Master thesis by Mette Eriksen

Activity of quantum dot bound T7 RNA polymerases - Ensemble and single molecule investigations

The RNA Polymerase is an enzyme, which copies the genetic sequence of DNA into RNA. As genes can be situated on opposite DNA strands and overlap, several RNA polymerases as well as other proteins can act on the DNA at the same time, which results in collisions as well as blockage. We aim at investigating how the RNA polymerase circumvents collisions with other RNA polymerases and with other DNA associated proteins. By the means of the techniques Video Fluorescence Microscopy and Optical Tweezers, the translocation of the RNA polymerase on the DNA can be studied in real time by the attachment of a fluorescent quantum dot to the enzyme.
A genetically modified phage T7 RNA polymerase is used as model and has been functionalized with either a polystyrene bead or a quantum dot and studied both on the ensemble and single molecule level. The presented results confirm that the functionalized enzyme stays active, which allowed us to initiate fluorescence microscopy experiments of DNA interacting quantum dot bound T7 RNA polymerases and the first findings of these experiments are presented.

Supervisor: Lene Oddershede