Structure of the Complete RNA Polymerase II Elongation Complex and its Interaction with the Elongation Factor TFIIS

This thesis describes crystal structures of complete, 12-subunit yeast RNA polymerase II (Pol II) in complex with a synthetic transcription bubble and product RNA, with an NTP substrate analogue, and in complex with the transcription elongation factor TFIIS. The structure of the Pol II-transcription bubble-RNA complex reveals incoming template and non-template DNA, a seven base-pair DNA-RNA hybrid, and three nucleotides each of separating DNA and RNA. Based on this structure, those parts of Pol II were identified which are involved in separating template DNA from non-template DNA before the active site, and DNA from product RNA at the upstream end of the DNA-RNA hybrid. In both instances, strand separation can be explained by Pol II-induced duplex distortions. Only parts of the complete transcription bubble present in the complexes are ordered in the crystal structure, explaining the way in which high processivity of Pol II is reconciled with rapid translocation along the DNA template. The presence of an NTP substrate analogue in a conserved putative pre-insertion site was unveiled in a Pol II-transcription bubble-RNA complex crystal soaked with the substrate analogue GMPCPP. The structure of the Pol II-TFIIS complex was obtained from Pol II crystals soaked with TFIIS. TFIIS extends from the Pol II surface to the active site and complements the active site with two essential and invariant acidic residues for hydrolytic RNA cleavage. TFIIS also induces extensive structural changes in Pol II that reposition nucleic acids, in particular RNA, near the active centre. These results support the idea that Pol II contains a single tuneable active site for RNA polymerisation and cleavage. The technical obstacles imposed by crystal structure determination of large, transient protein-DNA-RNA complexes were overcome by two novel, fluorescence-based assays to monitor and optimise the composition of the crystals. Both assays are not limited to Pol II complexes, but can serve as a general tool for the crystallographic community

Complete RNA polymerase II elongation complex structure and its interactions with NTP and TFIIS.

The crystal structure of the complete 12 subunit RNA polymerase (pol) II bound to a transcription bubble and product RNA reveals incoming template and non-template DNA, a seven base pair DNA/RNA hybrid, and three nucleotides each of separating DNA and RNA. The complex adopts the posttranslocation state and accommodates a cocrystallized nucleoside triphosphate (NTP) substrate. The NTP binds in the active site pore at a position to interact with a DNA template base. Residues surrounding the NTP are conserved in all cellular RNA polymerases, suggesting a universal mechanism of NTP selection and incorporation. DNA-DNA and DNA-RNA strand separation may be explained by pol II-induced duplex distortions. Four protein loops partition the active center cleft, contribute to embedding the hybrid, prevent strand reassociation, and create an RNA exit tunnel. Binding of the elongation factor TFIIS realigns RNA in the active center, possibly converting the elongation complex to an alternative state less prone to stalling

Development of a Bisphosphonate Delivering Hydrogel for the Augmentation of Impaired Peri-Implant Bone

