Multiple RNA Polymerase Transactions on Single DNA Templates

Abstract

We are studying the interactions of more than one RNA polymerase (RNAP) operating on single linear double-stranded DNA templates using imaging by atomic force microscopy (AFM) [1,2]. These templates include a short polynucleotide sequence of A, C, G or T forming a single-stranded end loop to discriminate polarity of the DNA molecules in the AFM [3]. Model templates include convergently and tandemly-aligned promoters for bacterial E. Coli RNAP.Measurement of the positions of the RNAP on the DNA before and after firing from the promoters allow us infer outcomes of “collision” events. The end-labelling ssDNA loop not only acts as a fiducial marker but also captures RNAPs that have fired from promoters directed towards and closest to the loop. This enables us to quantify different outcomes of these events between more than one RNAP operating in a convergent or tandem configuration. Interestingly, RNAPs which approach each other often stall before close contact is made, implying that transient DNA super-coiling between RNAP mediates their interactions. Furthermore, the distribution of outcomes for head-on (convergent transcription) and rear-end (tandem transcription) collision events are similar, suggesting that long range interactions mediated through the local DNA conformation serve to regulate RNAP activity and positioning on the templates. These experiments inform the regulation of genetic expression at the molecular level, including acting as simple models for nested genes.[1] Billingsley D.J., Bonass W.A, Crampton N., Kirkham J. and Thomson N.H. (2012) Physical Biology 9 021001.[2] Crampton N., Bonass W.A., Kirkham J., Rivetti C. and Thomson N.H. (2006) Nucleic Acids Research 34 (19) 5416-5425.[3] Billingsley D.J., Crampton N., Kirkham J., Thomson N.H., Bonass W.A (2012) Nucleic Acids Research 40 (13) e99.