Single-molecule studies of eukaryotic RNA Polymerase II

RNA transcription is the essential first step in the conversion of genetic information stored in DNA into fully functioning proteins. RNA polymerases read this genetic information and make a complimentary form, called RNA. RNA Polymerase II is responsible for read- ing and converting most of the protein coding genes. However, eukaryotic DNA is highly complex and contains multiple roadblocks to transcription including nucleosomes and DNA damage. If these damages are not resolved, mutations can arise causing genome in- stability and also cancers including melanoma and adenocarcinoma. How these roadblocks affect RNAPII on a molecular level has yet to be probed deeply at a single-molecule level due to the dynamic nature of this process. Here, I develop novel single-molecule based assays to directly monitor RNAPII elongation dynamics without the need to genetically modify the polymerase. Using the smFRET based assay I was able to visualise RNAPII elongation on different templates including AT-rich DNA. I showed that AT-rich DNA not only induces premature termination of transcription, but also that severe pausing pre- cedes this step. Addition of different damages into the template also caused destabilising effects on RNAPII to varying amounts. While abasic lesions caused complete stalling of RNAPII, UV-induced damage and 8-Oxo Guanine groups led to more dynamic pausing and backtracking behaviour, with RNAPII bypassing the latter damage. Finally, Rad26, a recovery factor, was shown to promote bypass of oxidative damage by physically block- ing the backtracked state and keeping the polymerase around the site of damage. The assays developed here can be utilised for a variety of purposes where other roadblocks, e.g. nucleosomes, can be inserted readily into the template. Furthermore, the results shown here will be important in increasing our understanding of how DNA damage and other roadblocks interfere with the transcription pathway.Open Acces