Research is focused on translation factors such as the elongation factor EF-Tu, a multifunctional protein that during protein synthesis alternately forms complexes with GDP or GTP, aminoacyl-tRNA, the ribosome and elongation factor EF-Ts (see Fig. 1). By hydrolysis of bound GTP into GDP and inorganic phosphate, the protein changes its conformation and switches over to a different interaction pattern. Antibiotics such as kirromycin, pulvomycin and GE2270A freeze EF-Tu in a single conformation and thus block the elongation cycle of the ribosome. The cycle can also become blocked in other ways such as by a cleavage in the mRNA. The stuck ribosome is rescued by interaction with Ala-tmRNA having both tRNA and mRNA properties. It makes the ribosome hop to a short internal reading frame terminated by a proper stop codon. In a way not yet understood, EF-Tu en EF-Ts also function as subunits in the replicase complex for bacteriophage Qβ RNA. The complex starts RNA synthesis with GTP on the 3'-CCA end of the phage RNA, a 3'-end structure as in all tRNAs.
Recent international progress in the field of cryo-electron microscopy and crystallographic analysis has yielded high-resolution 3D-structure models of all the macromolecular complexes of Fig. 1, the ribosome included. These static structures must now become animated by analysis of their functional interactions during RNA and protein synthesis. For this analysis various experimental approaches are used. Via mutagenesis methods, mutant macromolecules such as EF-Tu, EF-Ts, or tmRNA are isolated with a changed interaction behaviour. Via all kinds of kinetic assays the changes in the structure-function relationships are measured. The elongation cycle serves as a model for numerous processes of signal transduction in cells, that depend on GTP consumption and can be blocked by toxins or antibiotics. The recognition of tRNA-like structures is a central theme in a number of Genexpress projects.
Fig. 1 The elongation cycle of protein synthesis in bacteria.
Streptomycetes are Gram-positive bacteria from the soil and are remarkable for their special capacity to differentiate During growth and DNA replication the cells do not separate but elongate and branch, which produces a mycelium network. Influenced by external factors such as starvation or drying out, an aerial mycelium develops with curled hyphae from the tips of which spores are secluded. For more details, please refer to the Streptomyces pages of Dr. van Wezel. Streptomycetes are of special interest because they produce more than 60% of all known antibiotics. During the differentiation process, the regulation of the expression of certain genes depends on special sigma factors and a rare tRNALeu.
Antibiotic producing streptomycetes must be resistant against their own antibiotic. In the case of a translation inhibitor like tetracycline, the resistance factor resembles a specialised EF-G; we are currently investigating the resistance mechanism. We have discovered even more forms of specialised elongation factors and are searching for their particular function. On account of the great importance of antibiotics, the biotechnological industry is interested in increasing production efficiencies. The latter are negatively influenced by the high viscosity of the thready mycelium of streptomycetes in an industrial fermentor.
In close collaboration with the group of Dr. Gilles van wezel, we study the role of SsgA, a protein that can stimulate growth to high densities by inducing fragmentation of the mycelium to compartments of almost similar size as E. coli. The nature of the underlying mechanism is being investigated in collaboration with the Technical University of Delft and DSM Anti-Infectives. The specific role of EF-Tu and EF-Ts during replication of bacteriophage Qβ RNA is investigated by ir. Sander Mathu. Drs. Sharief Barends studies the functioning of the hybrid tmRNA molecule in interaction with elongation factors and the ribosome. Drs. Gertjan Hoogvliet studies the functioning of specialised elongation factors in antibiotic producing streptomycetes.