Objectives: To directly observe collision events between two E. coli RNA polymerases (RNAPs) acting on a single linear model DNA template that contains convergently-aligned promoters.
Methods: Stalled ternary complexes were imaged following incubation of the template, RNAPs and ribonucleosides triphosphates (NTPs) omitting CTP from the reaction buffer. Convergent transcription was initiated by addition of all 4 NTPs. Samples were taken at specified times for AFM imaging and analysis.
Results: The distance between the two RNAPs can be accurately measured by AFM, and addition of the NTPs showed that this distance decreased as expected. Where two RNAPs remained on the DNA, the RNAPs were stalled against each other on the template. There was also evidence that one RNAP was causing the other to back-track over longer time-scales.
Conclusions: The back-tracking effect observed here suggests that the collisions are between an actively transcribing RNAP and one which remains stalled, indicating that simultaneous transcription of nested genes, may be precluded at a single genetic locus. However, this time-lapse approach only focuses on collisions where both RNAPs remain on the template and we are therefore looking at a sub-set of all possible results. New fast scan AFM technology offers the possibility of studying these processes closer to real-time, overcoming this problem.