Biophysical and structural studies of a new type of transposases – RAYTs

Iva Nečasová, Tatsiana Chernovets, Jaroslav Nunvář and Bohdan Schneider

Institute of Biotechnology AS CR, v. v. i., Prague, Czech Republic


Genes of REP-associated tyrosine transposases (RAYTs) have been found in many bacterial species. Members of the RAYT enzyme transpose – recognize, bind, cleave, and reinsert specific single stranded DNA elements, so called repetitive extragenic palindromes, REPs. The nuclease activity of RAYTs has been proven only for the Escherichia coli RAYT [1], and for Haemophilus parasuis in our laboratory, and the mechanism of the complex process of transposition is understood very poorly, and up to now, only crystal structure of the complex between E. coli RAYT transposase and its REP DNA has been determined (PDB code 4er8, [2]). The novelty and challenging nature of the RAYT/REP complex inspired our study of this system. Bioinformatic analysis of genomic databases revealed several hundred of RAYT-associated REP sequences, we have characterized some of their biophysical properties, and discovered surprising conformational diversity of these palindromic DNA elements that reaches far beyond the expected hairpin stem-loop architecture. We further have identified RAYT genes in several bacteria and selected eight most promising candidates according to the values of sequence-calculated hydrophilicities. These eight RAYT genes have been cloned and expressed in E. coli. Production of most of these enzymes is challenging at the expression step, as their nuclease activity is deleterious for cells, they also have very low solubility, and extremely limited stability, they precipitate or aggregate shortly after purification. Despite these obstacles, we have succeeded in stabilizing the H. parasuis RAYT, been able to measure it various properties as thermostability, and CD spectrum, and also determine the binding affinity to the recognition REP oligonucleotide (dissociation equilibrium constant determined by the thermophoresis is about 6 nM). Crystallization trials and cleavage experiments with H. parasuis RAYT are under way.


1.         Ton-Hoang, B., et al., Structuring the bacterial genome: Y1-transposases associated with REP-BIME sequences. Nucleic Acids Res, 2012. 40(8): p. 3596-609.

2.         Messing, S.A., et al., The processing of repetitive extragenic palindromes: the structure of a repetitive extragenic palindrome bound to its associated nuclease. Nucleic Acids Res, 2012. 40(19): p. 9964-79.