by an extended major groove and a broadened minor groove generating an overall DNA bend of 20 degrees. Overall, contacts in these regions account for increased stability of the altered DNA shape and allow recognition of the appropriate Ter site [1][2].
Mechanism of action
The ability of Tus to terminate replication in E. coli in a polar manner is believed to involve the inhibition of DnaB helicase. This is achieved either through a “locked complex” model provided by Tus-Ter interactions providing a physical block, protein-protein interactions between Tus and DnaB, or through a combination of these two effects [2][3].
The Tus- Ter locked complex
It has been suggested that the affinity of Tus for Ter may contribute to the polar arrest of replication in E. coli demonstrated by a direct correlation between the affinity and replication termination [4]. Investigations of the affinity of Tus for partially unwound Ter DNA have provided crystal structures of Tus bound to Ter unwound at the of Ter. These crystal structures show the C6 of Ter flipped up into a of Tus forming a so called locked complex. This locking results in a dramatic increase in the affinity of Tus for Ter. In contrast, the progressive unwinding of Ter from the permissive face results in dissociation of Tus from Ter. It is interesting to note that this C6 is conserved amongst all Ter sequences, further demonstrating the likelihood of its importance in replication arrest. This leads to a model suggesting that DnaB approaching from the non-permissive face unwinds Ter until it reaches the C6. When C6 is unwound it flips to form a locked complex with Tus hence preventing any further progression of the replication machinery, i.e. a physical block to the DnaB. However when the DnaB approaches from the permissive face, the C6 is located at the opposite end of the Ter sequence and so is unable to form a locked complex with Tus leading to dissociation of Tus and progression of the replication fork. However, when E. coli Ter sequences are inserted into a plasmid in B. Subtillis expressing Tus, the replication fork arrest from the non-permissive end only occurs with 0.5% efficiency compared to 45.4% efficiency in a wild type E. coli system [5]. If only Tus-Ter interactions were important in the mediation of polar fork arrest, then the efficiency in the two systems should be similar. This highlights the importance of other factors in the mediation of polar fork arrest.
Tus-DnaB interactions
Numerous studies support a model for replication termination resulting specifically from Tus-DnaB protein interactions. Experimentation in the field has demonstrated that the within the L1 loop of the non-permissive face of Tus is important in the formation of protein-protein interactions with DnaB. When this glutamic acid is exchanged for lysine (E49K), an increase in affinity for Ter and a decrease in affinity for DnaB result [6][7]. Despite the increased affinity for Ter, this E49K mutatation results in a reduced capability of polar replication fork termination demonstrating the importance of Tus-DnaB interactions. In further confirmation of this helicase specific mechanism, the engineering of intra-strand covalent crosslinks introduced immediately upstream of the C6 of Ter prevent DnaB helicase from unwinding the C6 [8]. Despite this inability to unwind and from a locked complex with Tus, polar fork termination is still permitted indicating that the formation of a locked complex is unnecessary for replication termination.
Current Models
Recent models for the termination of replication in E. coli propose that when DnaB approaches the Tus-Ter complex from the permissive face there are no considerable protein-protein interactions between the DnaB and Tus resulting in the dislodgement of Tus from Ter and hence allowing for the progression of the replication fork. However, when DnaB approaches the non-permissive face, significant protein-protein interactions between the DnaB and Tus prevent the dislodgement of Tus, resulting in replication termination. If for any reason this mechanism may fail, DnaB will unwind Ter until it reaches C6 which would induce the formation of a locked complex and subsequent prevention of replication fork progression [3].
References
- ↑ 1.0 1.1 1.2 1.3 Neylon, C., Kralicek, A. V., Hill, T.M. and Dixon, N.E. (2005) Replication Termination in Escherichia coli: Structure and Antihelicase Activity of the Tus-Ter Complex. Microbiology and Molecular Biology, 69 (3): 501-526.
- ↑ 2.0 2.1 2.2 2.3 2.4 Kamada, K., Horiuchi, T., Ohsumi, K., Shimamoto, N. and Morikawa, K. (1996) Structure of a replication-terminator protein complexed with DNA. Nature 383 (6681): 598-603.
- ↑ 3.0 3.1 3.2 3.3 Kaplan, D. L. and Bastia, D. (2009) Mechanisms of polar arrest of replication fork. Molecular biology, 72 (2): 279-285.
- ↑ Mulcair, M. D., Schaeffer, P. M., Oakley, A. J., Cross, H. F., Neylon, C., Hill, T. M. and Dixon, N .E. (2006) A molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell 125: 1309-1319.
- ↑ Anderson, P., Griffith, A., Duggin, I. and Wake, R. (2000) Functional specificity of the replication fork-arrest complexes of Bacillus subtilis and Escherichia coli: significant specificity for Tus-Ter functioning in E.coli. Molecular Microbiology, 36 (6): 1327-1335.
- ↑ Henderson, T., Niles, A., Valjavec-Gratian, M. and Hill, T. (2001) Site-directed mutagenesis and phylogenetic comparisons of Escherichia coli Tus protein: DNA-protein interactions alone cannot account for Tus activity. Molecular Genetics and Genomics, 265 (6): 941-953.
- ↑ Mulugu, S., Potnis, A., Shamsuzzaman, T. J., Alexander, K. and Bastia, D. (2001) Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Science, USA, 98 (17): 9569-9574.
- ↑ Bastia, D., Zzaman, S., Krings, G., Saxena, M., Peng, X. and Greenberg, M. (2008) Replication termination mechanism as revealed by Tus-mediated polar arrest of a sliding helicase. Proceedings of the National Academy of Science, USA, 105 (93): 12831-12836.
