Imaging DNA - Polymerase Complexes with Single Molecule Resolution

The AMELX gene encoding amelogenin (the principle protein of the developing enamel matrix) is a nested gene, being located within (and in the opposite orientation to) the first intron of a larger gene, ARHGAP6, which encodes a GTPase activating protein. AMELX expression is spatially and temporally restricted whereas ARHGAP6 is widely expressed, raising important questions in respect of their simultaneous transcriptional control which cannot be easily answered by conventional molecular ensemble techniques. Atomic force microscopy (AFM), may provide a tool to investigate convergent simultaneous transcription at the single molecule level. Objectives: To optimise AFM conditions for imaging DNA-polymerase complexes with single molecule resolution while preserving faithful biological function. Methods: Suitable substrates for binding DNA to provide sample stability while avoiding kinetic trapping were assessed by comparing mica ± Mg++ and mica modified using 3-aminopropyltriethoxysilane (APTES-mica). A novel 1428 bp linear DNA template, containing convergently aligned T7 and T3 promoters was used with corresponding coliphage polymerases in in vitro transcription assays and for AFM imaging. Stalled DNA-polymerase complexes were imaged in the AFM following incubation of template, polymerases and appropriate mixtures of activated ribonuceleotides. Results: Inclusion of Mg++ ions was essential for successful imaging of DNA on mica under fluid in the AFM. DNA adopted an open conformation on mica substrates but was more closed after mica modification with APTES. In vitro assays demonstrated that both promoters were transcriptionally active. AFM imaging revealed the presence of globular structures, corresponding to single polymerases aligned at specific sites along the DNA templates. Conclusions: These preliminary results show that with design of suitable DNA templates and optimization of imaging conditions, AFM offers a solution to determining polymerase fate during simultaneous convergent transcription in a nested gene model, addressing the question of whether simultaneous expression of AMELX and ARHGAP6 is possible during tooth development.