Concurrent DNA transcription from convergent and tandem promoters studied by atomic force microscopy

With recent advances in sequencing and mapping of genomes, the occurrence of overlapping and nested transcription units is more common than previously thought in both eukaryotes and prokaryotes. The interleaved genome model means that transcriptional interference by collisions between concurrently transcribing RNA polymerases is more likely than ever before. This thesis presents a study of the outcomes of collisions between RNAPs transcribing concurrently from convergent and tandem promoters using AFM to provide a view of single populations seen after collisions. Through the development of an improved DNA end labelling method and incorporation of an inhibitor of RNAP non-specific binding the results of collisions can be viewed with more confidence than previously possible. It was seen that collisions from both convergent and tandem promoters resulted in both RNAPs remaining bound to the template in hard contact. These collisions occurred by two main mechanisms. Either between two active elongation complexes (ECs) or between an elongation complex and an inactive complex referred to as a sitting duck (SD). EC-EC collisions were found to be the most common for convergent promoters while with tandem promoters the distinction between the two is less clear. In the case of EC-SD collisions it is shown that shunting upstream of up to 100 bp by an EC is possible. By utilizing a linear template that is susceptible to supercoiling due to spin locking, it is shown that a region of highly positive supercoiled domain can prevent two convergently transcribing RNAPS coming into hard contact. It is also shown that topology of the DNA plays a role in the distribution of EC-EC and EC-SD collisions that occur for both promoter arrangements. This indicates that topology influences the outcomes of concurrent transcription and provides a mechanism by which RNAPs can sense one another via the DNA template.