Mechanistic Studies of Transcription in vitro Transcription is the fundamental process in all living organisms and the first step in gene expression. The protein machine at the heart of transcription is RNA polymerase (RNAP) which is also the direct or indirect target of most regulation of transcription. Bacterial RNAP is the most well characterised member of this protein family and we have chosen it as our preferred model system for detailed structural and mechanistic studies of transcription.
In our single-molecule approach, we can directly visualise structural states and transition between these, allowing us to follow the process of transcription in real time. This enables us to detect previously unobserved intermediates, extract kinetics of the individual steps involved in RNA synthesis, and further elucidate mechanism of action for small molecules or inhibitors targeting bacterial RNAP.
Our experiments are focusing in particular on the early stages of transcription, in particular open complex formation, initial transcription and promoter escape.
Development of new fluorescence methods and instruments A big part of the work in our group would be impossible to do without an ongoing effort to re-invent, refine, and develop new tools, methods and instruments.
A big part of this work has been the development of a compact wide-field/TIRF microscope, which is now sold as the Oxford Nanoimager. It allows benchtop single-molecule experiments and is a great asset in our daily work. In order to see the molecules we are looking for, we are constantly working on new labelling strategies both in vivo and in vitro. As part of these efforts, we have established a robust and easy-to-implement method for virus labelling using a Calcium-mediated interaction between the viral envelope and fluorescently labelled ssDNA. (1). We can now use this for virus detection and identification (2).
We further have a particular focus on developing robust labelling strategies using transiently binding ssDNA probes. Their specificity and interaction strength can easily be adjusted, they are biocompatible and commercially widely available. Depending on their hybridisation kinetics and experimental set-up, we use them to establish new applications of super-resolution microscopy (VIRUS-PAINT), establish bleaching-free single-molecule imaging (3), and pioneer a new single-molecule DNA-sequencing method (4).
