RSS

PDB:2VJV

Protein Name

IS608 transposase TnpA / DNA complex

Species

Helicobacter pylori

Biological Context

DNA transposition is a significant part of evolution. A transposable element, or transposon, can move from one position in a genome to another. Thus, it can disrupt the function or change the regulation of existing genes, promoting divergence among genomes. It is observed that many genomes are scattered with transposons.

Insertion sequences, ISs, are the smallest transposable elements. Their lengths are less than 2.5kb and they can only convey transposition related genes. Major ISs have inverted repeat sequences on both ends, and contain transposase genes. The transpositions of ISs is carried out by transposases. The IS in a donor DNA is cleaved, moved, and integrated in the target DNA.

The transposition mechanisms of the IS200/IS605 family is different from other IS families. First, IS200/IS605 members act on single-stranded DNAs. Besides, instead of inverted repeat sequences, they contain imperfect palindromes on both ends (LE_IP, RE_IP). DNA hairpin structures are constructed at the palindromes. IS608 belongs to the IS200/IS605 family and was identified from Helicobacter pylori. It encodes transposase TnpA. IS608 transposition is done by TnpA, and IS608 is moved from a donor sequence containing TTAC to a target sequence also containing TTAC. During transposition, the cleaved IS608 shows an intermediate structure as circular DNA, named transposon junction. TnpA also has a second role in the transposition reaction, in which a tyrosine residue works as a nucleophile for DNA cleavage.

IS608_transposition (Fig. 1) IS608 transposition
IS608 moves from a donor sequence (black) to a target sequence (gray) via a circular intermediate named transposon junction.

Structure Description

2vjv2vjv_x2vjv_y

TnpA works as a homo dimer. A complex of the TnpA dimer, a DNA fragment of IS608 left end (LE26), and another DNA fragment from LE donor flank (D6) has been determined (Fig. 2, Fig. 3).

Each subunit of TnpA contains one movable helix (αD), harboring the tyrosine nucleophile Tyr127, which is important for the cleavage of IS608. The remaining region of the subunit contains a His-hydrophobic-His (HUH) motif. In the active conformation of TnpA dimer, αD is in trans conformation. Thus, the Tyr127 of one subunit is close to the HUH motif of the other subunit, and they form the active site. TnpA has αD in cis conformation in its inactive state. However, in the reported structure αD helices are away from HUH motifs in a position different from the inactive state, which could come from artifacts by the crystal lattice contact. On the other hand, it is observed that TnpA/LE26 complex without D6 sequence forms appropriate active site with Tyr127 and HUH motif. TnpA dimer seems to catch the IS608 ends (LE_IP, RE_IP) at its active site, and cutting and rejoining of the ends are carried out spontaneously.

Although the D6 sequence itself does not bind to TnpA, the TnpA/LE26/D6 ternary complex is stable with three base pairs between TTAC on D6 and AAAG on LE26 (Fig.3, Fig. 4). Thus, the recognition of cleavage site (TTAC) by TnpA is not based on a kind of specific DNA binding motif, but on base-pair formation with a DNA region (AAAG) on LE26 which is bound by TnpA. The 3’-end of D6 is the cleavage position and is oriented towards the HUH motif (Fig. 3). The position and orientation of the TTAC on D6 is the same with the terminal TCAA sequence on RE in TnpA/RE35 complex, which is the other cleavage site.

Further analysis with TnpA/IS608 complex structure with longer LE region is expected to elucidate the whole mechanisms of IS608 transposition.

D6_and_LE26 (Fig. 2) LE26 and D6 sequences
A 26-mer DNA sequence (LE26) and a 6-mer DNA sequence (D6) are used as the left end of IS608 and the LE donor flank, respectively.The red underlined regions on LE26 form hairpin structure and the yellow region is able to form base pairs with TTAC on D6.
structure (Fig. 3) TnpA/LE26/D6 complex
Complex of TnpA dimer (green, orange), LE26 (red, yellow), and D6 (black).The hairpin structure of LE26 is colored red and the upstream four residues are colored yellow.

His64 and His66 belong to the HUH motif.
model (Fig. 4) Schematic view of TnpA/LE26/D6 complex
TnpA (orange) binds to the hairpin structure on LE26 (red) and the upstream AAAG forms base pairs with TTAC. Thus, TnpA/IS608 complex can recognize TTAC-containing donor and target sequences.

Orange rectangle represents the αD and Y is the tyrosine nucleophile.

Protein Data Bank (PDB)

References

Source

Barabas, O. Ronning, D.R. Guynet, C. Hickman, A.B. Ton-Hoang, B. Chandler, M. Dyda, F.; "Mechanism of is200/is605 Family DNA Transposases: Activation and Transposon-Directed Target Site Selection."; Cell(Cambridge,Mass.); (2008) 132:208- PubMed:18243097.

Others

author: Naoya Fujita


Japanese version:PDB:2VJV