Determining the biochemical basis of AlkBH4’s tRNA modifying activity

• Cassidy Driscoll, Biochemistry, University of Delaware

• Jeffrey Mugridge, Department of Chemistry and Biochemistry, University of Delaware

AlkBH4 is an Fe(II)-/2-oxoglutarate-dependent dioxygenase family enzyme that uses Fe(II), oxygen, and cofactor 2-oxoglutarate to oxidize many different substrates in the cell, such as protein, DNA, and RNA. AlkBH4 plays important roles in DNA replication and embryonic development and has been linked to numerous human diseases, including non-small lung cancers where its overexpression leads to tumor progression. Recently, it has been suggested that AlkBH4 catalyzes the hydroxylation of 5-methoxycarbonylmethyluridine (mcm5U) to (R)-5-methoxycarbonylhydroxymethyluridine ((R)-mchm5U) on tRNAs at the wobble position to help regulate protein synthesis. However, the data supporting this claim are relatively weak – there is not clear in vitro biochemistry showing that AlkBH4 forms the (R)-mchm5U modification and there is evidence that AlkBH4 reacts with (R)-mchm5U to form an unknown product. We aimed to express and purify AlkBH4 and produce various tRNA substrates for biochemical experiments to understand the tRNA modification activity of human AlkBH4. 

AlkBH4 was overexpressed in E. coli BL21 cells and then purified using NiNTA affinity chromatography, anion exchange chromatography, and size exclusion chromatography on the FPLC. SDS-PAGE was used to analyze the purity of the protein after each purification step, showing adequate purity to be used in further experimentation. In vitro transcription of tRNA (Arg)UCU and tRNA (Arg)UCG was performed to produce unmodified tRNA substrates. Using electrophoretic mobility shift assays, the binding affinity of AlkBH4 to these unmodified substrates was determined. The KD values for AlkBH4 to tRNA (Arg)UCU and tRNA (Arg)UCG were determined to be 3.389±0.151µM and 2.442±0.062µM, respectively. In the future, the modified tRNA substrates will be produced and activity assays will be performed to determine AlkBH4’s kinetic parameters.