Enhancing AlkBH8-tRNA Binding with Single-Point Mutations

• Samantha Plank, Biochemistry, University of Delaware

• Jeffrey Mugridge, Chemistry and Biochemistry, University of Delaware

AlkBH8 is an Iron(II)/2-oxoglutarate-dependent dioxygenase that catalyzes the methylation and subsequent hydroxylation of 5-carboxymethyluridine (cm 5 U) at the wobble position of transfer RNAs (tRNAs) to 5-methoxycarbonylmethyluridine (mcm 5 U) and (S)-5-methoxyhydroxycarbonylmethyluridine (S)-mchm 5 U. Loss of these modifications results in increased translation errors and slower translation rates as they are crucial for stabilizing codon-anticodon interactions.Additionally, AlkBH8 has been implicated in human bladder cancer and oxidative stress-based cognitive disease.However, there is very little biochemical and biophysical information on AlkBH8-tRNAinteractions and there are no structures of the complex. The goal was to express and purify AlkBH8 variants and run biochemical experiments to understand single-point mutation effects on tRNA binding. 

Single-point mutations were introduced to individual AlkBH8 domains that were predicted to enhance tRNA binding affinity using polymerase chain reaction (PCR). These AlkBH8 mutants were then overexpressed in E. coli BL21 cells and purified using NiNTA affinity chromatography, anion exchange chromatography, and size exclusion chromatography. Following each purification step, SDS-PAGE was used to analyze purity of the protein prior to further purification. In vitro transcription of tRNA (Gly)UCC was performed to produce tRNA substrates. Electrophoretic mobility shift assays (EMSAs) were used to test the binding affinity of the AlkBH8 variants to the tRNA. The KD values for MTase/TRMT112 domain T38E and W150K variants to tRNA (Gly)UCC were determined to be 1.284 µM and 1.887 µM, respectively. Comparing these KD values to that of WT MTase/TRMT112, 1.661 µM, there is no observable binding effect promoted by these mutations. In the future, more mutations to the full-length AlkBH8/TRMT112 and its individual domains will be generated to study AlkBH8-tRNA interactions.