Manfred Schäck

• Transfer RNAs are among the most posttranscriptionally modified RNA molecules in nature. Anticodon modifications at wobble position U34 are particularly important, since they help to modulate the codon - anticodon interaction and wobble recognition in the process of translation. The eukaryotic Elongator complex consists of 6 different proteins (Elp1-Elp6) and catalyzes the first step in the Elongator dependent U34 modification pathway. Elongator dysfuncion has severe effects on different cellular processes and Elongator mutants in yeast and Arabidopsis display specific stress phenotypes. This thesis presents the first investigation on Elongator dependent anticodon U34 modifications in the evolutionary supergroup of Amoebozoa. The first part of the thesis focused on the identity of the complex. Pulldown studies with ectopically overexpressed Elongator subunits revealed that the Dictyostelium discoideum Elongator complex also consists of 6 proteins. Furthermore, it was shown that the Elp3 protein is unstable. The second part of the thesis focused on the Elongator dependent modifications. A series of mutant strains was generated that interrupted the Elongator dependent modification pathway at all major steps. Different analytic techniques were applied to investigate the modifications. It could be shown that the Elongator dependent modifications ncm5U, mcm5U and mcm5s2U are conserved in D. discoideum. The effect of loss of the mcm5s2U modifications was tested on the decoding capability of tQ(UUG) towards glutamine codon translation with artificial gene constructs. PolyQ leaders with different CAA/CAG codon ratios fused to GFP were expressed in the background of different mutant strains. The expression differences were measured via flow cytometry and Western blot. Furthermore, the first unconditional tQ(CUG) null mutant in an eukaryotic model organism was generated. These findings provide evidence that wobbling of tQ(UUG) over CAG codon can occur in D. discoideum.