The application of antibiotics to the treatment of bacterial infections revolutionized modern medical practice. In the decades since, a combination of improperly controlled usage and the remarkable ability of bacterial populations to develop resistance to these drugs has severely restricted the clinical usefulness of many antibiotics. We are now at a critical juncture where the majority of useful antibiotics have known and sometimes extensive resistance, and few novel replacements or strategies to combat the resistance problem are in active development. Many clinically useful antibiotics target the bacterial ribosome. One increasingly prevalent form of resistance to these drugs is alteration of the modification status of the ribosomal RNA (rRNA) via acquired or intrinsic methyltransferase enzymes. While enzymes responsible for incorporating these antibiotic resistance- associated rRNA modifications are known, we understand far less about their mechanisms of action (such as specific substrate recognition), which might offer viable new targets to counter the resistance. Further, we also currently have a poor understanding of the molecular basis for how rRNA methylation affects ribosome-antibiotic interactions. The experiments proposed in this application will directly address these critical gaps in our fundamental knowledge of rRNA methylation and bacterial antibiotic resistance. In the first two aims we will define the molecular mechanisms of ribosome subunit recognition by two different rRNA modification enzymes, the acquired aminoglycoside-resistance 16S rRNA (m7G1405) methyltransferases (Aim 1) and the intrinsic Mycobacterium tuberculosis methyltransferase TlyA (Aim 2). Next, we will develop a new computational and experimental framework for understanding antibiotic-methylated rRNA interactions (Aim 3). Our goal is to explain at the molecular level how rRNA modifications limit drug efficacy and how these effects can be evaded. Collectively, the results of these three independent but complementary aims will deepen our fundamental understanding of the molecular strategies used by rRNA modification enzymes and the impacts of rRNA methylation on antibiotic resistance in bacteria. Our results will support future innovative strategies to counter the resistance conferred by these enzymes, for example, by facilitating the development of inhibitors of m7G1405 methyltransferase activity or 30S substrate binding, and could also lead to the rational design of novel antimicrobials capable of fully evading the effects of rRNA modification.