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Functional analysis of a heat shock inducible methyltransferase from Escherichia coli

RrmJ was originally discovered by Richmond and coworkers as a highly heat inducible E. coli gene. Previous studies by us and others have shown that RrmJ functions as a 2'-O-methyltransferase that methylates the universally conserved nucleotide U 2552 in the A-loop of the 23S rRNA. Deletion studies revealed that a strain lacking RrmJ shows a severely impaired growth as well as a strong ribosomal defect. This led to the conclusion that the U 2552 methylation is involved in either ribosome assembly or stability. Structural comparison of RrmJ with other 2'-O-methyltransferases revealed striking similarities with the bi-functional protein VP39 from Vacchinia virus. Because VP39 has been intensively investigated in the past this structural resemblance was extremely helpful for the functional characterization of RrmJ as it is presented in this work. Using extensive site directed mutagenesis, I investigated the active site as well as substrate binding site of RrmJ. Our results revealed that RrmJ's active site is composed of a catalytic triad KDK, allowing us to propose a S N 2 reaction mechanism for the methylation reaction. The amino acids that I have identified to be involved in substrate binding were found to build a positively charged ridge, which should be able to interact with the sugar phosphate backbone of the RNA substrate. Modeling studies performed in collaboration with Bart Staker suggested that the A-loop has to loop out of the tightly packed structure of the 50S ribosomal subunit in order to be accessible for RrmJ. Furthermore, modeling of the A-loop onto the surface of RrmJ underlined the proposed substrate binding site and suggested a base flipping mechanism for U 2552 in order to fit into the active site of RrmJ. Co-fractionation studies of RrmJ built the foundation for the major observation that RrmJ has a methyltransferase independent function as a ribosome assembly helper protein. For the binding of RrmJ to 50S ribosomal subunits, the N-terminus of RrmJ appeared to be crucial. I showed that the binding of RrmJ to the 50S ribosomal subunits supports the assembly process of the 70S ribosome. This second function of RrmJ appears to be responsible for the ribosomal defect observed in the rrmJ deletion strain, which has originally been attributed to the Um 2552 modification. This finding was supported by Caroline Kumsta, a diploma student in the lab, who showed that absence of U 2552 does not lead to a ribosome assembly defect. III Summary Lastly, …