Prof. David Dulin
(Interdisciplinary Center for Clinical Research, FAU, Erlangen-Nürnberg)
Single-molecule biophysics approaches to study gene machines
Every organism contains a piece of genetic information, in the form of either DNA, e.g. for any living organism, or RNA, e.g. for RNA virus. During any organism life cycle, nanoscopic molecular machines called enzymes express and maintain the genome. Enzymes are characterized by an underlying instability, where multiple free energy states separated by low activation energy, i.e. typically few kBT (kB being the Boltzmann constant) interconvert rapidly through complex kinetic pathways. Therefore, in the wet and hot environment of the cell, enzymatic activity is noisy, asynchronous and heterogeneous. To characterize precisely a given enzyme without averaging all the different kinetic states, it is therefore important to observe each enzyme individually, i.e. the single molecule level, and at the spatiotemporal resolution that matters, i.e. ~1 nm at 10-100 ms. Single molecule techniques, such as magnetic tweezers and total internal reflection fluorescence microscopy (TIRFM)-based single molecule fluorescence resonance energy transfer (smFRET), have been developed for this purpose. During my talk I will present two studies that illustrate the power of single molecule techniques. First, I will describe how high throughput magnetic tweezers helped to observe rare events such as nucleotide misincorporation during RNA virus genome replication by the viral polymerase. Second, I will show how TIRFM-based smFRET experiments helped to characterize the kinetics of an asynchronous multistep process such as bacterial initial transcription.