Structural studies on the thiostrepton resistance methyltransferase. (360G-Wellcome-079242_Z_06_Z)

We propose to study the structure and function of the RNA modifying enzyme thiostrepton resistance methyltransferase (TRMT). TRMT is expressed in the thiostrepton antibiotic producing organism, Streptomyces azureus, to confer resistance by ribose 2 -OH methylation of a single specific nucleotide (A1067) in the large ribosomal subunit rRNA. We will determine the X-ray crystallographic structures of TRMT in various functional states: from the apo enzyme to complexes with S-Adenosyl-L-methionine (A doMet) cofactor (or cofactor product) and/ or rRNA fragments. As a starting point, we have already obtained crystals of the TRMT-rRNA complex (using a 58 nucleotide rRNA domain) that diffract to better than 2.8 resolution, and can be indexed and processed with reasonable statistics. Crystals of other complexes will be obtained through systematic high-throughput crystallisation methods. These studies will also be supported by RNA and protein mutagenesis analysis and further structural studies of antibiotic-rRNA-protein complexes. Our results will address important questions of RNA-protein recognition in RNA modification (vital for selection of the correct target by the enzyme) and the mechanism of RNA modification. While thiostrepton is not a clinically important antibiotic, this work will also provide a good model system for understanding antibiotic resistance via rRNA modification.