Mechanism of Bacterial Ribosome 30S and 50S Subunit Recognition and Modification by M. tuberculosis TlyA

Abstract

RNA (rRNA) modification is important for correct ribosome assembly, can alter ribosome function, and can confer resistance to clinically important ribosome‐targeting antibiotics. While the activity of most ribosome‐targeting antibiotics is blocked by rRNA methylation, the tuberactinomycin antibiotic capreomycin requires ribose 2′‐OH methylation at C1409 of the 16S rRNA within the small ribosome subunit (30S) and C1920 of the 23S rRNA within the large ribosome subunit (50S). TlyA is the 2′‐O‐methyltransferase that incorporates both modifications using S‐adenosyl‐L‐methionine (SAM) as a methyl group donor. The X‐ray crystal structure of the C‐terminal domain (CTD) of TlyA showed that the domain adopts a Class I methyltransferase fold, while homology modeling suggests the N‐terminal domain (NTD) adopts an S4 ribosomal protein fold. Additionally, the TlyA CTD structural studies revealed that the short interdomain linker was able to adopt two different conformations and was unexpectedly critical for SAM binding within the CTD. This project will test the resulting hypothesis that the interdomain linker acts a “molecular switch,” altering the interaction between the NTD and the CTD to control TlyA activity upon correct recognition of its two structurally distinct substrates. Using a sequence alignment and structure‐guided mutagenesis approach coupled with fluorescence polarization (FP) TlyA‐30S binding and methyltransferase activity assays, the role of the NTD and individual residues in this domain in 30S and 50S binding will be defined. Using complementary studies of TlyA protein dynamics (using HDX‐MS) and structure (using X‐ray crystallography and cryo‐EM), the long‐term goal of this project is to determine TlyA's molecular mechanism of site‐specific ribosome subunit recognition and modification. Defining these molecular details will help complete the picture of TlyA activity and its role in bacterial drug sensitivity or resistance, as well as furthering our general understanding of rRNA modification processes that are prevalent in all domains of life.Support or Funding InformationThis work was supported in part by the Graduate Division of Biological and Biomedical Sciences (GDBBS) of the Laney Graduate School (LGS) of Emory University, NIH grant R01‐AI088025, and the Antibiotic Resistance and Therapeutic Discovery Training Program (ARTDTP) T32‐AI 106699.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.