Rio proteins are serine kinases that contain few of the conserved residues of typical protein kinases and are thus categorized as atypical protein kinases. Rio proteins are required for the cleavage of 20S rRNA to 18S rRNA. The rRNA synthesis begins with transcription of a 35S<br/>rRNA by RNA polymerase I. This 35S rRNA transcript contains 5.8S, 18S, and 25S rRNA. Proteins assemble onto 35S rRNA to direct cleavage of the rRNA to produce 20S rRNA. Further processing of the 20S rRNA is performed in the cytoplasm with proteins including Rio kinases to release the 18S rRNA. Once the cleavages are completed, these ribosomal assembly factors dissociate. The Rio kinases consist of four 4 subfamilies of proteins, Rio1, Rio2, Rio3, and RioB. Rio2 exist in archaea, eukaryotes and bacteria with high sequence and activity similarities. Canonical protein kinases are very similar to Rio2 proteins but are more active in ATPase and atuophosphorylation activity and they are onlyobserved in archaea.<br/>Computational studies have revealed that the arginine residue at position 228 highly affects the activity of canonical protein kinases. This residue in Rio family is replaced by either alanine or glycine. We were interested to study mutation at 228 of Rio2, and have found out how this mutation would affect the structure and activity of the Rio2 family. We have investigated the evolutionary relationship between Rio kinases and the canonical eukaryotic protein kinases. Our observation is that canonical eukaryotic protein kinases have evolved from Rio kinases. We have made several mutations in a key differentiating position of the kinase domain sequence of Ct- Rio2, G228, and have collected biochemical data to test the ability of the mutated Rio kinase to autophosphorylate and carry out ATP hydrolysis. We have also co-crystallized Ct-Rio2 G228 point mutants with ATP in order to determine the structural differences between the mutant and<br/>wild-type. Hence, wild type Ct-Rio2 and five mutants of Ct-Rio2 were purified and co-crystalized with ATP/Mg2+. These mutants include G228A, G228D, G228N, G228R, and G228V and x-ray diffraction data sets were collected.

Investigation of the evolutionary relationship between Rio2 kinases and the canonical eukaryotic protein kinases using X-ray crystallography

249th National Meeting (2015)

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