Osteoporosis is a major health problem in our aging society. This metabolic disease, which is characterized by a deterioration of the bone microarchitecture and a significant loss of bone mass, is affecting a rising number of mostly elderly patients. The main clinical consequences are typical fragility fractures resulting in severe pain, morbidity, and mortality for affected patients. Osteoporosis does not only cause fractures, but also complicates fracture treatment, as implants are difficult to anchor in the impaired bone structure. Therefore, many mechanical and pharmaceutical approaches have been developed over the last decades to target this issue and improve implant anchorage in osteoporotic bone. The overall goal of the present PhD thesis was the development of a bisphosphonate (BP) releasing hydrogel that can enhance implant fixation in osteoporotic bone. The project was divided into three major sections: the investigation of the BP effect on peri-implant bone, the development and testing of the drug delivering biomaterial, and the evaluation of its efficiency in terms of implant fixation improvement. An ovariectomized rat model of postmenopausal osteoporosis was utilized for the investigation of the spatio-temporal effect of locally released Zoledronate, the BP used in this study. The drug was delivered from a biodegradable hyaluronic acid (HyA) hydrogel matrix to the bone stock surrounding screws that were implanted in the femoral condyles of the rats. Static and dynamic bone histomorphometric parameters were monitored in four concentric bone layers around the screw with time-lapsed in vivo microCT scans. With this study, we were able to demonstrate a significant enhancement of early bone formation rate accompanied by an efficient inhibition of peri-implant bone resorption in a large range around the screw. In a second in vivo study, we incorporated Zoledronate-loaded hydroxyapatite nanoparticles in the HyA hydrogel resulting in an unexpected rapid mineralization of the hydrogel within 10 days after implantation. Histology performed 2 months after implantation revealed granule-shaped mineralized spots within the peri-implant bone serving as scaffolds for new bone formation. When using Zoledronate-loaded particles, we could demonstrate a strong inhibitory effect on both peri-implant bone resorption and mineralized hydrogel degradation. Finally, we used the in vivo microCT scans of the first animal study to create micro-finite element models for the analysis of the screw fixation time course. We were able to show that Zoledronate locally delivered from the hyaluronic acid hydrogel improved screw fixation significantly as soon as 17 days after implantation when compared to an untreated control group. This difference persisted until the end of the study at day 58. Taken together, the studies performed for the present PhD thesis demonstrated an excellent suitability of HyA hydrogels for the local delivery of BPs intended to improve implant fixation in impaired bone. A Zoledronate triggered enhancement of screw fixation occurred early and persisted over a prolonged period, an important aspect considering that osteosynthesis implants need a reliable bone anchorage from the time of implantation until complete fracture healing. Furthermore, it could be shown that an addition of hydroxyapatite particles to the HyA resulted in a rapid in vivo mineralization of the hydrogel, a promising feature for bone defect repair applications

Crystal Structure of an Anti-Ang2 CrossFab Demonstrates Complete Structural and Functional Integrity of the Variable Domain.

Bispecific antibodies are considered as a promising class of future biotherapeutic molecules. They comprise binding specificities for two different antigens, which may provide additive or synergistic modes of action. There is a wide variety of design alternatives for such bispecific antibodies, including the "CrossMab" format. CrossMabs contain a domain crossover in one of the antigen-binding (Fab) parts, together with the "knobs-and-holes" approach, to enforce the correct assembly of four different polypeptide chains into an IgG-like bispecific antibody. We determined the crystal structure of a hAng-2-binding Fab in its crossed and uncrossed form and show that CH1-CL-domain crossover does not induce significant perturbations of the structure and has no detectable influence on target binding

Vehicle Suspension Including a Link

A link of a vehicle suspension is elastic and includes three suspension points. A first point is adapted to be pivotally connected to a wheel carrier of the corresponding vehicle. A second point is adapted to be pivotally connected to a rigid chassis of the corresponding vehicle. The third point is adapted to be pivotally connected to a shackle, the shackle pivotally connecting the third point to the rigid chassis of the vehicle

In vitro and in vivo investigation of bisphosphonate-loaded hydroxyapatite particles for peri-implant bone augmentation

Locally applied bisphosphonates, such as Zoledronate, have been shown in several studies to inhibit peri-implant bone resorption and recently to enhance peri-implant bone formation. Studies have also demonstrated positive effects of hydroxyapatite particles on peri-implant bone regeneration and an enhancement of the anti-resorptive effect of bisphosphonates in the presence of calcium. In the present study, both hydroxyapatite nanoparticles (nHA) and Zoledronate were combined to achieve a strong reinforcing effect on peri-implant bone. The nHA-Zoledronate-combination was first investigated in vitro with a pre-osteoclastic cell assay (RAW 264.7) and then in vivo in a rat model of postmenopausal osteoporosis. The in vitro study confirmed that the inhibitory effect of Zoledronate on murine osteoclast precursor cells was enhanced by loading the drug on nHA. For the in vivo investigation, either Zoledronate-loaded nHA or pure nHA were integrated in hyaluronic acid hydrogel. The gels were injected in screw holes that were predrilled in rat femoral condyles before insertion of miniature screws. MicroCT-based dynamic histomorphometry and histology revealed an unexpected rapid mineralization of the hydrogel in vivo through formation of granules, which served as scaffold for new bone formation. The delivery of Zoledronate-loaded nHA further inhibited a degradation of the mineralized hydrogel as well as a resorption of the peri-implant bone as effectively as unbound Zoledronate. Hyaluronic acid with Zoledronate-loaded nHA, thanks to its dual effect on inducing a rapid mineralization and preventing resorption, is a promising versatile material for bone repair and augmentation

