L - Technische Universität Braunschweig

More documents

Recommendations

Info

10 APPLICATION DR. DINA GROHMANN elongation factors and RNAP) while others have to be disrupted (contacts between RNAP and the promoter DNA and initiation factors). During my postdoctoral work I established a fluorescently labelled version of the previously developed wholly recombinant transcription system derived from the hyperthermophilic archaeal model organism Methanocaldococcus jannaschii. With the possibility to site-specifically introduce fluorescent probes into the twelve-subunit archaeal RNAP as well as into the basal transcription factors TBP and TFE fluorescence-based technologies are employed in my lab to gain a deeper understanding of the organisation of large transcription complexes. Single molecule fluorescence measurements are an excellent tool to analyse dynamic protein-nucleic acid complexes and we were just able to describe the dynamic behaviour of the RNAP clamp domain throughout the transcription cycle (manuscript in preparation). Furthermore, my group is focusing on the transcription initiation factor TBP which induces a severe bend in the promoter DNA that can be quantitatively described using a FRET-based bending assay. Even though the structures of TBP from different organisms is highly conserved we found that the kinetics and of the bending process is dramatically different in the archaeal and eukaryotic domain of life (manuscript submited). Our data suggest that the TBP-promoter DNA interaction is highly adapted to environmental conditions resulting in fundamentally different factor requirements. RNA-induced silencing complex (RISC) Targeted gene silencing by RNA interference (RNAi) represents a very critical mechanism to control cellular transcript and protein levels and is therefore involved in a multitude of important cellular functions. All RNA-silencing processes are based on large ribonucleoprotein assemblies, termed RNAinduced silencing complexes (RISCs). At the functional core of RNA-silencing pathways, every RISC contains a member of the Argonaute family (Ago). This key protein is bound to a small non-coding RNA and is responsible to direct RISC to the target mRNA which then leads to Ago-catalysed degradation of the mRNA or translational inhibition. Misregulated RNAi-based processes cause cancer and it is of special interest to gain a detailed understanding of the molecular mechanisms that drive RNAi in order to develop specific and effective therapeutics. A description of the dynamics of the mobile PAZ-domain of Ago is still missing and the mechanisms that underlie the transfer of the RNA between the RISC proteins are poorly understood. I started to use single-molecule methods in conjunction with biochemical approaches to unravel the conformational changes of site-specifically fluorescently labelled Ago proteins (manuscript summarising our first results is currently under review). Here, we succeeded in a stochastical labelling of a protein with a fluorescent donor and acceptor via two unnatural amino acids. Our in vitro data are currently complemented by ex situ experiments that allow the direct immobilization of the endogenous RISC complex from cellular extracts on a cover slip making it amenable to single molecule studies (single-molecule pulldown assay). As the RISC complex has not been successfully reconstituted in larger amounts so far structural information of the complete complex could just be obtained using cryo-electron microscopy. Our approach potentially allows us to gain more insights into the structural organization and the dynamics of the complex.
11 APPLICATION DR. DINA GROHMANN RESEARCH ACCOMPLISHMENTS Whether I worked on proteins that are involved in the assembly of the photosynthetic complex II, the HIV-1 Reverse Transcriptase or currently on the more complex system of multisubunit RNA polymerases and transcriptional regulation I have always been highly interested in the molecular mechanisms that govern the function of a protein/enzyme interacting with other proteins or nucleic acids. This included very often a detailed characterization of not just a single protein but a whole protein-nucleic acid interaction network and a dissection of the factors that influence the activity of an enzyme. Instrumental to