A Few Strokes to the Family Portrait of Translational GTPases

Detta är en avhandling från Uppsala : Acta Universitatis Upsaliensis

Sammanfattning: Protein biosynthesis is a core process in all living organisms. Assembly of the protein chain from aminoacids is catalysed by the ribosome, ancient and extremely complex macromolecular machine. Several different classes of accessory molecules are involved in translation, and one set of them, called translational GTPases (trGTPases), was in the focus of this work. In this thesis properties of two trGTPases– EF-G and eRF3 - were studied by means of direct biochemical experiments. EF-G is a bacterial trGTPase involved in two steps of translation: translocation and ribosomal recycling. Translocation is a process of the ribosomal movement along the mRNA, and recycling as the step when upon completion of the protein ribosome is released from the mRNA via splitting in two ribosomal subunits. We found that off the ribosome EF-G has similar affinities to GDP and GTP, and thus given the predominance of the latter in the cell, EF-G should be present mostly in the complex with GTP. However, binding to the ribosome increases factors affinity to GTP drastically, ensuring that it is in the GTP-bound state. GDP can not promote neither translocation, not recycling, and GDPNP can not promote recycling. It can, however, promote translocation, but in so doing it results in an intermediate ribosomal state and translocation process can be reversed by addition of GDP, which is not the case for the EF-G•GTP-catalyzed reaction.The second trGTPase we investigated is eukaryotic termination factor eRF3. This protein together with another factor, eRF1, is involved translation termination, which is release of the synthesized protein from the ribosome. We demonstrateed, that eRF3 alone has basically no propensity to bind GTP and thus resides in the GDP-bound state. Complex formation between eRF1 and eRF3 promotes GTP binding by the latter, resulting in the formation of the ternary complex eRF1•eRF3•GTP, which in turn is catalyzing the termination event.Experimental investigations of trGTPases where rationalized within a generalized thermodynamical framework, accommoding the existent experimental observations, both structural and biochemical.