TAL effector
From Proteopedia
Transcription activator-like effector PthXo1 (TALE PthXo1)TALEs are proteins originally derived from the plant pathogene Xanthomonas spp. The gene encoding for the effector are Pthxo1 and Pxo_00227. Originally they were detected in Xanthomonas Oryzae but the TALE PthXo1 genes were transformed into Escherichia Coli to express the DNA-binding protein.
DNA recognition by TAL effectors is mediated by tandem repeats, each 33 to 35 residues in length, that specify nucleotides via unique repeat-variable diresidues (RVDs). The crystal structure of PthXo1 bound to its DNA target was determined by high-throughput computational structure prediction and validated by heavy-atom derivatization. Each repeat forms a left-handed, two-helix bundle that presents an RVD-containing loop to the DNA. The repeats self-associate to form a right-handed superhelix wrapped around the DNA major groove. The first RVD residue forms a stabilizing contact with the protein backbone, while the second makes a base-specific contact to the DNA sense strand. Two degenerate amino-terminal repeats also interact with the DNA. Containing several RVDs and noncanonical associations, the structure illustrates the basis of TAL effector–DNA recognition [1]
Contents |
3D structures of TALE PthXo1
2jg2 – PpSPT + aldimine – Pseudomonas paucimobilis
2jgt – PpSPT
2w8j - SpSPT + aldimine – Sphingomonas paucimobilis
2w8t, 2w8u, 2w8v, 2w8w - SpSPT (mutant) + aldimine
2xbn – SpSPT + pyridoxamine phosphate
3a2b – SPT + pyridoxal phosphate + serine – Sphingobacterium multivorum
About this Structure
DNA binding module with marked RVD
Tertiary structure of the RVD
Figure 1: Natural structure of TALEs derived from Xanthomonas sp. Each DNA-binding module consists of 34 amino acids, where the RVDs in the 12th and 13th amino acid positions of each repeat specify the DNA base being targeted according to the cipher NG = T, HD = C, NI = A, and NN = G or A. The DNA-binding modules are flanked by nonrepetitive N and C termini, which carry the translocation, nuclear localization (NLS) and transcription activation (AD) domains. A cryptic signal within the N terminus specifies a thymine as the first base of the target site.[2]
Figure 2: Domain organization of PthXo1 and structure of a single TAL effector repeat TAL effectors contain N-terminal signals for bacterial type III secretion, tandem repeats that specify the target nucleotide sequence, nuclear localization signals, and a C-terminal region that is required for transcriptional activation. PthXo1 contains 23.5 canonical repeats (color coded to match Figure 2) that contact the DNA target found in the promoter of the rice Os8N3 gene (17). Blue bases correspond to positions in the target where the match between protein and DNA differs from the optimal match specified by the recognition code (3,4). Arrows indicate the start and end of the crystallized protein construct. In the structure, repeats 22 to 23.5 are poorly ordered, as are the C-termini of the two N-terminal cryptic repeats. The sequence and structure of a representative repeat (#14) is shown; RVD residues (HD) that recognize cytosine are red.[3]
Engineering
Figure 3: Based on the RVD the construction of a so called GATE Assembly kit was performed by the iGem 2012 Team from Freiburg University. [4]
Reference
- Mak AN, Bradley P, Cernadas RA, Bogdanove AJ, Stoddard BL. The Crystal Structure of TAL Effector PthXo1 Bound to Its DNA Target. Science. 2012 Jan 5. PMID:22223736 doi:10.1126/science.1216211
- Sanjana NE, Cong L, Zhou Y, Cunniff MM, Feng G, Zhang F. A transcription activator-like effector toolbox for genome engineering. Nat Protoc. 2012 Jan 5;7(1):171-92. doi: 10.1038/nprot.2011.431. PMID:22222791 doi:10.1038/nprot.2011.431
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