the success of my work has been the development of cutting edge labelling strategies, e.g. the site-specific incorporation of fluorescent probes via unnatural amino acids into proteins. During my PhD thesis I developed alternative strategies for the inhibition of HIV focusing on the polymerase of the virus, the Reverse Transcriptase. With the emergence of drug-resistant HIV strains new inhibitory approaches are required and the HIV-1 Reverse Transcriptase is an ideal target protein as it is vital for viral replication copying the viral RNA into cDNA for subsequent integration into the host genome. I worked on the development of peptide, nucleic acid (aptamers, hexamers) and small molecule inhibitors that are targeted at the reverse transcriptase. Most notably, I characterized the first small molecule inhibitor that prevents the dimerization of the two Reverse Transcriptase subunits thereby inactivating the enzymatic function that is correlated with the dimeric form of the enzyme. Based on my mutational studies a structure-based ligand design combined with docking studies was carried out that led to the identification of several small molecules with the potential to disrupt RT dimerization. Using an in vitro dimerization assay I demonstrated that one of the selected molecules strongly reduced the association of the two RT subunits. Additionally, I showed that the compound simultaneously inhibited both the polymerase as well as the RNaseH activity of the enzyme. This study represented the first successful rational screen for a small molecule HIV RT dimerization inhibitor. Furthermore, I described the effect of an additional viral protein, the Nucleocapsid (NC)-protein on reverse transcription. NC is a so-called nucleic acid chaperon and is associated with the viral RNA and promotes various steps during reverse transcription. In order to describe in a quantitative fashion the effect of NC on reverse transcription I carried out single-turnover, single-nucleotide incorporation studies. I complemented these functional studies with time-resolved FRET experiments to determine the influence of NC on the dissociation of the RT-substrate complex. My studies revealed that NC considerably enhances the stability of RT-substrate complexes by reducing the observed dissociation rate constants, which more than compensates for the observed drop in the polymerization rate 1 . These data shed a new light on the activity spectrum of NC as it not only indirectly assists the reverse transcription process by its nucleic acid chaperoning activity but also positively affects the RTcatalysed nucleotide incorporation reaction by increasing polymerase processivity presumably via a physical interaction of the two viral proteins. During my work as Postdoctoral Fellow at the University College London I focused on the application of fluorescence techniques in combination with other biophysical methods to carry out structural and functional studies on one of the most complex cellular machines – the multi-subunit RNA
Page 1 and 2: 2013 Dr. Dina Grohmann Junior Resea
Page 3 and 4: 3 APPLICATION DR. DINA GROHMANN Flu
Page 5 and 6: 5 APPLICATION DR. DINA GROHMANN UNI
Page 7 and 8: 7 APPLICATION DR. DINA GROHMANN RES
Page 9: 9 APPLICATION DR. DINA GROHMANN oli
Page 13 and 14: 13 APPLICATION DR. DINA GROHMANN BO
Page 15 and 16: 15 APPLICATION DR. DINA GROHMANN 19
Page 17 and 18: 17 APPLICATION DR. DINA GROHMANN TE
Page 24 and 25: Nucleic Acids Research Advance Acce
Page 26 and 27: Nucleic Acids Research, 2012 3 (HJ)
Page 28 and 29: Nucleic Acids Research, 2012 5 corr
Page 30 and 31: Nucleic Acids Research, 2012 7 rate
Page 32 and 33: Nucleic Acids Research, 2012 9 (ii)
Page 34 and 35: Published on Web 04/12/2010 RNA-Bin
Page 36 and 37: Molecular Cell Article The Initiati
Page 38 and 39: Molecular Cell Mechanisms of TFE an
Page 48 and 49: REVIEWS Evolution of multisubunit R
Page 50 and 51: REVIEWS Assembly platform Wall Stal
Page 52 and 53: REVIEWS Table 1 | Conserved RNA pol
Page 54 and 55: REVIEWS Bacteria a σ4 σ3.2 σ3 σ
Page 56 and 57: REVIEWS Bacteria a RNAP flap RNAP c
Page 58 and 59: REVIEWS a b c d NGN RNAP-binding si
Page 60 and 61:
REVIEWS 1. Werner, F. Structural ev
show all

L - Technische Universität Braunschweig

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?