P 007 Probing the Mechanism of Convergent Transcription with Atomic Force Microscopy

Transcription initiated from two convergently aligned promoters is a feature of a number of gene structures. In " nested " genes, one gene is located within (and in the opposite orientation to) a larger gene. Such genes have been identified in bacteria and viruses as well as in higher organisms including humans. Such arrangements are likely to cause transcribing or initiating RNAP to collide. We aim to study the process of convergent simultaneous transcription in a nested gene model at the single molecule level using Atomic Force Microscopy (AFM). DNA:RNAP complexes are formed in vitro, immo-bilised on a surface and imaged by AFM. Through use of DNA templates with C-deficient sequences downstream of convergently aligned promoters, we have formed stalled elongation complexes by omission of CTP from the reaction buffer. Transcription is re-initiated with addition of CTP allowing the two RNAPs to interact on a single DNA template. The RNAPs may either pass or collide, where a collision will cause stalling or displacement. Initial analysis indicates that in the majority of cases they stall against each other and remain bound to the DNA template. This may be a result of asynchronicity in the system, and the single molecule real-time experiment will be required to investigate the outcome of collision of two active convergent elongating RNAPs.