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Lehrstuhl Mikrobiologie, Grohmann-Research

Fakultät für Biologie und Vorklinische Medizin
Institut für Biochemie, Genetik und Mikrobiologie
Lehrstuhl für Mikrobiologie & Archaeenzentrum
Prof. Dr. Dina Grohmann - Research


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Mechanisms of archaeal transcription

Transcription is one of the most fundamental processes in life carried out by RNA polymerases (RNAP). A plethora of basal and gene-specific transcription factors interact physically and functionally with RNAP which results in the execution of a highly fine-tuned genetic program that is at the very heart of biology. Our projects aim to expand our understanding of transcriptional regulation and transcription complex organisation and dynamics. Over the last years we were able to shed light on the dynamics of the archaeal transcription machinery using fluorescence-based single-molecule measurements in combination with classical biochemical approaches. To this end, we exploit our unique fluorescently labelled recombinant archaeal transcription system derived from the archaeal organism Methanocaldococcus jannaschii which allowed us for example to map the binding site of transcription factor E at the surface of the RNAP and to describe the factor swapping mechanism between transcription factor E and Spt4/5. Recently, we were able to monitor the conformational distortion of the promoter DNA induced by TBP (TATA-binding protein) for two archaeal and the eukaryotic transcription system in real-time and found that eukaryotic and archaeal TBP follow different DNA bending pathways translating into different regulatory modes during early steps of gene expression.

Grohmann Fig1

Figure 1: A fluorescently labeled archaeal transcription system (adapted from Schulz et al and Grohmann, Methods, 2015, Methods, 86:10-18). Top: The archaeal RNA polymerase from Sulfolobus shibatae (PDB: 2WAQ). The subunits are individually color-coded and the seven labeling sites used in a variety of studies are marked with a red sphere. Site-specific coupling of fluorophores to the large subunits Rpo1'(light grey) and Rpo2" (gold) is achieved via a bioorthogonal coupling reaction between the unnatural amino acid p-azidophenylalanine introduced at this site and a phosphine-derivative of a fluorescent dye. Bottom: Structural presentation of the basal transcription factors TBP (TATA-binding protein), transcription factor B (TFB) and E (TFE). Crystal structure of Methanocaldococcus jannaschii TBP (PDB: 2Z8U). Model of archaeal TFB presented in Nagy et al. based on the TBP-TATA-TFB structure from Pyrococcus woesei and the eukaryotic TFIIB structure (Nagy, Grohmann and Michaelis, 2015, Nature Communications, 6:6161). Homology model of TFE from Methanocaldococcus jannaschii TFE (Grohmann et al and Werner, 2011, Molecular Cell, 43:263-74).

Function and mechanism of archaeal Argonaute

In eukaryotic organisms, Argonaute is the functional core of the RNA-silencing machinery critically involved in the regulation of gene expression. Despite the mechanistic and structural similarities between archaeal, bacterial and eukaryotic Argonaute proteins, the biological function of bacterial and archaeal Argonautes is only poorly understood. We especially focus on an archaeal Argonaute variant from the organism Methanocaldococcus jannaschii. We were able to show that this Argonaute variant is the only prokaryotic Argonaute that exclusively utilizes DNA substrates. We are especially interested in the structural features and dynamic behaviour of Argonaute that promote substrate recognition and cleavage, thereby revealing differences and similarities in eukaryotic and prokaryotic Argonaute biology.

Grohmann Fig2

Figure 2: Fluorescence-aided single molecule sorting can be of crucial advantage when dissecting a complex biological sample illustrated for an Argonaute-nucleic acid complex. Argonaute, a protein involved in the fundamental cellular process of gene silencing, is a multi-domain protein containing the flexible PAZ domain. In order to understand the conformational transitions upon loading of the target strand a donor-acceptor pair (Atto550/Alexa647) was engineered into the protein-nucleic acid complex and the resulting FRET signal should informs about conformational changes in the complex. Single-molecule measurements gave access to the 1 % of molecules that exhibit a stable FRET signal. Ultimately, this revealed that Argonaute adopts a different conformation (E=0.42) when the target strand is loaded as compared to the Argonaute-guide DNA complex, which is characterized by a high FRET value (0.84) [Zander et al and Grohmann, RNA Biology, 2014, 11:45-56).


Fluorescence-based single-molecule microscopy

My lab combines classical biochemical methods with sophisticated biophysical techniques like single molecule fluorescence microscopy. Single-molecule fluorescence techniques are ideally suited to provide information about the structure-function-dynamics relationship of a biomolecule as static and dynamic heterogeneity can be easily detected. Currently, we use total internal reflection fluorescence (TIRF) microscopy which allows us to carry out e.g. single-molecule FRET measurements and single-molecule pulldown assays in real-time. Many biophysical techniques require a modification of the biomolecule with a reporter group (e.g. fluorescent dye, spin label, etc). We have a long-standing expertise in the chemical modification of biomolecules via biorthogonal reactions which allows us to site-specifically engineer fluorescent dyes, spin labels or biotin moieties into complex biomolecular machineries enabling us to carry out detailed biophysical studies.

Grohmann Fig3

Figure 3:  Single molecule fluorescence resonance energy transfer (FRET) experiments probe the TBP-promoter DNA interaction (figure adapted from Gietl and Grohmann, Biochemical Society Transactions, 2012, 41:368-73). Single-molecule FRET measurements require the labelling of the promoter DNA with a donor and an acceptor fluorophore adjacent to the TATA box. DNA bending by TBP shortens the distance between the two fluorophores leading to an increase in FRET i.e. the donor intensity (green) decreases while the acceptor intensity (red) increases (left). Single-molecule FRET measurements inform about the conformational distortion of the promoter DNA induced by TBP and the bending mechanism for two archaeal and the eukaryotic transcription system in real-time (right). TBP and TFB/TF(II)B are highly conserved in structure and function among the eukaryotic and archaeal domains of life but intriguingly have to operate under vastly different conditions. We found that eukaryotic and archaeal TBP follow different DNA bending pathways translating into different regulatory modes during early steps of gene expression (figure adapted from Gietl et al and Grohmann, Nucleic Acids Research, 2014, 42:6219-31).

  1. Fakultät für Biologie und Vorklinische Medizin
  2. Faculty Research

Lehrstuhl für Mikrobiologie & Archaeenzentrum

Prof. Dr. Dina Grohmann


Lehrstuhl für Mikrobiologie
Universitätsstraße 31
93053 Regensburg

Phone: +49 941 9433147
Fax: +49 941 9432403
Mail: dina.grohmann@ur.de