Structure–function studies of the RNA polymerase II elongation complex

X-ray crystallographic and complementary functional studies have contributed significantly to the current understanding of gene transcription. Here, recent structure–function studies on various aspects of the elongation phase of transcription are summarized

Time course of bone screw fixation following a local delivery of zoledronate in a rat femoral model – a micro-finite element analysis

A good fixation of osteosynthesis implants is crucial for a successful bone healing but often difficult to achieve in osteoporotic patients. One possible solution to this issue is the local delivery of bisphosphonates in direct proximity to the implants, A critical aspect of this method, that has not yet been well investigated, is the time course of the implant fixation following the drug release. Usual destructive mechanical tests require large numbers of animals to produce meaningful results. Therefore, a micro-finite element (microFE) approach was chosen to analyze implant fixation. In vivo micro computed tomography (microCT) scans were obtained, first weekly and later bi-weekly, after implantation of polymeric screws in the femoral condyles of ovariectomized rats. In one half of the animals, Zoledronate was released from a hydrogel matrix directly in the peri-implant bone stock, the other animals were implanted only with screws as control. The time course of the implant fixation was investigated with linear elastic microFE models that were created based on in vivo microCT scans. The numerical models were validated against experimental pullout-tests measurements in an additional cadaver study. The microFE analysis revealed a significant increase in force at yield of the Zoledronate treated group compared to the control group. The force of the treated group was 28% higher after 17 days of screw implantation, 42% higher after 31 days. The significant difference persisted until the end of the in vivo study at day 58 (p<0.01). The early onset and prolonged duration of the implant anchorage improvement that was found in this study indicates the great potential of Zoledronate-loaded hydrogel for an enhancement of osteosynthesis implant fixation in impaired bone

The role of energy dissipation of polymeric scaffolds in the mechanobiological modulation of chondrogenic expression

Mechanical stimulation has been proposed to induce chondrogenesis in cell-seeded scaffold. However, the effects of mechanical stimuli on engineered cartilage may vary substantially between different scaffolds. This advocates for the need to identify an overarching mechanobiological variable. We hypothesize that energy dissipation of scaffolds subjected to dynamic loading may be used as a mechanobiology variable. The energy dissipation would furnish a general criterion to adjust the mechanical stimulation favoring chondrogenesis in scaffold. Epiphyseal chondro-progenitor cells were then subject to unconfined compression two hours per day during four days in different scaffolds, which differ only by the level of dissipation they generated while keeping the same loading conditions. Scaffolds with higher dissipation levels upregulated the mRNA of chondrogenic markers. In contrast lower dissipation of scaffolds was associated with downregulation of chondrogenic markers. These results showed that energy dissipation could be considered as a mechanobiology variable in cartilage. This study also indicated that scaffold with energy dissipation level close to the one of cartilage favors chondrogenic expression when dynamical loading is present

Crosslinking-MS analysis reveals RNA polymerase I domain architecture and basis of rRNA cleavage

RNA polymerase (Pol) I contains a 10-subunit catalytic core that is related to the core of Pol II and includes subunit A12.2. In addition, Pol I contains the heterodimeric subcomplexes A14/43 and A49/34.5, which are related to the Pol II subcomplex Rpb4/7 and the Pol II initiation factor TFIIF, respectively. Here we used lysine-lysine crosslinking, mass spectrometry (MS) and modeling based on five crystal structures, to extend the previous homology model of the Pol I core, to confirm the location of A14/43 and to position A12.2 and A49/34.5 on the core. In the resulting model of Pol I, the C-terminal ribbon (C-ribbon) domain of A12.2 reaches the active site via the polymerase pore, like the C-ribbon of the Pol II cleavage factor TFIIS, explaining why the intrinsic RNA cleavage activity of Pol I is strong, in contrast to the weak cleavage activity of Pol II. The A49/34.5 dimerization module resides on the polymerase lobe, like TFIIF, whereas the A49 tWH domain resides above the cleft, resembling parts of TFIIE. This indicates that Pol I and also Pol III are distantly related to a Pol II–TFIIS–TFIIF–TFIIE complex