Work plan

Having gathered first experiences with this thermophilic eukaryote, we consider C. thermophilum as an ideal model organism to systematically study the structure and function of the following machines: RNA pol I and pol III and their general transcription factors (Müller, Beck, Hurt, Kos, Russell, Gavin, Bork), complexes involved in co- or post-transcriptional processing (Meinhart, Carlomagno, Koš, Hurt, Fischer, Bork, Gavin, Lemke), nuclear transport (Lemke, Beck, Hurt), ribosome biogenesis factors (Hurt, Koš, Sinning, Wade, Bork, Lemke, Schroeder) and factors operating on mature ribosomal subunits during protein folding and targeting (Sinning, Bukau, Mayer, Wade, Lemke, Schroeder). Given the overall conservation of these molecular machineries in eukaryotes, insights gained from C. thermophilum can be easily transferred to more classical model organisms (yeast, fly, human), where further detailed functional studies will be carried out.

 

(1) RNA transcription and post-transcriptional processing: In eukaryotes transcriptional activity is divided between three RNA polymerases. Pol I and pol III contribute up to 80% of the total transcriptional activity in growing cells. However, there is only limited structural information available for pol I and pol III and their general transcription factors. A detailed structurefunction analysis will be pursued using a combination of X-ray crystallography, single particle electron microscopy, NMR and different mass spectroscopy techniques (native MS, MS of cross-linked peptides). Spatial and temporal information will be combined to establish a dynamic interaction network of the protein complexes involved in pol I and pol III transcription. Molecular machines that are physically and/or functionally linked to these transcription machineries will also be studied from the thermophile, which includes post-transcriptional RNA processing, polyadenylation and nuclear export.

 

(2) Ribosome biogenesis: Although conserved in evolution, eukaryotic ribosomes and their biogenesis differ significantly from prokaryotic ribosomes. Accordingly, most of the eukaryotic biogenesis factors cannot be obtained from prokaryotic thermophiles. Since it may be difficult to crystallize pre-ribosomal subunits, due to low yield and homogeneity, the 3D EM structures of pre-60S and pre-40S particles will be determined at intermediate resolution (e.g. by cryo- EM) and the location of associated factors on the pre-ribosomal surface will be identified by antibody labeling or UV crosslinking. X-ray structures of pre-ribosomal factors and modules will be used to fit the overall EM structure of the nascent ribosomes. Thus, in vitro reconstitution of purified C. thermophilum pre-ribosomal factors into stable subcomplexes will be performed, which is crucial to dissect the interaction network between the numerous preribosomal factors on the newly forming subunits. A Reinhard-Koselleck grant to Ed Hurt to study ribosome biogenesis further supports this activity. (3) Nascent chain folding and processing: Eukaryotic ribosomes associate with numerous factors that mediate ribosome biogenesis and dictate the fate of the nascent chain during protein synthesis. The emerging nascent chains become co-translationally folded with the help of ribosome-associated chaperones, modified by ribosome associated enzymes and targeted to the ER by the signal recognition particle (SRP). Despite the pivotal roles of these ribosomeassociated factors, our understanding of their action on nascent chains in eukaryotes is very limited. We will utilize the C. thermophilum genome to express these factors for structure determination using X-ray crystallography and cryo-EM. Together with biochemical and biophysical analyses using e.g. HX-MS, fluorescence and EPR spectroscopy, these studies will allow dissecting the network of interactions experienced by newly synthesized proteins.

 

(4) From structural insight to dynamic interaction networks: In addition to structural studies, large-scale biochemistry and computational approaches will enable us to put the structural work in a systemic context. Time and space resolved interaction networks will be deduced to arrive at a highly resolved biological understanding of the processes studied. For example, global transcriptomics and proteomics, coupled with large-scale biochemical purifications of selected processes will, by state-of-the-art bioinformatic analysis, reveal adaptation of networks to thermophily beyond the individual proteins, will capture fingerprints of different states of individual processes, and place it into the context of eukaryotic evolution. Budget: Funding is requested for 4 postdocs and 4 PhD students with appropriate consumables. A committee representing the consortium will recruit candidates on the basis of scientific excellence and collaborations between the participating groups. In addition, support for an annual retreat to exchange results and ideas is requested.