Cloning and production of bait proteins for characterization of pre-ribosomal substrates of rRNA methyltransferases

Sammanfattning

Abstract The ribosome is an important component in the cell, responsible for protein synthesis, made of RNA and proteins. It is composed of a small and a large subunit which assemble together. During its assembly process, many different modifications are made. Some of these modifications are methylated nucleotides in the rRNA. rRNA methyltransferases are proteins that perform the modifications with the help of methyl donors. RlmE and EmtA are two of these proteins. They both act on the rRNA of the large subunit and methylate two specific sites. RlmE modification of U2552 is essential for ribosome assembly and EmtA modification of G2470 provides antibiotic resistance. In this study, the two methyltransferases were each cloned to contain two sequential affinity tags, a His tag facilitating protein purification and a twin-strep tag for ribosome pulldown experiments. Both RlmE and EmtA were successfully expressed and purified. Thermal shift assays were used to test the functionality of the two methyltransferases in S-adenosylmethionine (SAM) binding. The RlmE showed significant increase in stability whereas EmtA had a decrease in melting point, which indicates co-factor binding. Future work should include ribosomal pulldown experiments followed by Cryo-electron microscopy (cryo-EM). This could lead to structures of methyltransferases in complex with their respective substrate, ultimately revealing their binding sites, the potential role of RlmE in ribosome assembly, and the function of EmtAs in antibiotic resistance.