Aim 1

To visualize and mechanistically understand mammalian translocation.

Translocation is one of the least understood processes in protein biosynthesis. Its correct completion is crucial for the continuation of the translation elongation cycle, and ultimately for protein synthesis. As upon translocation, the contacts between the ribosome and the tRNA2•mRNA-module extensively rearrange, it is a process which requires utmost accuracy. Efficient translocation depends on the action of the specialized GTPase eEF2; however, detailed insights into the mechanism, by which eEF2 catalyzes translocation is lacking and opposing hypotheses are vividly discussed: The Brownian ratchet model, in which eEF2 is supporting intrinsic conformational changes that lead to translocation, and the power stroke model, according to which eEF2, being a motor protein, actively moves the tRNAs in the direction of translocation (Chen et al., 2016; Liu et al., 2014; Rodnina et al., 1997; Spirin, 2009).

Moreover, structural knowledge on tRNA translocation is dominated by studies carried out in the bacterial system, and structural data on mammalian tRNA translocation does not exist. Therefore, this work is dedicated to studying how translocation is performed in the mammalian system, using an in vitro reconstitution of a rabbit 80S•tRNA2•mRNA•eEF2•GMPPNP complex. The goal is to obtain structures of mammalian translocation intermediates to characterize mammalian translocation and compare it to translocation in the bacterial and yeast system.