Duss Group

Assembly mechanisms and function of protein-RNA complexes at the single-molecule level

The Duss group uses single-molecule methods in combination with integrative structural biological and biochemical approaches to understand how protein-RNA complexes are assembled and how macromolecular machines cooperate with each other, providing new opportunities to fight diseases and to create new functional molecular assemblies.


What we are interested in

Gene expression is coordinated by multiple interconnected processes including transcription by the RNA polymerase, simultaneous co-transcriptional RNA folding, RNA modification and RNA processing, the dynamic interaction of the nascent RNA with proteins and other ligands, and the translation of the mRNA into proteins by the ribosome (Fig. 1; Qureshi & Duss, FEBS letters, 2023). For some of these processes structural information is available, but we critically lack an understanding of dynamics and therefore the mechanisms of how these processes are functionally coupled to each other.

The challenge: While studying these mechanisms in vivo provides physiological relevance and an environment where coupling between the processes can occur, the cellular complexity makes it difficult to dissect the (dynamic) role of individual components in these interconnections. In contrast, in vitro reconstitution experiments provide a controlled environment allowing the manipulation of each component in the system but they often do not recapitulate the in vivo situation entirely because of their limited complexity.

Our approach: Our lab bridges the gap between controllable, quantitative and high-resolution experiments from in vitro reconstitutions with in vivo physiological relevance. We reconstitute complex and active transcription, translation, mRNA processing & modification, and DNA repair machineries and investigate whether their activities and behaviors differ when studied in isolation versus when all components are present and the processes are simultaneously active. In order to directly visualize how various processes central to gene expression cooperate with each other, we take advantage of the recent explosion of high-resolution cryo-electron microscopy and cryo-electron tomography structures, representing numerous static states of different complexes involved in transcription, RNA processing & modification, translation, and DNA repair and use cutting-edge multi-color single-molecule fluorescence methods (Fig. 2) to place these various structural snapshots along a reaction trajectory. Together with integrative structural biology, biochemistry and in vivo single-molecule tracking, this combination of methods (Gor & Duss, 2023) allows us to dissect how various macromolecular machines cooperate and work in context.

Figure 1: The formation of protein–RNA complexes is complicated and heterogeneous, and involves several coupled processes such as transcription, nascent RNA folding, RNA processing, RNA modification, protein and small molecule ligand binding, and the action of transiently binding assembly factors.
Figure 2: Using multi-color single-molecule fluorescence microscopy, several processes occurring during protein–RNA complex assembly can simultaneously be visualized in real-time, which allows us to dissect the complicated molecular mechanisms of intrinsically heterogeneous assembly processes. Figure adapted from Duss et al., Cell 2019, and Duss et al., Nat Commun 2018.

Current & future projects and goals

  • Investigating the molecular interconnections between the bacterial transcription, translation and DNA repair machineries and how the underlying processes are regulated by non-coding RNAs (Duss et al., Nature, 2014; DFG grant 2023-2025), RNA binding proteins and small molecule ligands (FEBS excellence award 2022). For example, we have tracked transcription-translation coupling in real-time (Qureshi & Duss, bioRxiv, 2023; Movie on twitter/X).
  • Obtaining a quantitative and dynamic understanding of how the transcription machinery and cellular assembly factors (e.g. RNA modification and RNA processing enzymes) cooperate to guide bacterial ribosome assembly (Duss et al., Cell, 2019).
  • Studying how eukaryotic transcription is interconnected with early splicing and m6A RNA modification (Höfler & Duss, Life Science Alliance, 2023).
  • Generating a dynamic molecular understanding on how prokaryotic and eukaryotic transcription condensates are formed and modulate transcription activity (SPP2191 DFG grant 2023-2025).
  • Developing in vitro and in vivo single-molecule fluorescence microscopy methods to track nascent RNA folding, RNP assembly and function.
Visualizing the dynamic process of transcription-translation coupling in real-time (Qureshi & Duss, bioRxiv, 2023)