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CeNS Colloquium

Kleiner Physik-Hörsaal N 020, Fakultät für Physik
Date: 02.08.2019, Time: 15:30h

CAS Fellowship Talks 2019

Talk 1) Prof. Shimon Weiss, University of California, Los Angeles

Dynamic structural biology: Two decades of smFRET applied to transcription initiation

Classical structural biology can only provide static snapshots of bio-macromolecules. Single-molecule Förster resonance energy transfer (smFRET) paved the way for studying dynamics in macromolecular structures under biologically relevant conditions. Since its first implementation in 1996, smFRET experiments have confirmed previously hypothesized mechanisms and provided new insights into many fundamental biological processes, such as DNA maintenance and repair, transcription, translation, and membrane transport. We will review two decades contributions of smFRET to our understanding of transcription initiation. Transcription by bacterial DNA-dependent RNA polymerase is a multistep process that uses genomic DNA to synthesize complementary RNA molecules. Transcription initiation is a highly regulated step in E. coli, but it has been challenging to study its mechanism because of its stochasticity and complexity. We describe how single molecule approaches have contributed to our understanding of transcription and have uncovered mechanistic details that were not observed in conventional assays because of ensemble averaging. Additionally, building on current state-of-the-art implementations of smFRET, we highlight possible future directions for smFRET in transcription and in other applications.


Talk 2) Prof. Eitan Lerner,  The Hebrew University of Jerusalem, Jerusalem

Milestones towards smFRET empirically-restrained structural modeling of conformations

Structural modeling using empirical spatial restraints is gaining slow yet steady populrity in the study of the ensemble structures of conformations. Bringing the Förster Resonance Energy Transfer (smFRET) ruler to the single-molecule level allows distinguishing different conformations according to specific inter-residue distances. Performing such single-molecule FRET (smFRET) experiments probing multiple pairs of residues provides enough spatial restraints for the structural modeling of the ensemble of structures that define a given conformation. It is therefore clear that the retrieval of accurate inter-residue distance information is of the utmost important. The structural assessment is based on the Förster relation between the efficiency of energy transfer between two dyes and the distance between them. It is known, however, that inter-dye distance fluctuations, on the timescale of the fluorescence lifetime (or shorter), can increase the observed FRET efficiency and thus give the impression of an overall decreased inter-dye distance. Although the information on diffusion-enhancement of FRET could in principle be retrieved from model fitting to fluorescence decays, in single-molecule fluorescence measurements fluorescence decays are too noisy to be accurately fitted with such complex models. Here we introduce Monte-Carlo diffusion-enhanced photon inference (MC-DEPI). MC-DEPI recolors photons of smFRET measurements taking into account dynamics of inter-dye distance fluctuations, multiple interconverting states and photoblinking. Using this approach, we show that the distance interpretation of smFRET experiments of even molecules as simple as doubly-labeled dsDNA is nontrivial and requires decoupling the effects of rapid inter-dye distance fluctuations on FRET in order to avoid systematic biases in the estimation of the inter-dye distance distribution. MC-DEPI can hence be used for the retrieval of accurate and unbiased distance information that is required for proper structural modeling.