| Popis: |
σ70 is an essential bacterial transcription initiation factor in E. coli. It recruits RNA polymerase core to promoter sequences on DNA. σ70 was originally thought to dissociate from RNA polymerase immediately upon transcription initiation. However, it has been shown in vitro that histidine-tagged σ70 can remain on the polymerase during transcript elongation. It is not clear whether untagged σ70 exhibits the same behavior in living cells. In this thesis I investigated whether the histidine tag is responsible for σ70 retention on the transcription elongation complex. I expressed, purified, and fluorescently labelled σ70 constructs with and without the histidine tag. I examined transcription with both constructs using colocalization single-molecule spectroscopy experiments on a total internal reflection microscope. My experiments showed essentially no difference in σ70 retention on elongation complexes with and without the histidine tag on σ70. These data indicate that σ70 retention is not an artifact of the histidine tag. |
