LINE1 retrotransposon is the only autonomous mobile genetic element in the human genome. The LINE1 sequence encodes proteins ORF0p, ORF1p and ORF2p. In its active state, the ORF1p protein forms homotrimers which, together with ORF2p, carry out the entire process of retrotransposition. ORF1p binds its own coding mRNA and functions as a nucleic acid chaperone. It is responsible for the formation of the ribonucleoprotein particle (RNP). LexA and ToxR screening systems were developed for the detection of protein homodimerization. They are based on bacterial transcription factors LexA and ToxR. Both of them are active in their homodimeric forms and consist of DNA binding domain and a dimerization domain. By replacing the dimerization domain of LexA or ToxR with the coding sequence of any protein of interest, it is possible to determine whether the inserted protein homodimerizes or not. This is done based on the signal produced by a reporter protein in the systems. To analyse homodimerization degree of the ORF1p, the corresponding coding sequence was cloned into the LexA and ToxR systems. For comparison, the coding sequence of a mutant form of the protein, designated JMIII, was also cloned into both systems. Furthermore, truncated versions of ORF1p and JMIII were cloned into the systems. In comparison to the original sequences of ORF1p and JMIII, truncated versions lacked the coding regions for the flexible parts of the proteins.
The results revealed differences between the LexA and ToxR systems. In the LexA system, ORF1p exhibited a certain degree of homodimerization, whereas the mutant JMIII did not. In the ToxR system, neither ORF1p nor JMIII showed homodimerization. In following studies, removal of the flexible regions from ORF1p and JMIII improved the performance of the systems. Truncated ORF1p variant exhibited a higher degree of homodimerization in the LexA system, while JMIII, same as before, failed to form homodimers. The differences observed between ORF1p and JMIII in the LexA system suggest that the mutations are located within structurally important regions of the protein, which are essential for assembly into higher order structures. In the ToxR system, truncation of protein sequences resulted in homodimerization of both ORF1p and JMIII. We suggested that the differences in results between both systems were most likely caused by a mutation within ToxR promoter of the ToxR system. The mutation was later confirmed by sequence alignment analysis. Because of that further studies would be required to achieve deeper characterization of the used systems for our proteins. In addition, the method used for detecting homodimerization should be validated using another homotrimeric protein.
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