Journal: Nucleic Acids Research

Article Title: Single-turnover kinetic analysis of non-long terminal repeat retrotransposition defines the pathway and rate constants leading to second-strand synthesis

doi: 10.1093/nar/gkag223

Figure Lengend Snippet: R2Bm mechanism cartoon, gene structure, and 28S DNA target. ( A ) Cartoon of the R2 retrotransposition pathway. R2 protein, bound to its mRNA, binds the 28S DNA locus and nicks the first strand . This is followed by transfer of the nicked DNA from the endonuclease active site (EN) to the polymerase active site (RT) . TPRT then occurs using the R2 mRNA as a template and the nicked lower strand as a primer . The second strand is then nicked (using the 5′RNA portion of the RNA shown in B), and the new 3′ end is used as a primer to copy the cDNA from the TPRT reaction (, , and ). Finally, host factors seal the nicks, leaving an R2 element integrated in the host genome . For simplicity in drawing, the diagram contains one R2 enzyme, but the reaction could involve a dimer. Only the polymerase and endonuclease domain of the protein is drawn. Sequence-specific DNA-binding domains are also present but are not shown due to ambiguity of their position in the second half of the reaction. ( B ) R2Bm gene structure and RNAs used in this study. The two regions of the elements whose RNA sequence is known to be recognized by the R2 enzyme correspond to the 5′RNA segment , and the 3′UTR. RNAs used in this study are diagrammed below, along with their length. The truncated ORF, containing the 5′RNA region, is 582 nt compared to a full R2Bm ORF of 3342 nt. ( C ) 28S DNA target. Regions of the 28S DNA target that are recognized by the R2Bm protein are annotated based on findings by Deng et al. . A 3′ phosphate blocking group was added to both strands to prevent non-specific addition to the 3′ ends of the starting DNA substrate. Top and bottom strands are 5′ labelled with 6-FAM and Cy3, respectively, and the 3′ ends are phosphorylated to prevent extensions by the R2 polymerase. Created in BioRender. Dangerfield, T. (2026) https://BioRender.com/e1lrntp ..

Article Snippet: In vitro transcription reactions were set up in a volume of 320 μl with 8 μg of purified PCR product, 5 mM DTT, 2 mM NTPs, and 1600 Units of T7 RNA polymerase in T7 RNA Polymerase reaction buffer (NEB).

Techniques: Sequencing, Binding Assay, Blocking Assay